Hcv Infection and Cryoglobulinemia

HCV Infection and Cryoglobulinemia

Franco Dammacco Editor

HCV Infection and Cryoglobulinemia Foreword by Jay H. Hoofnagle

Editor Franco Dammacco Department of Internal Medicine and Clinical Oncology University of Bari Medical School Bari, Italy

ISBN 978-88-470-1704-7 e-ISBN 978-88-470-1705-4 DOI 10.1007/978-88-470-1705-4 Springer Milan Heidelberg Dordrecht London New York Library of Congress Control Number: 2011929637 © Springer-Verlag Italia 2012 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, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the Italian Copyright Law in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the Italian Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

To my wife Tecla, lifetime partner. Without her unfailing inspiration, I would have been unable to read even a single page of the endless, puzzling and fascinating Book of Nature.

Foreword

Major breakthroughs in biomedical research are often followed by paradigm shifts in our understanding of diseases. This was particularly true after the landmark discovery of hepatitis C virus (HCV). The identification of a small but viral-specific RNA sequence in the serum of patients with non-A, non-B hepatitis led directly to the development of tests for antibody and viral RNA. Moreover, it fostered recognition of several facts: (1) that hepatitis C is the most common cause of chronic liver disease, cirrhosis, and liver cancer in most countries of the world; (2) that post-transfusion hepatitis can be prevented by screening for antibody; (3) that the implementation of simple public health measures would markedly decrease the rate of new infections with this virus; and (4) that a therapy with beneficial effects on the disease could actually cure the infection and permanently eradicate the virus. Another paradigm shift, perhaps less well known but just as ground-breaking, was the recognition of hepatitis C as the major cause of the uncommon and poorly understood “autoimmune” disease known as essential mixed cryoglobulinemia. Immediately obvious was that the name “essential mixed cryoglobulinemia” was no longer appropriate. The syndrome was not “essential” but instead due to hepatitis C, and it was not always “mixed.” Importantly, cryogloblins were detectable in low amounts in a large proportion of patients with chronic hepatitis C, not all of whom had vasculitis. Perhaps a better term for the condition is HCV-related cryoglobulinemic vasculitis. This change in terminology points out that the mere presence of cryoglobulins is not adequate; rather, the diagnosis also requires clinical signs and symptoms of vasculitis. What is the nature of the cryoglobulins found in HCV-related vasculitis? They appear to be circulating immune complexes and to consist of intact hepatitis C virions bound by IgG anti-HCV. These immune complexes are then aggregated into large macromolecular complexes by pentameric rheumatoid factor, that is, IgM antibody to IgG. The ability of the large viral-IgG-IgM complexes to precipitate in the cold (thus “cryo”globulins) is well known but they can also precipitate in tissues, such as skin, joints, kidneys, lung, intestine and nerves, in response to cold or to other, less well defined stresses (perhaps including mechanical pressure, hypoxia, minor tissue damage, local immune activation, or immunoglobulin receptors). Precipitation of these viral-antibody complexes gives rise to the clinical signs and symptoms of the disease, which most commonly presents as an episodic cutaneous vasculitis over the lower extremities that is often painful and pruritic and may be accompanied by local edema, joint or muscle aches, and fatigue. More serious forms of cryoglobulinemia result in injury to the lungs (interstitial pneumonitis), intestine (intestinal infarction or perforation), kidney (glomerulonephritis), and peripheral nerves (neuropathy), probably as a result of local vascular injury. These complications can be severe, vii

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Foreword

disabling, or indeed fatal. Fortunately, the clinical syndrome of cryoglobulinemic vasculitis is rare; but its infrequency does not help the unfortunate affected individuals. Therapies directed at HCV eradication can result in remission of the clinical syndrome, but not all patients respond to the current antiviral regimens, and others respond but have an incomplete remission of the vasculitis and its complications. HCV-related cryoglobulinemic vasculitis itself represents a special paradigm for understanding complex diseases. It is basically an uncommon complication of a common disease. What do we know of its pathogenesis? It is more frequently seen in women than men with hepatitis C and it arises during the chronic phase of the illness, after years if not decades of infection. The severity of injury correlates only roughly with the levels of serum cryoglobulins and rheumatoid factor and with the degree of complement activation. The production of cryoglobulins apparently derives from an overactivation of B cells, resulting in the production of clones that secrete anti-HCV and rheumatoid factor. The prolonged stimulation and activation of B cells can give rise to genetic alterations that may cause unregulated, self-sustaining clonal B cell proliferation and even B cell lymphoma. Thus, a common chronic viral infection of the liver appears to be a cause of both an autoimmune disease and cancer. Further elucidation of the pathogenesis of HCV-related cryoglobulinemic vasculitis may thus also lead to fundamental discoveries regarding the pathogenesis of other autoimmune diseases (rare complications of common infections?) and cancer (unregulated cell growth caused by chronic stimulation by microbial antigens or toxins?). Recently, important inroads have been made in understanding the natural history of HCV-related cryoglobulinemic vasculitis as well as its treatment. Antiviral therapies that reduce or eradicate HCV also improve the vasculitis, with sustained viral clearance usually followed by long-term remission of the cryoglobulinemia and the disappearance of serum cryoglobulins. As new therapies for hepatitis C become available (particularly the new direct-acting antiviral agents), those for cryoglobulinemia will likely become more effective. In patients in whom eradication of hepatitis C is not possible, therapies directed at B cell overactivity (particularly rituximab) may nonetheless be effective, at least in the short-term. Combined approaches of anti-B cell followed by potent antiviral therapy may represent the best therapeutic strategy for patients with advanced or resistant disease. The current monograph, “Hepatitis C Virus Infection and Cryoglobulinemia,” brings together an international group of investigators from the fields of basic virology, clinical medicine, rheumatology, hematology, nephrology, oncology, immunology, and genetics to focus on perhaps the most unusual manifestation of this chronic viral infection. The editor and authors should be congratulated for this most welcomed and combined effort at understanding and improving the management of HCV-related cryoglobulinemic vasculitis. A broader and more complete understanding of this complex disease is likely to bring further paradigm shifts in our understanding of how the intricate interactions between an infectious agent and susceptible host are responsible for clinical disease. Bethesda, MD, USA

Jay H. Hoofnagle Director, Liver Disease Research Branch National Institutes of Health

Contents

1

Introductory Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Franco Dammacco and Domenico Sansonno

Part I 2

1

Hepatitis C Virus Infection and the Role of the Immune System

Natural History, Pathogenesis, and Prevention of HCV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Edgar D. Charles, Lynn B. Dustin, and Charles M. Rice

11

3

Immune Control of HCV Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lynn B. Dustin

4

B Cell Activation: General to HCV-Specific Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vito Racanelli and Claudia Brunetti

37

Organ-Specific Autoimmunity in HCV-Positive Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Corrado Betterle and Fabio Presotto

43

5

Part II 6

Cellular Compartments of HCV Infection (and Replication)

HCV and Blood Cells: How Can We Distinguish Infection from Association? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lynn B. Dustin and Charles M. Rice

7

Mechanisms of Cell Entry of Hepatitis C Virus . . . . . . . . . . . . . . . . . . . Franco Dammacco and Vito Racanelli

8

HCV Infection of Hematopoietic and Immune Cell Subsets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tram N.Q. Pham and Tomasz I. Michalak

Part III 9

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55 63

69

Cryoglobulinemia and the Complement System

Cryoglobulinemia and Chronic HCV Infection: An Evolving Story . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jürg A. Schifferli and Marten Trendelenburg The Complement System in Cryoglobulinemia . . . . . . . . . . . . . . . . . . . Marten Trendelenburg

79 85

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Contents

The Pivotal Role of C1qR in Mixed Cryoglobulinemia . . . . . . . . . . . . . Domenico Sansonno, Loredana Sansonno, and Franco Dammacco

Part IV

12

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Structural and Genetic Features, Cytokines and Chemokines in Cryoglobulinemia

Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance . . . . . . . . . . . . . Peter D. Gorevic

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13

Molecular Insights into the Disease Mechanisms of Type II Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Valli De Re and Marica Garziera

14

The Role of VCAM-1 in the Pathogenesis of Hepatitis-C-Associated Mixed Cryoglobulinemia Vasculitis . . . . . . 113 Gilles Kaplanski

15

Up-Regulation of B-Lymphocyte Stimulator (BLyS) in Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . 119 Martina Fabris and Salvatore De Vita

16

Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis . . . . . . . . . . . . . . . . . . . . 127 Sabino Russi, Silvia Sansonno, Gianfranco Lauletta, Domenico E. Sansonno, and Franco Dammacco

17

Serum a-Chemokine CXCL10 and b-Chemokine CCL2 Levels in HCV-Positive Cryoglobulinemia . . . . . . . . . . . . . . . . . 137 Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, IIaria Ruffilli, Caterina Mancusi, Ele Ferrannini, and Poupak Fallahi

Part V

Clinical Manifestations of Cryoglobulinemia

18

Experimental Models of Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . 145 Charles E. Alpers, Tomasz A. Wietecha, and Kelly L. Hudkins

19

The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . 155 Clodoveo Ferri, Alessandro Antonelli, Marco Sebastiani, Michele Colaci, and Anna Linda Zignego

20

Classification of Cryoglobulinemic Vasculitis. . . . . . . . . . . . . . . . . . . . . 163 Salvatore De Vita and Luca Quartuccio

21

Demographic and Survival Studies of Cryoglobulinemic Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Giuseppe Monti, Francesco Saccardo, and Laura Castelnovo

22

HCV-Associated Membranoproliferative Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Christos P. Argyropoulos, Sheldon Bastacky, and John Prentiss Johnson

Contents

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23

Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Gianfranco Ferraccioli, Francesca Faustini, and Elisa Gremese

24

Endocrine Manifestations of HCV-Positive Cryoglobulinemia . . . . . . 191 Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, Alda Corrado, Andrea Di Domenicantonio, and Poupak Fallahi

25

Cutaneous Cryoglobulinemic Vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . 195 Konstantinos Linos, Bernard Cribier, and J. Andrew Carlson

26

Peripheral Neuropathy and Central Nervous System Involvement in Cryoglobulinemia . . . . . . . . . . . . . . . . 209 Salvatore Monaco, Sara Mariotto, and Sergio Ferrari

27

Long-Term Course of Patients with Mixed Cryoglobulinemia . . . . . . . 219 Damien Sene and Patrice P. Cacoub

28

HBV/HCV Co-infection and Mixed Cryoglobulinemia . . . . . . . . . . . . . 227 Massimo Galli and Salvatore Sollima

29

Clinical and Immunological Features of HCV/HIV Co-infected Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . 233 David Saadoun and Patrice P. Cacoub

30

HCV-Negative Mixed Cryoglobulinemia: Facts and Fancies . . . . . . . . 239 Massimo Galli, Salvatore Sollima, and Giuseppe Monti

31

Cryoglobulinemia in HCV-Positive Renal Transplant and Liver Transplant Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Lionel Rostaing, Hugo Weclawiak, and Nassim Kamar

Part VI

HCV Infection, Cryoglobulinemia and Non-Hodgkin’s Lymphomas

32

Chromosome Abnormalities in HCV-Related Lymphoproliferation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 Cristina Mecucci, Gianluca Barba, and Caterina Matteucci

33

Molecular Features of Lymphoproliferation in Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 Valli De Re and Maria Paola Simula

34

The Higher Prevalence of B-Cell Non-Hodgkin’s Lymphoma in HCV-Positive Patients with and Without Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 Franco Dammacco and Domenico Sansonno

35

Incidence and Characteristics of Non-Hodgkin’s Lymphomas in HCV-Positive Patients with Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Pietro Enrico Pioltelli, Giuseppe Monti, Maurizio Pietrogrande, and Massimo Galli

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Contents

Waldenström’s Macroglobulinemia Associated with Cryoglobulinemia: Pathogenetic, Clinical, and Therapeutic Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 Meletios A. Dimopoulos and Efstathios Kastritis

Part VII

Therapy of Cryoglobulinemia

37

Should HCV-Positive Asymptomatic Patients with Mixed Cryoglobulinemia Be Treated with Combined Antiviral Therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 José Luis Calleja Panero, Juan de la Revilla Negro, and Fernando Pons Renedo

38

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 Francesco Zaja, Stefano Volpetti, Stefano De Luca, and Renato Fanin

39

Rituximab in Cryoglobulinemic Vasculitis: First- or Second-Line Therapy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Peter Lamprecht and Paul Klenerman

40

PIRR Therapy in HCV-Related Mixed Cryoglobulinemia . . . . . . . . . . 315 Franco Dammacco and Domenico Sansonno

41

Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications . . . . . . . . . . . . 325 Franco Dammacco, Cinzia Conteduca, and Domenico Sansonno

42

Active or Indolent Cutaneous Ulcers in Cryoglobulinemia: How Should They Be Treated? . . . . . . . . . . . . . . 335 Maurizio Pietrogrande

43

Double Filtration Plasmapheresis: An Effective Treatment of Cryoglobulinemia. . . . . . . . . . . . . . . . . . . . . 337 Alfonso Ramunni and Paola Brescia

44

Emergency in Cryoglobulinemia: Clinical and Therapeutic Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Francesco Saccardo, Laura Castelnovo, and Giuseppe Monti

45

Novel Therapeutic Approaches to Cryoglobulinemia: Imatinib, Infliximab, Bortezomib, and Beyond . . . . . . . . . . . . . . . . . . . 349 Giampaolo Talamo and Maurizio Zangari

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Contributors

Charles E. Alpers Department of Pathology, University of Washington, Seattle, WA, USA Division of Nephrology, Department of Medicine, University of Washington, Seattle, WA, USA Alessandro Antonelli Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Christos P. Argyropoulos Renal and Electrolyte Division, Department of Internal Medicine, University of Pittsburgh, Pittsburgh, PA, USA Gianluca Barba Hematology and Clinical Immunology Unit, Clinical and Experimental Medicine, University of Perugia, Perugia, Italy Sheldon Bastacky Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA Corrado Betterle Unit of Endocrinology, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy Paola Brescia Division of Nephrology, Department of Internal and Public Medicine, University of Bari, Bari, Italy Claudia Brunetti Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Patrice P. Cacoub UMR 7211 (UPMC/CNRS), U 959 (INSERM), Université Pierre Marie Curie, Paris, France Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France José Luis Calleja Panero Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain J. Andrew Carlson Division of Dermatology and Dermatopathology, Department of Pathology, Albany Medical College, Albany, NY, USA Laura Castelnovo Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy Edgar D. Charles Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA Michele Colaci Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy xiii

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Cinzia Conteduca Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Alda Corrado Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Bernard Cribier Dermatologique Clinique, Les Hopitaux Universtaires de Strasbourg, Strasbourg, France Franco Dammacco Department of Biomedical Sciences and Clinical Oncology, University of Bari Medical School, Bari, Italy Stefano De Luca Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Valli De Re Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy Salvatore De Vita Clinic of Rheumatology, Department of Medical and Biological Sciences, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy Andrea Di Domenicantonio Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Meletios A. Dimopoulos Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece Lynn B. Dustin Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA Martina Fabris Clinical Pathology and Clinic of Rheumatology, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy Poupak Fallahi Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Renato Fanin Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Francesca Faustini Division of Rheumatology, Institute of Rheumatology and Affine Sciences(IRSA), CIC – Catholic University of the Sacred Heart, Rome, Italy Gianfranco Ferraccioli Division of Rheumatology, Institute of Rheumatology and Affine Sciences(IRSA), CIC – Catholic University of the Sacred Heart, Rome, Italy Ele Ferrannini Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Silvia Martina Ferrari Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Sergio Ferrari Department of Neuroscience, University of Verona, Verona, Italy Clodoveo Ferri Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy

Contributors

Contributors

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Massimo Galli Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, Università di Milano, Milan, Italy Marica Garziera Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy Peter D. Gorevic Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA Elisa Gremese Division of Rheumatology, Institute of Rheumatology and Affine Sciences(IRSA), School of Medicine, CIC – Catholic University of the Sacred Heart, Rome, Italy Kelly L. Hudkins Department of Pathology, University of Washington, Seattle, WA, USA John Prentiss Johnson Renal and Electrolyte Division, Department of Internal Medicine, University of Pittsburgh, Pittsburgh, PA, USA Nassim Kamar Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France INSERM U858/I2MR, Equipe 10, CHU Rangueil, Toulouse, France Gilles Kaplanski Service de Médecine Interne, Hôpital de la Conception, Marseille, France Efstathios Kastritis Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece Paul Klenerman Peter Medawar Building for Pathogen Research and National Institute for Health Research Biomedical Research Centre, University of Oxford, Oxford, UK Peter Lamprecht Department of Rheumatology, Vasculitis Center UKSH & Clinical Center Bad Bramstedt, University of Lübeck, Lübeck, Germany Gianfranco Lauletta Department of Internal Medicine and Human Oncology, University of Bari Medical School, Bari, Italy Konstantinos Linos Department of Pathology, Albany Medical College, Albany, NY, USA Caterina Mancusi Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Sara Mariotto Department of Neuroscience, University of Verona, Verona, Italy Caterina Matteucci Hematology and Clinical Immunology Unit, Clinical and Experimental Medicine, University of Perugia, Perugia, Italy Cristina Mecucci Hematology and Clinical Immunology Unit, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy Tomasz I. Michalak Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Center, Memorial University, St. John’s, NL, Canada

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Salvatore Monaco Department of Neuroscience, University of Verona, Verona, Italy Giuseppe Monti Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy Tram N.Q. Pham Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Center, Memorial University, St. John’s, NL, Canada Maurizio Pietrogrande Internal Medicine Unit, Policlinico San Marco, Osio Sotto, University of Milan, Milan, Italy Pietro Enrico Pioltelli Hematology Unit, Ospedale San Gerardo, Monza, Italy Fabio Presotto Department of Medical and Surgical Sciences, Medical University of Padua, Padua, Italy Unit of Internal Medicine, General Hospital of Este (Padua), Padua, Italy Luca Quartuccio Clinical Pathology and Clinic of Rheumatology, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy Vito Racanelli Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy Alfonso Ramunni Division of Nephrology, Department of Internal and Public Medicine, University of Bari, Bari, Italy Fernando Pons Renedo Department of Gastroenterology and Hepatology, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain Juan de la Revilla Negro Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain Charles M. Rice Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA Lionel Rostaing Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France INSERM U563, IFR 30, CHU Purpan, Toulouse, France Ilaria Ruffilli Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy Sabino Russi Department of Internal Medicine and Human Oncology, University of Bari Medical School, Bari, Italy David Saadoun Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France Francesco Saccardo Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy Domenico E. Sansonno Liver Unit, Department of Biomedical Sciences and Clinical Oncology, University of Bari Medical School, Bari, Italy

Contributors

Contributors

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Loredana Sansonno Laboratory of Genetics, Department of Biomedical Sciences, University of Foggia Medical School, Foggia, Italy Silvia Sansonno Clinical and Experimental Pharmacology – DOMERT, Centro di Riferimento Oncologico, Aviano, Italy Jürg A. Schifferli Division of Internal Medicine, Department of Medicine, University Hospital Basel, Basel, Switzerland Marco Sebastiani Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy Damien Sene Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France Maria Paola Simula Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy Salvatore Sollima Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, University of Milan, Milan, Italy Giampaolo Talamo Division of Hematology-Oncology, Penn State Hershey Cancer Institute, Hershey, PA, USA Marten Trendelenburg Clinic for Internal Medicine and Laboratory for Clinical Immunology, University Hospital Basel, Basel, Switzerland Stefano Volpetti Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Hugo Weclawiak Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France Tomasz A. Wietecha Department of Pathology, University of Washington, Seattle, WA, USA Francesco Zaja Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy Maurizio Zangari Division of Hematology, Blood/Marrow Transplant and Myeloma Program, University of Utah, Salt Lake City, UT, USA Anna Linda Zignego Department of Internal Medicine, University of Florence Medical School, Florence, Italy

Part I Hepatitis C Virus Infection and the Role of the Immune System

1

Introductory Remarks Franco Dammacco and Domenico Sansonno

The Strength of the Truth: Where there is truth there is also true knowledge. And where there is no truth there cannot be true knowledge. This is why the word knowledge is associated to the name of God. And where there is true knowledge there is always happiness. Mohandas Karamchand (‘Mahatma’) Gandhi

The first observation of cold-induced precipitation of serum proteins dates back to 1933, when Prof. Maxwell Myer Wintrobe, the legendary physicianscientist who laid the foundations of modern hematology, described, in collaboration with Dr. M. V. Buell, an unusual case of multiple myeloma in a woman whose serum reversibly precipitated at cold temperatures [1]. Fourteen years later, Lerner and Watson [2] showed cold-precipitable proteins to be gammaglobulins and named them “cryoglobulins.” They also coined the term “cryoglobulinemia” to indicate the corresponding clinical condition. At that time, cryoglobulins were thought to be structurally formed by a single protein. In 1962, testing isolated and purified cryoglobulins by anion-exchange chromatography, Lospalluto et  al. [3] used analytical ultracentrifugation to demonstrate that solubilized cryoproteins indeed contained two components, ­designated at that time as 7S and 19S on the basis of their sedimentation coefficients. These fractions ­corresponded to the immunoglobulins that we now call IgG and IgM, respectively. They were also able to show that a ­number of positive reactions for ­rheumatoid factor (RF) were associated with the 19S fraction and that, while the 7S (IgG) fraction could indifferently F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_1, © Springer-Verlag Italia 2012

belong to the patient’s serum or derive from pooled normal donors (Cohn’s faction II), replacement of the patient’s 19S (IgM) with a ­normal  IgM counterpart resulted in the disappearance of the ­cryoprecipitating properties. Thus, the 19S ­gammaglobulin component behaved as an incomplete cryoglobulin. Meltzer and Franklin [4] and Meltzer et  al. [5], in 1966, provided an accurate description of the typical clinical symptoms associated with cryoglobulinemia. They also confirmed that cryoglobulins consisted of two different globulin components and were consistently endowed with RF activity. Due to ignorance regarding its etiology, they called this clinical condition “essential” mixed cryoglobulinemia (MC). In the following years, many investigators carried out immunochemical studies of a number of isolated cryoglobulins and demonstrated their structural heterogeneity. Based on these studies and on a review of 86 patients, Brouet et al. [6] classified cryoglobulinemia into three main types, a classification that is still largely accepted. Type I cryoglobulins are single monoclonal immunoglobulins, typically IgM or IgG, but rarely also IgA and even Bence Jones proteins. Types II and III cryoglobulins are characterized by polyclonal IgG associated with monoclonal (type II) or polyclonal (type III) IgM. Since RF activity is regularly associated with the IgM fraction, immune complexes are formed through the binding of IgM-RF to autologous IgG. The immunochemical characterization of ­different cryoglobulins and their possible significance as ­inducers of tissue lesions were emphasized 1

2

F. Dammacco and D. Sansonno

Table 1.1  Important chronological advances (milestones) in the study of cryoglobulinemia Year 1933 1947 1962 1966 1968–1970 1973…

1974 1977… 1987 1990–1994 1994… 2003

2004 2005 2002–2005

2010

2010 2011…

Observations A cold-precipitable protein is discovered in the serum of a patient with multiple myeloma The terms “cryoglobulin” and “cryoglobulinemia” are proposed and the phenomenon is observed in various diseases Chromatographic separation of cryoglobulins shows their mixed (7 + 19S) nature and ­rheumatoid factor (RF) activity is found to be associated to the 19S fraction The clinical picture and the structural heterogeneity of so-called “essential” mixed ­cryoglobulinemia (MC) are clearly defined Different cryoglobulins are immunochemically characterized and their significance as inducers of tissue lesions is emphasized A number of IgMk RFs from patients with MC are shown to display a major cross-reactive idiotype (CRI) designated WA. The prevalence of CRIs has subsequently been the object of an extensive review aimed to assess the relationship of MC to rheumatic and lymphoproliferative disorders Cryoglobulins are classified into three main types The possible etiological role of hepatitis B virus in “essential” MC is claimed, though not confirmed in later studies Therapy with recombinant interferon-a (IFN-a) is shown to be effective in patients with “essential” MC The large majority of patients with MC are found to be hepatitis C virus (HCV)-infected Potential progression from MC to overt non-Hodgkin’s lymphoma (NHL) is reported by different groups, though with wide geographic variations Rituximab, a chimeric anti-CD20 monoclonal antibody, is found to be clinically effective in patients with relapsing or refractory MC, but it often results in an increase in the serum HCV RNA levels A B-cell clonal expansion involving RF-secreting cells is demonstrated to be the biological hallmark of MC By analogy with non-cryoglobulinemic chronic HCV infection, pegylated IFN-a plus ribavirin is proposed as the standard of care in HCV-positive MC IFN-a (plus RBV) anti-viral therapy is demonstrated to induce a ³75% complete remission of low-grade NHL in HCV-positive MC patients and similar results are reported in a later systematic review In HCV-infected asymptomatic patients, WA B-cells bearing the WA cross-idiotype are defined as a marker for the development of cryoglobulinemic vasculitis and associated B cell malignancies, and should therefore represent a specific therapeutic target The combination of pegylated IFN-a, ribavirin and rituximab (PIRR therapy) is shown to provide a high percentage of long-term responses Administration of new protease inhibitors and new anti-CD20 monoclonal antibodies (Ofatumumab) is likely to result in better results than PIRR therapy

Referencesa [1] [2] [3] [4, 5] [7, 8] [9, 10]

[6] [12–14] [45] [15–23] [35–40] [47, 48]

[25, 26] [49] [41, 42]

[44]

[50, 51] Clinical trials in progress

 eferences are an accurate selection of the oldest pertinent publications dealing with each observation, but do not necessarily reflect R strict chronological priority

a

by Wager et al. [7] and Barnett et al. [8]. In  ­addition, ­several IgMk RFs purified from MC patients were shown to have private idiotypes, hence a unique primary amino acid sequence and two major cross-reactive idiotypes (CRIs), although the best characterized of them were Wa-positive RFs [9, 10]. These historical data in the developing story of cryoglobulinemia and a number of subsequent advances up to the present time are summarized in Table 1.1. It soon became evident that to put forward a wellgrounded clinical suspicion of cryoglobulinemia was

not a difficult task in patients complaining of: (a) recurrent episodes of palpable purpura, usually confined to the lower limbs and often to the buttocks as well, which frequently result in chronic dyschromia, namely, a brownish pigmentation over the legs and the ankles secondary to hemosiderin deposits in the sites previously affected with the purpuric eruptions; leg ulcers and cold urticaria may also be seen, though much less often; (b) arthralgias affecting the small and/or large joints to a variable extent; (c) intense asthenia and chronic fatigability. The occurrence of these symptoms

1  Introductory Remarks

is indeed so frequent that the triad purpura/asthenia/ arthralgia is considered a practically unfailing stigma of this clinical condition [11]. However, since MC is a systemic vasculitis, along with liver, skin and joint involvement, there may be additional manifestations, including nephropathy and sensory-motor neuropathy, the latter being particularly resistant to treatment. As already shown by previous studies [3–5], the occurrence in the serum of RF and the low circulating levels of the complement fractions C3 and even more strikingly C4 strongly argue that cryoglobulinemia should be considered an immune complex-mediated vasculitis. However, the inciting agent capable of inducing immune-complex formation has remained unknown for a long time. In 1977, Levo et  al. [12] claimed they had found a correlation between cryoglobulinemia and hepatitis B virus (HBV) on the basis of clinical and serological features, which drew attention to the ­relatively frequent liver involvement in MC patients and the electron microscopy evidence of structures resembling the spheres, tubules and Dane particles ­characteristic of HBV infection in isolated and purified cryoprecipitates. Although the same authors hypothesized that other, at that time undetermined viruses were also involved, evidence of HBV infection in most of the patients with MC was not confirmed by other groups [13, 14], suggesting that this presumed correlation indeed reflected the prevalence of HBV infection in the geographic area in which the research was carried out. When at the beginning of the 1990s, in step with the availability of new reagents, it became possible to detect the serum occurrence of antibodies to the hepatitis C virus (HCV) and shortly after of HCV RNA, many groups in Europe [15–21] and the USA [22, 23] consistently reported in the space of <4  years that a high proportion of patients with “essential” MC were indeed HCV-infected. Based on these results, the already mentioned data by Levo et al. [12] on the possible role of HBV infection in MC were further disproved. On the contrary, the hallmark of a significantly high prevalence of HCV infection in MC has challenged its taxonomy, leaving the definition of an “essential” disorder to the small percentage (possibly <10%) of HCV-negative patients, whereas over 90% of cases should be considered “HCV-related.” But even in the minority of patients apparently lacking evidence of HCV infection, an occult infection is sometimes demonstrated by the detection of HCV

3

RNA genomic sequences in the relapse phase ­following therapy or during a flare of cryoglobulinemia [24], further narrowing the number of patients with truly “essential” MC. The awareness that MC is to be considered an undisputable extrahepatic manifestation of HCV infection has generated new impetus and great enthusiasm in carrying out additional research. It has now been established that an underlying VH1-69 positive B-cell clonal expansion mainly involving RF-secreting cells is the biological hallmark of MC [25, 26]. Immune complexes have been shown to consist of HCV core protein linked to IgG molecules with specific anti-core reactivity, which in turn are bound to IgM molecules with RF activity. The ensuing multi-molecular complexes easily bind the C1q protein, which results in their specific binding to the C1q receptors expressed on the membranes of endothelial cells [27]. However, it seems likely that structurally heterogeneous coldprecipitating immune complexes reflect different pathogenetic mechanisms and are capable of variable tissue damage. This is schematically depicted in Fig.  1.1. Although the exact mechanism(s) by which these immune complexes acquire cold-insoluble properties in a minority of HCV-infected patients are poorly understood, their deposition on the walls of small- and medium-sized arteries and veins, with consequent activation of the complement cascade, is the source of the vascular injury and hence a variable spectrum of clinical features. The model in Fig.  1.1 (viremic phase: Fig.  1.1a–d) implies that HCV proteins are a seemingly unfailing structural component of such complexes. Even so, the fact that serum cryoglobulins may sometimes be detectable in patients achieving a sustained or a long-term virologic response following treatment indicates that structurally different, HCVlacking but nonetheless cold-insoluble and vasculitisinducing immune complexes are still formed in these patients (Fig. 1.1, non-viremic phase: Fig. 1.1e, f). Thus, cryoglobulinemic vasculitis is probably a multi-factorial process in which the actions of immune complexes are the better studied “half of the moon,” whereas the other half is so far largely unexplored. For example, the critical role played by HCV in a wide variety of inflammatory processes seems to be mediated by the expression of transcription factors and proinflammatory genes, such as interleukins and chemokines, members of the tumor necrosis factor (TNF) superfamily. Available evidence indicates that

4

IgM anti-IgG F(ab’)2

a

HCV particle

HCV particle E2 Antigen IgG IgG

IgM RF

HCV particle

VIREMIC PHASE

CD81

Cell Membrane

b

HCV Core protein

HCV Core protein

c

IgG

C1qR

Integrin IgM C1q

d

Cell Membrane

C1q HCV Core protein

C1qR

Integrin

IgM

C1qR

Cell Membrane

e C4

NON-VIREMIC PHASE

Fig. 1.1  Proposed working model for cryoprecipitating immune complex (IC)-mediated vascular damage in HCV-related mixed cryoglobulinemia. Viremic phase: The ICs include HCV particles and non-enveloped HCV core-encoded protein as constitutive antigens. (a) IC-containing IgM molecules directed against Fc and F(ab¢)2 determinants of IgG with anti-E2 antigen reactivity bind to the endothelial cell surface through the HCV E2-CD81 tetraspanin protein interaction. (b) Free non-enveloped HCV core protein binds to the endothelial cell membrane via the C1q receptor (C1qR). HCV core protein is linked to the IgG molecule, bearing specific anti-core reactivity; IgG in turn is bound to IgM, which has rheumatoid factor (RF) activity. These multimolecular ICs are good acceptors of C1q protein, resulting in binding to endothelial cells via the collagen-like domain of C1qR. (c) HCV core directly binds to C1qR through a globular-head domain. The HCV core-C1qR interaction has been assumed to play a critical role in cell-activating mechanisms by intracellular signaling via integrins. (d) Globular C1qR, proteolytically cleaved and released from the cell surface, circulates bound to HCV core protein, IgM RF molecule, and C1q protein. In all instances, activation of the classical pathway of the complement cascade is dependent on IgM or IgG molecules present within the ICs. Binding of C1q induces conformational changes in the C1 complex and leads to activation of C1r and C1s serine protease subunits. Non-viremic phase: In this clinical phase, which usually follows antiviral treatment, ICs are not composed of detectable HCV particles or HCVencoded proteins, suggesting that viral components are not essential for IC formation. These ICs, however, retain the biological properties and potential pathogenicity roughly similar to those described for ICs of the viremic phase. In-situ-fixed (e) or circulating (f) ICs are capable of activating the complement cascade and determining tissue damage

F. Dammacco and D. Sansonno

C5b

IgG

Cell Membrane IgM C1q

C1qR

Integrin

f

C1q

IgM

C1qR Cell Membrane

1  Introductory Remarks

chemokines are essential to the regulation of T and B cell migration and sequestration in HCV-infected compartments [28]. Our group has particularly studied the chemokine CXCL13 (also referred to as B-cellattracting chemokine-1, BCA-1) which, by interaction with its receptor CXCR5 expressed on circulating B cells and a subset of memory CD4+ T cells, regulates the homeostatic trafficking of B cells. Serum levels of BCA-1 were found to be significantly higher in MC patients than in healthy controls and in HCV-infected non-cryoglobulinemic patients. Interestingly, the highest levels of BCA-1 in MC were directly related to active cutaneous vasculitis. In addition, specific CXCL13 mRNA expression in liver and skin biopsy tissues was also up-regulated in MC patients with active cutaneous vasculitis [29]. Whether these two biologic compartments are inter-related remains to be ascertained. The immunobiology of HCV and the intriguing virus/immune response relationships are presently the matter of intensive investigations [30]. A puzzling phenomenon is the observation that, although the virus is recognized and targeted by innate as well as cellular and humoral immune mechanisms, it is capable of establishing a persistent infection in the large majority of patients. The mechanisms are probably manifold. First of all, in addition to the obvious involvement of hepatocytes, HCV RNA may also target B cells, dendritic cells, monocytes and even cells of the digestive tract. Thus, it cannot be excluded that HCV persists for years in one or more of these cell types in a latent form and with an extremely low number of viral genomes, in spite of its clearance from the serum following antiviral therapy; this condition may nonetheless potentially give rise to overt recurrence. Probably, HCV is recognized through innate immunity, which results in the induction of a rapid interferon (IFN) response [31]. However, viral evasion strategies may involve a number of mechanisms: (a) mutational escape; (b) inhibition of the innate immune response through blockage of the antiviral effector functions of IFN-induced target genes; (c) defects in NK cell stimulation of dendritic cell maturation and hence nonspecific impairment in dendritic cell function; (d) deficient T-cell help and CD8+ T-cell exhaustion; (e) a rapid response by memory T cells that may block the primary activation of naïve T cells endowed with higher-affinity receptors specific for HCV antigens (the “original antigenic sin” phenomenon [32]); (f) a reduced function of regula-

5

tory T cells; (g) failure of neutralizing antibodies to clear established infection because of continued viral mutation and antibody-dependent selection; and no doubt others. These complex and possibly interwoven features, which are under intensive investigation in non-cryoglobulinemic HCV-infected patients, are obviously of the utmost importance in cryoglobulinemic patients as well and are discussed in detail in the following chapters. In addition to MC, a foreseeable effect deriving from HCV infection of cells of the lymphoid system is the stimulation of B-cell proliferation, resulting in immune-complex formation and the production of autoantibodies along with the onset of lymphoproliferative disorders. Indeed, HCV infection has been implicated in the pathogenesis of autoimmune ­disorders as it occurs in a subset of patients with membrano-proliferative glomerulonephritis, autoimmune hepatitis, Sjögren’s syndrome, and thyroiditis [33], but also in triggering the mechanisms leading to monoclonal gammopathies and non-Hodgkin’s lymphomas (NHLs) [34]. Additional infectious, genetic, and environmental factors may similarly play a role. Since MC frequently shares clinical, serological, and pathological features with the above-mentioned disorders, it has been suggested as “a cross-road between autoimmune and lymphoproliferative disorders” [33]. A most intriguing feature stemming from the evidence that HCV has tropism for hepatocytes and lymphocytes is the potential transformation of HCV infection into B-cell NHL, which is seen in a minority of infected patients. Obviously, infection with HCV (and HBV) implies a much higher relative risk of developing hepatocellular carcinoma rather than NHL. Nevertheless, a causal relationship between HCV infection and a small subset of NHLs is gaining  convincing support. Though HCV-positive MC should be considered a benign lymphoproliferative condition, in a subset of patients it may precede (sometimes by several years) the development of NHL. Figure 1.2 is an attempt to outline the possible relationships of HCV infection with NHL and MC. Striking geographic variations have been described in the frequency of this association, underscoring the importance of genetic and environmental factors and possibly the existence of lymphotropic HCV variants. Beginning in 1994 [35, 36], an increasing number of reports, including multi-center studies [37], recent reviews [38, 39] and population-based cohort studies

6 Fig. 1.2  A rough estimate of the relationships between chronic HCV infection and lymphoproliferative disorders. The percentages indicated reflect the experience of our group, but large variations may be found according to geographic, genetic, and environmental factors. Given the relatively high worldwide prevalence of HCV infection (a reservoir of approximately 170 million people), an important question that remains to be answered is how many chronically HCV-infected patients will eventually develop nonHodgkin’s lymphoma

F. Dammacco and D. Sansonno

HCV-POSITIVE PATIENTS ≥ 90%

∼ 12%

∼ 10%

? TYPE II MIXED B-CELL NON-HODGKIN’S LYMPHOMA (B-NHL)

∼ 7%

CRYOGLOBULINEMIA

(∼ 2% OF ALL B-NHLs)

[40], have consistently emphasized the potential oncogenic role of HCV, with the virus acting in concert with largely undefined host factors. Since the HCV genome is a single-stranded RNA molecule and given the current understanding of its replication pathways, identification of the possible mechanisms underlying the onset of HCV-induced B-cell NHL represents one of the most stimulating challenges of current onco-hematology. The role of HCV in lymphomagenesis is strongly suggested by the results of trials showing complete or partial remission of NHL in HCV-positive patients (but not in HCV-negative patients) following antiviral ­therapy [41, 42]. In addition, HCV-positive patients who achieve a treatment-induced sustained virologic response seem to have a lower risk of developing NHL than untreated patients, indicating that clearance of the virus exerts a preventive effect on lymphomagenesis [43]. However, a crucial but still undefined point is clarification of the mechanisms whereby chronic HCV infection may eventually induce the onset of NHL. The most logical conjecture is that lymphomagenesis is indeed a polyfactorial process in which chronic B-cell stimulation by HCV antigens (possibly HCVE2), and/or interaction of HCV-E2 with the tetraspanin CD81 receptor, and/or direct infection and replication of HCV into permissive (such as CD5-positive) B cells play a role. Starting from the observation that monoclonal RFs bearing the Wa cross-idiotype are detected in the large majority of HCV-infected patients with cryoglobulinemic vasculitis, it has been suggested that

Wa B-cells in asymptomatic patients should be considered as a marker for the development of vasculitis and B-cell malignancies [44]. If these patients develop a B-cell malignancy unresponsive to anti-viral therapy, then treatment with anti-WA cross-idiotype should be a logical consequence. As an immune-complex vasculitis, for several years MC has been treated with corticosteroids, often in association with immunosuppressive drugs in patients with highly active and resistant disease. However, responses have been poor and transient in most cases. Based on the double assumption that some clinical features of MC are suggestive of a viral etiology and that the consistent occurrence of a monoclonal RF is indicative of a lymphoproliferative process, the use of recombinant IFN-a in the therapy of MC was proposed by Bonomo et al. [45] in 1987 – a few years before the association between MC and HCV infection was clearly established. Indeed, these authors observed a noteworthy clinical improvement associated with a remarkable reduction of circulating cryoglobulins in seven out of seven MC patients who were treated with this anti-viral drug. A number of subsequent reports (reviewed in [46]) confirmed the efficacy and safety of this treatment, though relapse occurred in a sizable proportion of patients a few months after its discontinuation. In 2003, two consecutive papers published in the same journal [47, 48] showed that in 70–80% of HCV-positive MC patients resistant to IFN-a, the administration of rituximab, an anti-CD20 monoclonal antibody, resulted in rapid

1  Introductory Remarks

improvement of clinical, biological, and immunological features, although HCV RNA increased to roughly twice the baseline levels in the responders. Thus, pegylated IFN-a plus ribavirin has been proposed as the standard of care in HCV-related systemic vasculitis [49], but in “difficult” situations, represented by patients undergoing severe flare-ups and/or refractory to the standard of care, the combination of IFN-a plus ribavirin plus rituximab has been found to significantly improve overall results [50, 51]. Hopefully, even better clinical results will derive from the use of new protease inhibitors and more advanced anti-CD20 monoclonal antibodies, such as ofatumumab. Against this background, it was felt that times were ripe to produce a state-of-the-art survey of the multi-faceted picture of cryoglobulinemia. The most qualified authors have been invited to contribute critical articles reviewing significant developments related to each of the seven sections in which this volume has been divided: from basic mechanisms governing ­interactions between HCV and the immune system to the immunochemical characterization of cryoglobulins and the frequently concomitant serological ­abnormalities; from genetic features and the role of certain ­cytokines and chemokines to the cellular compartments of HCV infection and replication; from the clinical ­manifestations of cryoglobulinemic patients and their potential susceptibility to develop NHL to conventional treatment of the syndrome and the newer, promising therapeutic advances. It is hoped that a multi-disciplinary approach to this striking and ­fascinating disease will lead, in the not too distant future, to the identification of effective preventive and therapeutic measures.

References 1. Wintrobe MM, Buell MV (1933) Hyperproteinemia associated with multiple myeloma with report of a case in which an extraordinary hyperproteinemia was associated with thrombosis on the retinal veins and symptoms suggesting Raynaud’s disease. Bull Johns Hopkins Hosp 52:156–165 2. Lerner AB, Watson CJ (1947) Studies of cryoglobulins. I.  Unusual purpura associated with the presence of a high concentration of cryoglobulin (cold precipitable serum globulin). Am J Med Sci 214:410–415 3. Lospalluto J, Dorward B, Miller W Jr et al (1962) Cryoglobu­ linemia based on interaction between a gamma macroglobulin and 7S gamma globulin. Am J Med 32:142–147

7 4. Meltzer M, Franklin EC (1966) Cryoglobulinemia – a study of twenty-nine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 40: 828–836 5. Meltzer M, Franklin EC, Elias K et  al (1966) Cryoglobu­ linemia – a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med 40: 837–856 6. Brouet JC, Clauvel JP, Danon F et al (1974) Biological and clinical significance of cryoglobulins. Am J Med 57: 775–788 7. Wager O, Mustakallio KK, Räsänen JA (1968) Mixed IgAIgG cryoglobulinemia. Immunological studies and case reports of three patients. Am J Med 44:179–187 8. Barnett EV, Bluestone R, Cracchiolo A III et  al (1970) Cryoglobulinemia and disease. Ann Intern Med 73:95–107 9. Kunkel HG, Agnello V, Joslin FG et  al (1973) Crossidiotypic specificity among monoclonal IgM proteins with anti-globulin activity. J Exp Med 137:331–342 10. Gorevic PD, Frangione B (1991) Mixed cryoglobulinemia cross-reactive idiotypes: implications for the relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 28:79–94 11. Dammacco F, Sansonno D, Piccoli C et  al (2001) The ­cryoglobulins: an overview. Eur J Clin Invest 31:628–638 12. Levo Y, Gorevic PD, Kassab HJ et  al (1977) Association between hepatitis B virus and essential mixed cryoglobulinemia. N Engl J Med 297:946–947 13. Galli M, Monti G, Invernizzi F et  al (1992) Hepatitis B virus-related markers in secondary and in essential mixed cryoglobulinemias: a multicentric study of 596 cases. The Italian Group for the Study of Cryoglobulinemias (GISC). Ann Ital Med Int 7:209–214 14. Christodoulou DK, Dalekos GN, Merkouropoulos MH et al (2001) Cryoglobulinemia due to chronic viral hepatitis infections is not a major problem in clinical practice. Eur J Intern Med 12:435–441 15. Pascual M, Perrin L, Giostra E et al (1990) Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 162:569–570 16. Ferri C, Greco F, Longombardo G et al (1991) Antibodies against hepatitis C virus in mixed cryoglobulinemia patients. Infection 19:417–420 17. Dammacco F, Sansonno D (1992) Antibodies to hepatitis C virus in essential mixed cryoglobulinaemia. Clin Exp Immunol 87:352–356 18. Misiani R, Bellavita P, Fenili D et al (1992) Hepatitis C virus infection in patients with essential mixed cryoglobulinemia. Ann Intern Med 117:573–577 19. Cacoub P, Musset L, Lunel Fabiani F et al (1993) Hepatitis C virus and essential mixed cryoglobulinaemia. Br J Rheumatol 32:689–692 20. Dammacco F, Sansonno D, Cornacchiulo V et  al (1993) Hepatitis C virus infection and mixed cryoglobulinemia: a striking association. Int J Clin Lab Res 23:45–49 21. Marcellin P, Descamps V, Martinot-Peignoux M et al (1993) Cryoglobulinemia with vasculitis associated with hepatitis C virus infection. Gastroenterology 104:272–277 22. Agnello V, Chung RT, Kaplan LM (1992) A role for ­hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327:1490–1495

8 23. Levey JM, Bjornsson B, Banner B et  al (1994) Mixed ­cryoglobulinemia in chronic hepatitis C infection. A clinicopathologic analysis of 10 cases and review of recent literature. Medicine (Baltimore) 73:53–67 24. Casato M, Lilli D, Donato G et al (2003) Occult hepatitis C virus infection in type II mixed cryoglobulinaemia. J Viral Hepat 10:455–459 25. Sansonno D, Lauletta G, De Re V et al (2004) Intrahepatic B cell clonal expansions and extrahepatic manifestations of chronic HCV infection. Eur J Immunol 34:126–136 26. Vallat L, Benhamou Y, Gutierrez M et al (2004) Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection. Arthritis Rheum 50:3668–3678 27. Sansonno D, Dammacco F (2005) Hepatitis C virus, cryoglobulinaemia, and vasculitis: immune complex relations. Lancet Infect Dis 5:227–236 28. Sitia G, Isogawa M, Iannacone M et  al (2004) MMPs are required for recruitment of antigen-nonspecific mononuclear cells into the liver by CTLs. J Clin Invest 113:1158–1167 29. Sansonno D, Tucci FA, Troiani L et  al (2008) Increased serum levels of the chemokine CXCL13 and up-regulation of its gene expression are distinctive features of HCV-related cryoglobulinemia and correlate with active cutaneous vasculitis. Blood 112:1620–1627 30. Dustin LB, Rice CM (2007) Flying under the radar: the immunobiology of hepatitis C. Annu Rev Immunol 25:71–99 31. Wieland SF, Chisari FV (2005) Stealth and cunning: hepatitis B and hepatitis C viruses. J Virol 79:9369–9380 32. Klenerman P, Zinkernagel RM (1998) Original antigenic sin impairs cytotoxic T lymphocyte responses to viruses bearing variant epitopes. Nature 394:482–485 33. Ferri C, La Civita L, Longombardo G et  al (1998) Mixed cryoglobulinaemia: a cross-road between autoimmune and lymphoproliferative disorders. Lupus 7:275–279 34. Dammacco F, Sansonno D, Piccoli C et al (2000) The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinemia, and overt B-cell malignancy. Semin Liver Dis 20:143–157 35. Ferri C, Caracciolo F, Zignego AL et al (1994) Hepatitis C virus infection in patients with non-Hodgkin’s lymphoma. Br J Haematol 88(2):392–394 36. Pozzato G, Mazzaro C, Crovatto M et al (1994) Low-grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia. Blood 84(9):3047–3053 37. Monti G, Pioltelli P, Saccardo F (2005) Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 165:101–105

F. Dammacco and D. Sansonno 38. Marcucci F, Mele A (2011) Hepatitis viruses and non-Hodgkin lymphoma: epidemiology, mechanisms of tumorigenesis and therapeutic opportunities. Blood 117(6):1792–1798 39. Libra M, Polesel J, Russo AE et al (2010) Extrahepatic disorders of HCV infection: a distinct entity of B-cell neoplasia? Int J Oncol 36:1331–1340 40. Omland LH, Farkas DK, Jepsen P et al (2010) Hepatitis C virus infection and risk of cancer: a population-based cohort study. Clin Epidemiol 2:179–186 41. Hermine O, Lefrere F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after ­treatment of hepatitis C virus infection. N Engl J Med 347: 89–94 42. Gisbert JP, García-Buey L, Pajares JM et al (2005) Systematic review: regression of lymphoproliferative disorders after treatment for hepatitis C infection. Aliment Pharmacol Ther 21:653–662 43. Kawamura Y, Ikeda K, Arase Y et al (2007) Viral elimination reduces incidence of malignant lymphoma in patients with hepatitis C. Am J Med 120:1034–1041 44. Knight GB, Gao L, Gragnani L (2010) Detection of WA B cells in hepatitis C virus infection: a potential prognostic marker for cryoglobulinemic vasculitis and B cell malignancies. Arthritis Rheum 62:2152–2159 45. Bonomo L, Casato M, Afeltra A et al (1987) Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 83:726–730 46. Saadoun D, Delluc A, Piette JC et al (2008) Treatment of hepatitis C-associated mixed cryoglobulinemia vasculitis. Curr Opin Rheumatol 20:23–28 47. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 48. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101:3827–3834 49. Cacoub P, Saadoun D, Limal N et  al (2005) PEGylated ­interferon alfa-2b and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 52:911–915 50. Saadoun D, Resche Rigon M, Sene D et al (2010) Rituximab plus Peg-interferon-alpha/ribavirin compared with Peginterferon-alpha/ribavirin in hepatitis C-related mixed cryoglobulinemia. Blood 116:326–334 51. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a longterm study. Blood 116:343–353

2

Natural History, Pathogenesis, and Prevention of HCV Infection Edgar D. Charles, Lynn B. Dustin, and Charles M. Rice

2.1 Epidemiology Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV), but most are unaware of the infection. The global seroprevalence of anti-HCV antibodies is estimated to be 3%, with a higher prevalence in countries in the Mediterranean Basin, Far East, Africa, and Central America [1]. Strikingly, 14% of the general population in Egypt is infected with HCV [2, 3]; this high prevalence is due to contaminated syringes used during an anti-schistosomiasis campaign in the 1950s. Transmission of HCV primarily occurs via blood-toblood contact with an infected person. Since the implementation of screening of blood donors for HCV in Western countries, in 1991, the risk of HCV transmission by blood-product transfusion has been reduced to <1 per one million transfusion events. In the USA, the number of acute HCV infections fell from a peak of 180,000/year in the mid-1980s to 28,000/year in 1995 [4–9], corresponding to 12% of all cases of viral hepatitis. It has been estimated that the number of people in the USA who have been infected for 20 years or more will peak in 2015 [5], which implies that the rate of cirrhosis, hepatocellular carcinoma (HCC), and extrahepatic manifestations of HCV will not start to decline in Western countries before 2025, given the long incubation time for these sequelae. Currently, the most

E.D. Charles (*) Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_2, © Springer-Verlag Italia 2012

f­ requent cause of ongoing HCV transmission occurs through sharing of contaminated needles or syringes by injection drug users. Although sexual transmission of HCV is very rare, there appears to be an emerging pattern of HCV acquisition in non-drug-injecting, HIVinfected men who have sex with men [6, 7]. The risk of perinatal transmission of HCV is 5–6% [8, 9] and is correlated with maternal HCV RNA level, HIV status, and premature rupture of membranes [9]. Importantly, HCV is not spread by breast feeding, sneezing, coughing, sharing of eating utensils, or casual contact [10].

2.2 Pathogenesis 2.2.1 HCV Life Cycle HCV is an enveloped positive-strand RNA virus of the genus Hepacivirus and the family Flaviviridae. The 9.6-kb RNA genome contains large 5¢ and 3¢ noncoding regions (NCRs) that are essential to translation and replication. Entry into the cell is dependent upon interaction with a defined array of entry factors (CD81, SR-BI, claudin-1, and occludin) that play a major role in species and tissue tropism. HCV translation and replication occur in association with a membranous web believed to derive from the endoplasmic reticulum. Translation of the single, long open reading frame yields a polyprotein of approximately 3,000 amino acids (Fig.  2.1). Cleavage of this polyprotein by host and viral proteases releases the ten individual proteins that comprise the viral particle and replication machinery. HCV replication likely follows the strategy used by other positive-strand RNA viruses. The viral genome is transcribed in the cytoplasm to yield 11

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E.D. Charles et al. ~9600 nucleotides 5’

Structural

NCR F

◊

Core

*

NCR

3’

* *

* E1

Non-structural

E2

Envelope glycoproteins E1 signal peptide Genome encapsidation

p7 NS2

NS3

4A 4B

NS5A

NS5B

Phosphoprotein Serine protease RNA helicase Membrane RNA-dependent RNA polymerase Cysteine protease alterations Ion Serine protease channel cofactor

Fig. 2.1  HCV genes and gene products. Shown is the structure of the viral genome, including the long open reading frame encoding structural and nonstructural genes, an alternative reading frame protein (F), and the 5¢ and 3¢ non-coding regions (NCRs). The single asterisks refer to signal peptidase cleavage

sites, the diamond to the signal peptide peptidase cleavage site, and the arrows to the cleavage sites of the NS2 autoprotease (NS2/3 junction) and the NS3-4A serine protease (all downstream sites)

a complementary negative-strand RNA that serves as a template for positive-strand RNA molecules. HCV has no reverse-transcriptase activity; rather, NS5B, an error-prone RNA-dependent RNA polymerase lacking proofreading capacity, synthesizes both negative and positive strand RNA. Infectious particles are thought to consist of HCV RNA associated with the core structural protein and surrounded by a lipid envelope spiked with E1 and E2 structural proteins. Non-structural proteins from the p7-NS5B region are not likely to be present in viral particles. The major site of HCV replication is the liver, but the high rate of extrahepatic manifestations during chronic HCV infection have led some to suggest that other tissues are also infected. Other cell types reported to contain HCV RNA include B cells, dendritic cells, monocytes, and gut mucosal and sperm cells. It has been proposed that latent infection persists in many of these cells after elimination of virus from the peripheral blood. However, the vast majority of accumulating clinical data suggests that such an infectious reservoir, if it exists, does not pose a significant risk for viral reinfection or transmission.

half lives for these particles of 3 h in the plasma and 1–70 days for productively infected cells [10]. Estimates of NS5B’s nucleotide mis-incorporation rate range from 0.5 to 2/10,000 bases, implying that on a daily basis genomes are generated with a mutation at any given nucleotide. It is apparent that the rapid generation of high levels of viral genetic diversity has important implications for immune escape and de  novo and/or acquired resistance to drugs with specific viral targets.

2.2.2 Viral Dynamics Infected patients produce up to 1012 HCV particles per day, and mathematical modeling suggests in  vivo

2.2.3 HCV Genotypes and Viral Diversity HCV is classified into six major genotypes (numbered 1–6) with at least 11 common subtypes (designated a, b, c, etc.). These genotypes differ in their geographical distribution, with genotypes 1a and 1b, which account for ~60% of worldwide infections, predominating in northern Europe, North America, and in southern and eastern Europe and Japan. Genotype 3 is endemic in Southeast Asia. Genotype 4 is chiefly present in the Middle East, Egypt, and central Africa. Genotype 5 is primarily found in South Africa [11]. The core gene and the NCRs of the genome are relatively conserved; the particular conservation of the 5¢ NCR makes this region most suitable for detection by PCR [12]. Genotype-specific changes in the 5¢ NCR are commonly used to differentiate the six main genotypes,

2  Natural History, Pathogenesis, and Prevention of HCV Infection

but NS5B-based assays are more precise, especially in areas with high viral diversity [13, 14]. The nonstructural proteins have similar degrees of variability compared to the average genome values. The genes encoding the E1 and E2 proteins are more variable, with E2 containing two hypervariable regions of 20–30 amino acids that vary considerably between genotypes and within subtypes. Within an individual, HCV is present as a heterogeneous quasi-species. Evidence suggests that a genetic bottleneck exists during viral transmission [15, 16]. Subsequent immune pressure is likely responsible for diverse quasi-species generation [17], and it may be associated with an improved early virological response to antiviral treatment [18].

2.2.4 Histopathology HCV is believed to be primarily non-cytopathic, with the natural killer and cytotoxic T lymphocyte response (see following chapter) likely responsible for the majority of liver damage. Infection frequently causes a periportal infiltration of inflammatory cells, interface hepatitis, lobular hepatocellular injury, and hepatic steatosis consisting of large lipid droplets within the cytoplasm of hepatocytes. Lymphoid aggregates and B-cell-containing lymphoid follicles have been observed. HCV progression is characterized by increased hepatic fibrosis, in which collagen deposition begins within the portal triads and progresses to form bridges between portal areas or with terminal hepatic venules (bridging fibrosis) [19]. Evidence suggests that fibrosis is mediated by activated hepatic stellate cells. Cirrhosis is characterized by widespread collagen deposition with resultant distortion of hepatic architecture and, eventually, loss of hepatic function. Liver biopsy has proven to be invaluable in the clinical determination of the severity and prognosis of liver disease. However, it has two main drawbacks: it is invasive, and it is prone to sampling error when the liver is not homogenously diseased. The development of non-invasive tests that match or exceed the sensitivity and specificity of the liver biopsy is a major area of clinical research. There are several scoring systems to evaluate HCV-related histopathology. These include the Knodell, Scheuer, Ishak, and METAVIR systems, all of which assign a score to the degree of necroinflammation and a stage to the degree of fibrosis.

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2.3 Clinical Features 2.3.1 Acute and Chronic HCV The clinical manifestations of acute and chronic hepatitis C are non-specific and, when present, are often not distinguishable from other forms of viral hepatitis. HCV RNA can be detected in blood within 1–3 weeks of exposure [20, 21], but antibodies are not detected until 7–10 weeks later [22, 23]. The onset of infection is often unrecognized, as acute HCV is frequently asymptomatic. Symptoms include jaundice, anorexia, nausea, vomiting, and malaise. Fulminant hepatitis C is exceedingly rare; instead, 15–25% of patients will spontaneously clear HCV within 18  months. Viral clearance is associated with broadened cellular immune responses and the generation of non-sterilizing, yet cross-neutralizing, antibodies. Re-infection with subsequent exposure may be associated with reduced viremia [24] and an increased rate of clearance [25]. Evidence is accumulating that innate immune responses are crucial for early clearance. A single nucleotide polymorphism upstream of the IL-28B gene (which encodes interferon-l3) strongly affects the rate of spontaneous HCV clearance [26] and may partly explain why African–Americans have less successful virological responses to interferon-a [27]. Patients who fail to spontaneously clear HCV develop chronic HCV infection, the symptoms of which are also variable (in the absence of extrahepatic disease) and can include fatigue and right upper quadrant pain. Serum alanine aminotransferase (ALT) levels increase shortly before the onset of clinical symptoms. Peak ALT levels may reach levels tenfold higher than the upper limit of normal, but they usually remain only moderately elevated. In patients whose infections spontaneously resolve, ALT levels return to baseline and HCV RNA becomes undetectable. In patients who fail to clear virus, ALT levels and HCV RNA may fluctuate widely during the ensuing disease course.

2.3.2 Natural History and Prognosis Chronic HCV has a variable outcome, given the wide spectrum of disease, ranging from chronic mild hepatitis to decompensated cirrhosis and liver failure

14 Fig. 2.2  Natural history of HCV infection

E.D. Charles et al. Years - Decades

HCV exposure ?%

Acute hepatitis 75−90%

Chronic hepatitis

Risk Factors for Fibrosis Progression: Age > 40 years at time of infection Male gender Alcohol > 50 g/d HIV, HBV co-infection Hepatic steatosis

(Fig.  2.2). Most often, decades of infection occur before the development of cirrhosis, HCC, or extrahepatic manifestations of HCV. Even after the development of cirrhosis, survival is 91% and 79% at 5 and 10  years, respectively [28]. Once cirrhosis is established, the risk of decompensation (e.g., jaundice, ascites, variceal bleed, or hepatic encephalopathy) is cumulatively 4–5% per year and the risk of HCC 1–4% per year. With the onset of decompensation, 5-year survival declines to <50%. Most prospective natural history studies have shown that only 5–20% of individuals with chronic HCV will eventually progress to end-stage liver disease [29–33]. Subsets of individuals will have an accelerated progression of fibrosis or early extrahepatic manifestations within 10  years of HCV acquisition. The rate of fibrosis progression has been difficult to assess, because it may not be linear. The factors impacting progression are still incompletely understood, but they have been suggested to comprise both host and environmental factors. Most studies have found no association between HCV-specific factors such as viral load or genotype and the risk of fibrosis progression [34, 35]; however, the adverse effects of several host factors have been identified. Age at the time of infection appears to be one of the strongest predictors of outcome: the 20-year risk of cirrhosis is very low (<5%) in those infected before the age of 20, but this risk grows to >50% for those infected after age 50 [36]. Male gender is also a strong predictor of progression, as is heavy

10−50%

Extrahepatic manifestations

10−20%

Cirrhosis

4−5% / year

Decompensated cirrhosis

1−4% / year

Hepatocellular carcinoma

(>50  g/day) alcohol consumption. Faster progression to cirrhosis also occurs in patients who are co-infected with HIV [37]. Low CD4 T cell counts and HIV viremia adversely affect prognosis whereas suppression of viremia following antiretroviral therapy decreases the rate of fibrosis progression [38] and the risk of liverrelated mortality [39]. Co-infection with either active or occult HBV may also increase the risk of cirrhosis [40, 41]. Emerging evidence suggests that hepatic steatosis is increased with increased fibrosis [42], yet it remains to be seen whether control of metabolic factors attenuates fibrogenesis in such individuals.

2.3.3 Extrahepatic Manifestations It is increasingly recognized that chronic HCV infection is a cause of extrahepatic disease, including mixed cryoglobulinemia (MC), non-Hodgkin lymphoma (NHL), membranoproliferative glomerulonephritis, thyroid disease, porphyria cutanea tarda, lichen planus, and type 2 diabetes mellitus [43, 44]. Of these, MC has the strongest association with HCV. These disorders are discussed separately below.

2.3.3.1 B Cell Lymphoproliferative Disorders Chronic HCV infection elicits a spectrum of B cell lymphoproliferative diseases, ranging from the nonmalignant clonal B cell expansion usually seen in MC to NHL that is primarily of a low-grade, marginal

2  Natural History, Pathogenesis, and Prevention of HCV Infection

zone subtype, although associations with other, more aggressive, subtypes have been reported [45–48]. Since the identification of HCV, many reports have linked HCV infection to the development of B cell NHL [49]. However, given the low incidence of NHL during HCV infection it has been difficult to ascertain the additive risk HCV confers for NHL. A large retrospective trial conducted among USA veterans confirmed that HCV is a risk factor for NHL as well as for other B cell lymphoproliferative disorders, such as Waldenström macroglobulinemia and monoclonal gammopathy of undetermined significance (MGUS) [45]. It is likely that HCV MC represents an antigen-driven, relatively benign clonal B cell lymphoproliferation that, with continued antigenic stimulation, occasionally progresses towards overt NHL. Pre-malignant B cell clones can be detected in the bone marrow or in the liver several years before the development of frank lymphoma [50]. Typically, the NHL that arises in HCV MC patients is low-grade and characterized by clonal rheumatoid factor (RF)-bearing B cells that may be present in the liver, spleen, peripheral lymph nodes, peripheral blood, and/or bone marrow. It has been proposed that a subset of these low-grade NHL evolves to a high-malignancy phenotype. The most frequent NHL histiotypes are immunocytoma, splenic marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, and aggressive diffuse large B cell lymphoma [46–48], suggestive of the expansion of a germinal center or post-germinal center B cell during or after antigenic stimulation. It has been reported that higher-grade diffuse large B cell lymphoma is predominantly associated with MC-negative, rather than MC-positive, HCV patients [51]. Accordingly, it is possible that a fundamentally different etiologic mechanism underlies the development of aggressive NHL in the absence of MC. Consistent with the hypothesis that continued antigenic presence is required for ongoing clonal B cell proliferation, the eradication of HCV leads to the disappearance of HCV-associated MC [52] and low-grade NHL [53], similar to what has been observed with Helicobacter pylori-induced MALT lymphomas. An aggressive subset of H. pylori MALT contains t(11;18) translocations and does not regress after eradication of the bacterium. In contrast, there is no convincing evidence that chromosomal translocations are frequent in HCV-related NHL. Although several groups have detected t(14;18) in B cells from HCV MC patients

15

[54–56], these translocations have been identified in healthy controls [57] as well as in HCV-negative NHL patients [58].

2.3.3.2 Mixed Cryoglobobulinemia Among individuals chronically infected with HCV, 10–50% have MC and >90% of these patients have HCV [59–61]. Non-HCV causes of MC include infectious agents (e.g., HIV, HBV) and autoimmune disorders (e.g., systemic lupus erythematosis, Sjögren’s syndrome, and systemic sclerosis) [62]. A shared feature of these disorders is chronic inflammation in the setting of high antigenic load, suggesting that antigendriven B cell dysregulation is a prerequisite for the development of MC [63]. Only a minority of patients develops the classical symptoms of palpable purpura, joint pains, and fatigue. Cryoglobulins are classified as type I (monoclonal immunoglobulin (Ig) only), type II (mixture of monoclonal Ig, usually IgM RF, and polyclonal IgG) and type III (mixture of polyclonal Ig, usually IgM, and polyclonal IgG) [64]. HCV is primarily associated with type II (which typically has an IgMk RF with anti-idiotypic activity [65]), and to a lesser extent, with type III MC. Type I MC is rarely seen in HCV. Serum levels of RF are often increased in the setting of HCV MC, while levels of complement, particularly C4, may be profoundly decreased. It has been hypothesized that C1q bound to IgM RF engages C1q receptors on vascular endothelium, leading to neutrophil recruitment and vasculitis [66]. HCV MC vasculitis primarily affects the small and medium-sized vessels of the skin, kidneys, and peripheral nerves. Histology typically reveals a leukocytoclastic vasculitis, with deposition of IgM RF, IgG, C3, and neutrophils in the vessel wall. A necrotizing vasculitis, with fibrinoid necrosis of the intima and inflammation of the entire vessel wall and perivascular space, may also occur. Palpable purpura, primarily of the lower legs, is seen in >90% of patients with symptomatic HCV MC, is frequently intermittent, and is often the cardinal manifestation of HCV MC [61]. These purpuric lesions may occasionally progress to chronic ulcers and frank gangrene. 2.3.3.3 Membranoproliferative Glomerulonephritis (MPGN) The renal involvement in HCV-MC is usually type I MPGN [67], and it frequently heralds a poor clinical course. Manifestations range from isolated proteinuria

16

to overt nephritic or nephritic syndrome with variable progression towards chronic renal insufficiency. The MPGN is characterized by endocapillary mesangial cell proliferation, monocytic infiltration, double contour membranes, glomerular IgM, IgG, and C3 deposition, eosinophilic PAS-positive intraluminal deposits, and vasculitis of the small and medium-sized renal arteries [68, 69].

2.3.3.4 Peripheral Neuropathy The incidence of neurological involvement is variable. Sensorimotor neuropathy arises from cryoglobulin deposition in the vasa vasorum. Painful paresthesias and concomitant weakness, particularly in the lower limbs may occur [70], as may isolated mononeuritis, manifested by foot or wrist drop. 2.3.3.5 Other Extrahepatic Manifestations Infection with HCV has been associated with thyroid disease (hyper- or hypothyroidism) that in turn is often associated with interferon therapy and pre-existing anti-thyroid peroxidase antibodies. In addition, there is increasing recognition of a relationship between HCV and steatosis, insulin resistance, and overt type 2 diabetes mellitus [71]. The relationship between HCV and lichen planus is controversial; meta-analysis of primarily case-control studies suggests a significant positive association with wide geographical variation [72]. HCV is also associated with increases in nonorgan-specific autoantibodies [73], the clinical significance of which is unclear.

2.4 Prevention of HCV Infection 2.4.1 Primary and Secondary Prevention 2.4.1.1 Identification of HCV-Infected Individuals As HCV infection is usually asymptomatic, a large reservoir of infected individuals is at risk of unwittingly transmitting HCV to others. A major public health goal is to screen those at high risk for HCV. Screening programs should prioritize those people who have a history of intravenous drug use, a history of blood-product transfusion or organ transplantation before 1992, and individuals infected with HIV or HBV. Screening should also be carried out among the newly and chronically incarcerated, given the high prevalence of HCV infection among prisoners [74, 75]

E.D. Charles et al.

and the risk of transmission among inmates via injection drug use or tattooing.

2.4.1.2 Secondary Prevention Once HCV-infected individuals are identified, they should be counseled to not donate blood, semen, or tissues, and to avoid the sharing of personal items that may be exposed to blood. It is not necessary to avoid close contact with family members or to avoid sharing meals or utensils. Given the low rate of heterosexual transmission, there is no need for people in monogamous long-term heterosexual relationships to change sexual practices [76]. However, as HCV may be transmitted sexually among men who have sex with men, condom usage is advisable in such cases. HCV-infected individuals should be referred to medical practitioners for assessment of liver function, ­detection of viremia by HCV RNA, and treatment consideration. A discussion of HCV treatment is beyond the scope of this chapter. Currently, the standard of care is combination therapy with an HCV-specific protease inhibitor, pegylated interferon and ribavirin, with duration of treatment dependent upon HCV genotype and HCV RNA response [77]. Treatment options are expected to dramatically expand as additional HCVspecific protease inhibitors, as well as polymerase inhibitors, and perhaps other medications come to market [78].

2.4.2 Management of Percutaneous or Mucosal Exposure to HCV Individuals who have percutaneous or mucosal exposure to blood at high risk for HCV viremia should have baseline anti-HCV and ALT measurements. HCV RNA may be measured at 4–6  weeks; follow-up anti-HCV and ALT should be done at 4–6 months. Immu­noglobulins and antiviral agents are not recommended for post-­ exposure prophylaxis [79]. As antiviral therapy may be beneficial in patients with acute HCV, symptomatic patients and patients with positive HCV RNA should be referred to a physician for treatment consideration.

2.4.3 Children Born to HCV-Positive Women Studies have failed to demonstrate a reduced risk after cesarean delivery compared to vaginal ­delivery.

2  Natural History, Pathogenesis, and Prevention of HCV Infection

Immunoglobulin and antiviral agents are not recommended for neonatal post-exposure prophylaxis. Breastfeeding is not contraindicated as there is no ­documented ­transmission by this route [76], although caution should be exercised in the setting of chafed nipples with skin breakdown. Testing for HCV RNA may be performed 2–3  months after delivery, and ­anti-HCV should be performed no sooner than 12 months. Children with either positive anti-HCV or HCV RNA should be referred to a physician for further management.

2.5 Concluding Remarks HCV is a major cause of worldwide morbidity and mortality. As infection is usually asymptomatic, there is a large reservoir of people who are unaware that they harbor virus. Complications of cirrhosis and extrahepatic disease occur in a minority of individuals, and often take decades to develop. Consequently, estimates of the natural history of disease vary widely and it is difficult to predict the clinical course following infection. It is clear that HCV exerts deleterious effects on organs beyond the liver, and a major goal of continuing research is to clarify such relationships from the epidemiologic to the cellular level.

References 1. WHO (1997) Hepatitis C: global prevalence. Wkly Epidemiol Rec 72:341–344 2. Frank C, Mohamed MK, Strickland GT, Lavanchy D et  al (2000) The role of parenteral antischistosomal therapy in the spread of hepatitis C virus in Egypt. Lancet 355:887–891 3. Abdel-Aziz F, Habib M, Mohamed MK, Abdel-Hamid M et al (2000) Hepatitis C virus (HCV) infection in a community in the Nile Delta: population description and HCV prevalence. Hepatology 32:111–115 4. Alter MJ (1997) Epidemiology of hepatitis C. Hepatology 26:62S–65S 5. Armstrong GL, Alter MJ, McQuillan GM, Margolis HS (2000) The past incidence of hepatitis C virus infection: implications for the future burden of chronic liver disease in the United States. Hepatology 31:777–782 6. van de Laar TJ, van der Bij AK, Prins M, Bruisten SM et al (2007) Increase in HCV incidence among men who have sex with men in Amsterdam most likely caused by sexual transmission. J Infect Dis 196:230–238 7. Danta M, Brown D, Bhagani S, Pybus OG et al (2007) Recent epidemic of acute hepatitis C virus in HIV-positive men who have sex with men linked to high-risk sexual behaviours. AIDS 21:983–991

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8. Ohto H, Terazawa S, Sasaki N, Hino K et  al (1994) Transmission of hepatitis C virus from mothers to infants. The Vertical Transmission of Hepatitis C Virus Collaborative Study Group. N Engl J Med 330:744–750 9. Mast EE, Hwang LY, Seto DS, Nolte FS et al (2005) Risk factors for perinatal transmission of hepatitis C virus (HCV) and the natural history of HCV infection acquired in infancy. J Infect Dis 192:1880–1889 10. Mast EE, Alter MJ, Margolis HS (1999) Strategies to prevent and control hepatitis B and C virus infections: a global perspective. Vaccine 17:1730–1733 11. (1999) Hepatitis C – global prevalence (update). Wkly Epidemiol Rec 74:425–427 12. Simmonds P (1999) Viral heterogeneity of the hepatitis C virus. J Hepatol 31(Suppl 1):54–60 13. Simmonds P, Bukh J, Combet C et al (2005) Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 42:962–973 14. Bouchardeau F, Cantaloube JF, Chevaliez S et  al (2007) Improvement of hepatitis C virus (HCV) genotype determination with the new version of the INNO-LiPA HCV assay. J Clin Microbiol 45:1140–1145 15. Liu CH, Chen BF, Chen SC et al (2006) Selective transmission of hepatitis C virus quasi species through a needlestick accident in acute resolving hepatitis. Clin Infect Dis 42:1254–1259 16. Quer J, Esteban JI, Cos J et al (2005) Effect of bottlenecking on evolution of the nonstructural protein 3 gene of hepatitis C virus during sexually transmitted acute resolving infection. J Virol 79:15131–15141 17. Ray SC, Fanning L, Wang XH et al (2005) Divergent and convergent evolution after a common-source outbreak of hepatitis C virus. J Exp Med 201:1753–1759 18. Fan X, Mao Q, Zhou D et al (2009) High diversity of hepatitis C viral quasispecies is associated with early virological response in patients undergoing antiviral therapy. Hepatology 50:1765–1772 19. Goodman ZD (2007) Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J Hepatol 47:598–607 20. Farci P, Alter HJ, Wong D et al (1991) A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Med 325:98–104 21. Courouce AM, Le Marrec N, Girault A et al (1994) Antihepatitis C virus (anti-HCV) seroconversion in patients undergoing hemodialysis: comparison of second- and thirdgeneration anti-HCV assays. Transfusion 34:790–795 22. Sulkowski MS, Ray SC, Thomas DL (2002) Needlestick transmission of hepatitis C. JAMA 287:2406–2413 23. Netski DM, Mosbruger T, Depla E et  al (2005) Humoral immune response in acute hepatitis C virus infection. Clin Infect Dis 41:667–675 24. Osburn WO, Fisher BE, Dowd KA et al (2009) Spontaneous control of primary hepatitis C virus infection and immunity against persistent reinfection. Gastroenterology 138: 315–324 25. Page K, Hahn JA, Evans J et  al (2009) Acute hepatitis C virus infection in young adult injection drug users: a prospective study of incident infection, resolution, and reinfection. J Infect Dis 200:1216–1226 26. Thomas DL, Thio CL, Martin MP et al (2009) Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 461:798–801

18 27. Ge D, Fellay J, Thompson AJ et al (2009) Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461:399–401 28. Fattovich G, Giustina G, Degos F et al (1997) Morbidity and mortality in compensated cirrhosis type C: a retrospective follow-up study of 384 patients. Gastroenterology 112:463–472 29. Seeff LB (2002) Natural history of chronic hepatitis C. Hepatology 36:S35–S46 30. Kenny-Walsh E (1999) Clinical outcomes after hepatitis C infection from contaminated anti-D immune globulin. Irish Hepatology Research Group. N Engl J Med 340: 1228–1233 31. Wiese M, Berr F, Lafrenz M et al (2000) Low frequency of cirrhosis in a hepatitis C (genotype 1b) single-source outbreak in Germany: a 20-year multicenter study. Hepatology 32:91–96 32. Vogt M, Lang T, Frosner G et  al (1999) Prevalence and clinical outcome of hepatitis C infection in children who underwent cardiac surgery before the implementation of blood-donor screening. N Engl J Med 341:866–870 33. Seeff LB, Miller RN, Rabkin CS et al (2000) 45-year follow-up of hepatitis C virus infection in healthy young adults. Ann Intern Med 132:105–111 34. Poynard T, Bedossa P, Opolon P (1997) Natural history of liver fibrosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 349:825–832 35. Kleter B, Brouwer JT, Nevens F et  al (1998) Hepatitis C virus genotypes: epidemiological and clinical associations. Benelux Study Group on Treatment of Chronic Hepatitis C. Liver 18:32–38 36. Poynard T, Ratziu V, Charlotte F et al (2001) Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis c. J Hepatol 34:730–739 37. Graham CS, Baden LR, Yu E et  al (2001) Influence of human immunodeficiency virus infection on the course of hepatitis C virus infection: a meta-analysis. Clin Infect Dis 33:562–569 38. Macias J, Berenguer J, Japon MA et al (2009) Fast fibrosis progression between repeated liver biopsies in patients coinfected with human immunodeficiency virus/hepatitis C virus. Hepatology 50:1056–1063 39. Qurishi N, Kreuzberg C, Luchters G et al (2003) Effect of antiretroviral therapy on liver-related mortality in patients with HIV and hepatitis C virus coinfection. Lancet 362: 1708–1713 40. Zarski JP, Bohn B, Bastie A et al (1998) Characteristics of patients with dual infection by hepatitis B and C viruses. J Hepatol 28:27–33 41. Cacciola I, Pollicino T, Squadrito G et al (1999) Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. N Engl J Med 341:22–26 42. Leandro G, Mangia A, Hui J et  al (2006) Relationship between steatosis, inflammation, and fibrosis in chronic hepatitis C: a meta-analysis of individual patient data. Gastroenterology 130:1636–1642 43. Gumber SC, Chopra S (1995) Hepatitis C: a multifaceted disease. Review of extrahepatic manifestations. Ann Intern Med 123:615–620 44. Zignego AL, Ferri C, Pileri SA et  al (2007) Extrahepatic manifestations of hepatitis C virus infection: a general over-

E.D. Charles et al. view and guidelines for a clinical approach. Dig Liver Dis 39:2–17 45. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297:2010–2017 46. Ferri C, Caracciolo F, Zignego AL et al (1994) Hepatitis C virus infection in patients with non-Hodgkin’s lymphoma. Br J Haematol 88:392–394 47. Zuckerman E, Zuckerman T, Levine AM et  al (1997) Hepatitis C virus infection in patients with B-cell nonHodgkin lymphoma. Ann Intern Med 127:423–428 48. Talamini R, Montella M, Crovatto M et  al (2004) NonHodgkin’s lymphoma and hepatitis C virus: a case-control study from northern and southern Italy. Int J Cancer 110: 380–385 49. Matsuo K, Kusano A, Sugumar A et  al (2004) Effect of hepatitis C virus infection on the risk of non-Hodgkin’s lymphoma: a meta-analysis of epidemiological studies. Cancer Sci 95:745–752 50. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 43:94–102 51. De Vita S, Sacco C, Sansonno D et al (1997) Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection. Blood 90:776–782 52. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa2a therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 330:751–756 53. Hermine O, Lefrere F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after ­treatment of hepatitis C virus infection. N Engl J Med 347:89–94 54. Zuckerman E, Zuckerman T, Sahar D et al (2001) bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection. Br J Haematol 112:364–369 55. Zignego AL, Ferri C, Giannelli F et al (2002) Prevalence of bcl-2 rearrangement in patients with hepatitis C virus-related mixed cryoglobulinemia with or without B-cell lymphomas. Ann Intern Med 137:571–580 56. Libra M, Gloghini A, Malaponte G et al (2008) Association of t(14;18) translocation with HCV infection in gastrointestinal MALT lymphomas. J Hepatol 49:170–174 57. Limpens J, Stad R, Vos C et al (1995) Lymphoma-associated translocation t(14;18) in blood B cells of normal individuals. Blood 85:2528–2536 58. Libra M, De Re V, De Vita S et al (2003) Low frequency of bcl-2 rearrangement in HCV-associated non-Hodgkin’s lymphoma tissue. Leukemia 17:1433–1436 59. Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327:1490–1495 60. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33:355–374 61. Dammacco F, Sansonno D, Piccoli C et al (2001) The cryoglobulins: an overview. Eur J Clin Invest 31:628–638 62. Gorevic PD (1995) Cryopathies: cryoglobulins and cryofibrinogenimia. In: Frank MM, Austen KF, Claman HN (eds)

2  Natural History, Pathogenesis, and Prevention of HCV Infection Samter’s immunologic diseases. Little Brown, Boston, p 951 63. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76:818–824 64. Brouet JC, Clauvel JP, Danon F et  al (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 57:775–788 65. Agnello V (1997) The etiology and pathophysiology of mixed cryoglobulinemia secondary to hepatitis C virus infection. Springer Semin Immunopathol 19:111–129 66. Sansonno D, Dammacco F (2005) Hepatitis C virus, cryoglobulinaemia, and vasculitis: immune complex relations. Lancet Infect Dis 5:227–236 67. Saadoun D, Landau DA, Calabrese LH, Cacoub PP (2007) Hepatitis C-associated mixed cryoglobulinaemia: a ­crossroad between autoimmunity and lymphoproliferation. Rheumatology (Oxford) 46:1234–1242 68. Johnson RJ, Gretch DR, Yamabe H et  al (1993) Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. N Engl J Med 328:465–470 69. D’Amico G (1998) Renal involvement in hepatitis C ­infection: cryoglobulinemic glomerulonephritis. Kidney Int 54:650–671 70. Authier FJ, Pawlotsky JM, Viard JP et al (1993) High incidence of hepatitis C virus infection in patients with cryoglobulinemic neuropathy. Ann Neurol 34:749–750 71. Lonardo A, Adinolfi LE, Loria P et al (2004) Steatosis and hepatitis C virus: mechanisms and significance for hepatic and extrahepatic disease. Gastroenterology 126:586–597

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72. Shengyuan L, Songpo Y, Wen W et  al (2009) Hepatitis C virus and lichen planus: a reciprocal association determined by a meta-analysis. Arch Dermatol 145:1040–1047 73. Cacoub P, Renou C, Rosenthal E et al (2000) Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d’Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l’Hepatite C. Medicine (Baltimore) 79:47–56 74. Hennessey KA, Kim AA, Griffin V et al (2009) Prevalence of infection with hepatitis B and C viruses and co-infection with HIV in three jails: a case for viral hepatitis prevention in jails in the United States. J Urban Health 86:93–105 75. McGovern BH, Wurcel A, Kim AY et al (2006) Acute hepatitis C virus infection in incarcerated injection drug users. Clin Infect Dis 42:1663–1670 76. (1998) Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. Centers for disease control and prevention. MMWR Recomm Rep 47:1–39 77. Heathcote EJ (2007) Antiviral therapy: chronic hepatitis C. J Viral Hepat 14(Suppl 1):82–88 78. Meier V, Ramadori G (2009) Hepatitis C virus virology and new treatment targets. Expert Rev Anti Infect Ther 7: 329–350 79. U.S. Public Health Service (2001) Updated U.S. public health service guidelines for the management of ­occupational exposures to HBV, HCV, and HIV and recommendations for postexposure prophylaxis. MMWR Recomm Rep 50:1–52

3

Immune Control of HCV Infection Lynn B. Dustin

3.1

Introduction

The purpose of this chapter is to summarize our current understanding of the ways the immune system fights the hepatitis C virus (HCV), and how the virus avoids elimination in the majority of those who contract it. According to the most recent World Health Organization estimates, between 130 and 170 million people worldwide are persistently infected with HCV [1, 2]. Long-term sequelae of HCV infection can include cirrhosis, liver failure, and hepatocellular carcinoma [3]; these conditions make HCV a leading indication for liver transplantation [4]. Despite recognition by innate antiviral pathways, and despite stimulating adaptive immune responses, HCV establishes persistent infection in the majority of those who contract it [5, 6]. While there have been encouraging developments, prophylactic and therapeutic vaccines are not yet available [7, 8]. Antiviral regimens based on pegylated interferon (IFN)-a2 and ribavirin produce sustained control of HCV infection in about one-half of those treated [9]; thus, new drugs are actively sought to expand coverage [10, 11]. In this chapter, we discuss our current understanding of the battle between HCV and innate and adaptive immune responses: how these responses target HCV and how HCV manages to persist in a majority of those who become infected.

L.B. Dustin Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_3, © Springer-Verlag Italia 2012

3.2

Hepatitis C Virus

HCV is an enveloped RNA virus in the family Flaviviridae. Its genomic structure and replication pathways have been reviewed [12–15]. The HCV genome is a single, positive-sense, 9.6-kb RNA encoding a polyprotein of about 3,000 amino acids. Host and virus-encoded proteases process the polyprotein to release the ten individual proteins making up the viral particle and replication machinery. One or more additional proteins may be produced by translational frame-shifting; however, the functions of these proteins are poorly understood. HCV replication and assembly take place in association with membranous structures and lipid droplets in the cytosol of infected cells [15]. The structural proteins (core (capsid), E1 and E2) are at the amino terminus of the polyprotein. It is believed that HCV RNA associates with multiple copies of the core protein to form the nucleocapsid, which is packaged in a lipid envelope bearing the E1 and E2 glycoproteins. The infectious form of the virus in vivo is still incompletely understood, but it is believed that HCV associates with low- or very lowdensity lipid particles. The nonstructural proteins, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B, are present in infected cells but are probably not required components of the viral particles. The known roles of these proteins in the HCV lifecycle are reviewed elsewhere [11, 14, 15]. Briefly, p7 is a membrane-spanning protein possessing cation-channel activity; it is required for productive virus infection but not for HCV RNA replication. NS2 is a cysteine protease that plays an essential, but poorly understood, role in virus assembly. NS3 has NTPase/helicase and serine protease activities, the 21

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latter dependent on association with NS4A. NS3/4A catalyzes the release of the four downstream proteins (NS4A, NS4B, NS5A and NS5B) from the HCV polyprotein and, as discussed below, targets cellular proteins. NS4B may serve as a scaffold for the organization of the replication complex and causes rearrangement of cellular membranes. NS5A’s phosphorylation status may act as a switch regulating RNA replication and virus assembly. NS5B is the RNA-dependent RNA polymerase, required for synthesis of negative-sense (minus strand) and positive-sense RNA from an RNA template. HCV can evolve rapidly as its replication yields an impressive 1012 HCV particles per day in the infected liver. Newly produced HCV particles have an estimated serum half-life of 3 h [16, 17]. HCV’s NS5B RNA-dependent RNA polymerase lacks proofreading capacity. The high rate of virus production coupled with this error-prone replication mechanism permits rapid evolution under selection by the immune system and antiviral drugs. Within the infected host, HCV exists as a quasispecies, or swarm of related viral sequences.

3.3

HCV and Innate Antiviral Responses

Interferon response pathways are induced in the liver early in infection regardless of the outcome of infection [18–21]. Type I IFNs produced in response to HCV may induce an antiviral state in the surrounding cells. However, specific HCV gene products have been reported to target different steps in IFN induction or response. This may enable the virus to persist in the face of ongoing recognition by pathogen-associated molecular pattern receptors – and, indeed, in the face of IFN-based antiviral therapy. Acute HCV infection has been studied in experimentally infected chimpanzees. During the first 1–2 weeks of HCV infection, serum viral loads increase rapidly, with a mean doubling time half-life of 0.5 days [22, 23]. Based on measurements of viral RNA during acute infection, one study estimated that up to 10% of hepatocytes support HCV replication [18]. The rate of viral increase then slows abruptly to a mean doubling time half-life of 7.5 days, coinciding with evidence of an IFN response in the liver. It is believed that an initial IFN response establishes an antiviral state in most cells

of the liver, and that reduction in the number of susceptible cells slows the rate of virus production [22, 23]. The cellular source responsible for IFN production has not been identified. Autocrine production by infected hepatocytes may activate subsequent paracrine production by other cells. Alternatively, immune cells including plasmacytoid dendritic cells may produce the first IFNs after recognizing the presence of HCV-infected hepatocytes. Innate mechanisms, both within the infected cell and in patrolling immune cells, recognize RNA structures associated with viral infections such as HCV (reviewed in [24]). HCV targets innate virus-sensing mechanisms. HCV RNA replicates in the cytoplasm, and this process generates molecular patterns detected by the cytoplasmic DEx/D/H-Box RNA helicases, RIG-I and MDA-5. RIG-I may recognize HCV RNA due to HCV’s 5¢ triphosphate group [25] and a conserved uridine-rich sequence near the 3¢ end of HCV genomic RNA [26]. MDA-5 may recognize higher-order RNA structures generated during replication [27]. RNAactivated RIG-I and MDA-5 interact via their caspase activation and recruitment domains (CARDs) with the CARD of the mitochondrial membrane-tethered molecule, IPS-1 (reviewed in [28]; also known as VISA, MAVS, and Cardif). This binding induces IPS-1 to signal through at least two kinase complexes to stimulate the activation and translocation of latent IFN regulatory factor-3 (IRF-3) and other transcription factors (reviewed in [24, 29, 30]). These activate transcription of IFNb [28] as well as IFNl [31, 32] and other targets [33]. By binding to the IFNa receptor, IFNb activates a positive feedback loop in which IRF-3 and IRF-7 activate multiple IFNa genes. Similarly, IFNl1 (IL29) activates transcription of IFNl2 (IL28A) and IFNl3 (IL28B) genes [31, 32]. In a hepatoma line, HCV infection induces rapid – but transient – IRF-3 activation [34]. However, HCV can specifically inactivate the RIG-I-IPS-1 pathway of IFN induction. Thus, the NS3/4A protease was found to block RIG-I-mediated activation and translocation of IRF-3 [35]. NS3/4A specifically cleaves IPS-1 at a cysteine near the carboxy terminus of the protein [36, 37], releasing it from the mitochondrion and preventing its function [38]. In liver biopsies from patients with chronic HCV infection, HCV-infected cells (identified by NS3 immunostaining) show aberrant IPS-1 localization [34], supporting the importance of this observation in vivo. On a cautionary note, however, the

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Immune Control of HCV Infection

level of cleaved IPS-1 was not found to correlate with the level of HCV RNA in biopsy samples, suggesting that other mechanisms also mediate IPS-1 cleavage in the infected liver [39]; importantly, IPS-1 is targeted by cellular caspases [40].

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strongly associated with genetic polymorphisms in the IL28B locus, suggesting a role for IL28B in control of HCV infection (reviewed in [53]).

3.3.3 3.3.1

Does HCV Interfere with the Detection of Extracellular Virus?

Extracellular HCV RNA, in the form of viral particles or debris from infected cells, may activate Toll-like receptors (TLRs) [41]. TLR signal transduction can activate the production of IFNs and thereby inhibit HCV replication in infected cells [42]. TLR3 detects double-stranded RNA in endosomes, for example after capture of viral RNA by endocytic uptake of debris from infected cells. Hepatocytes express TLR3 [43]; dendritic cell (DC) subsets may express TLR3 (mDCs) or TLR7/8 (pDCs). Signal transduction through TLR3 is dependent on the adaptor, TRIF, which is cleaved and inactivated by NS3/4A [44]. Thus, cells expressing NS3/4A may be impaired in their ability to detect extracellular viral RNA through TLR3. If mDCs were infected with HCV, this mechanism could impair their ability to produce IFNs; however, as discussed below, there is little strong evidence for widespread HCV infection in DCs. Signal transduction by TLR7 and TLR8, which detect single-stranded RNA in endosomal compartments, does not depend on TRIF. pDCs may detect infected hepatocytes by a mechanism dependent on TLR7, with consequent production of IFNa and IFNb [45].

3.3.2

Type III IFNs

Recent evidence from genome-wide association studies of HCV patients highlights the importance of the l or type III IFNs (IL29 or IFNl1, IL28A or IFNl2, and IL28B or IFNl3) in control of HCV infection [46–50]. IFNl expression can be induced by viral infections [31]. Hepatocytes and many other cell types express the IFNl receptor, which is related to the IL10 receptor. The IFNl receptor activates a signal transduction cascade qualitatively similar to that of the type I IFN receptor [51]. In a cell culture model, IFNl was shown to inhibit HCV replication [52]. Spontaneous and treatment-induced clearance of HCV infection are both

HCV May Target IFN-Stimulated Antiviral Programs

While a number of mechanisms have been proposed for the impairment of IFNa/b induction in HCV infected cells, those observations do not explain how HCV persists in the liver in the presence of a robust IFN response – and during IFNa-based antiviral therapy. HCV proteins are reported to subvert type I IFN receptor signal transduction and the function of downstream IFN effector pathways [54, 55]. Type I IFNs signal through the IFNa receptor to activate the kinases Tyk2 and Jak1, which phosphorylate STAT1 and STAT2. These translocate to the nucleus and associate with IRF-9, forming the transcription factor ISGF-3 (reviewed in [56, 57]). Type III IFNs signal through the IFNl receptor, a heterodimer of the IL10 receptor b chain and the IL28 receptor a chain [51, 58]. Activation of the IFNl receptor also stimulates the Jak1 and Tyk2 kinases, leading to phosphorylation of STATs 1 and 2 [59]. While IFNa/b and IFNl stimulate transcription of a similar set of genes in liver cells [60], gene activation in response to type I IFNs is more rapid and less sustained than that observed in response to type III IFNs [52]. HCV’s core protein was reported to activate the expression of suppressors of cytokine signaling (SOCS) proteins [61], which limit JAK-STAT signaling downstream of IFN receptors, in a study of core-transfected cells. However, this result should be interpreted with caution as it has not been reproduced in cells supporting HCV infection. ISGF-3 stimulates the transcription of 300 or more IFN-stimulated genes (ISGs), many with direct antiviral activities. ISGs with direct antiviral activity include 2¢, 5¢ oligoadenylate synthetase (OAS), RNAse L, protein kinase R (PKR), and ISG56 [56, 57]. Several reports suggest that HCV gene products inhibit the function of some ISGs. However, not all of these effects have been confirmed with HCV-infected cells. 2¢, 5¢OAS polymerizes ATP into 2¢, 5¢-linked oligoadenylates, which in turn activate RNAse L to degrade viral RNA. NS5A was reported to inhibit 2¢, 5¢OAS activity [62]. IFNa-based antiviral treatments may select for RNAse L-resistant HCV sequences [63].

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PKR is activated by double-stranded RNA; activated PKR can phosphorylate and inactivate the eukaryotic translation initiation factor, eIF2-a, thus inhibiting protein synthesis [56]. It was reported that by activating PKR to phosphorylate eIF2-a, HCV may block the translation of mRNA encoding other IFN-stimulated genes [64]. In contrast, both NS5A and the envelope glycoprotein, E2, were reported to bind and inhibit PKR [65–67]. E2 binding of PKR presents a topological problem, since the E2 domain required for such an interaction is not believed to localize to the cytosol. Although IFNa receptor signal transduction is reportedly impaired in cells expressing HCV core or the complete HCV polyprotein (reviewed in [54, 55]), IFNs can abolish HCV replication in cell lines. Furthermore, ISG expression is readily detected in the liver of chronically infected patients and in chimpanzees with acute HCV infection. Interestingly, elevated hepatic expression of ISGs in patients with chronic HCV infection is associated with failure of subsequent IFN-based antiviral therapy [68, 69]. High levels of the IFN-induced chemokine CXCL10 (IP10) in the blood of chronic HCV patients before treatment presage treatment failure [70–74].

3.4

Innate Immune Cells and HCV Clearance

The liver is rich in innate immune cells including natural killer (NK) cells, NKT cells, and myeloid subsets including Kupffer cells (liver-resident macrophages) and dendritic cells (DCs) [75, 76]. Little information is available about the roles played by NKT cells in resolution or persistence of HCV infection, although some studies indicate that their levels or activation state are altered in the liver [77] or in the peripheral blood [78] during persistent infection.

3.4.1

Natural Killer Cells

Natural killer cells, abundant in the liver [76], are key early responders in viral infection. They may mediate lysis of infected cells, produce IFNg to directly control viral replication [79], and promote local accumulation of lymphoid and inflammatory cells [80]. Activated NK cells stimulate dendritic cell (DC) maturation, in part through TNFa and IFNg, and thus provide a direct link between innate and acquired immunity [81].

One study reported that the NK cells of HCV patients had decreased ability to promote DC maturation [82]. NK cell cytokine production has the potential to impact HCV replication in larger numbers of cells [83, 84]. However, NK cells from HCV patients may be biased toward cytotoxic rather than cytokine-mediated mechanisms [85], which would limit their antiviral efficacy and at the same time promote tissue damage. The importance of NK cells in the resolution of HCV infection is underscored by observations that genetic polymorphisms in HLA and NK cell inhibitory/activating receptors, which affect NK cell activation thresholds [84], are associated with the outcome (spontaneous clearance vs. chronicity) of HCV infection [86, 87]. Some studies have demonstrated a reduced frequency of circulating NK cells as well as alterations in their cytotoxic and cytokine secretion activities (reviewed in [83]), possibly resulting in a lower antiviral activity overall for NK cells in HCV patients than in healthy controls. Some groups have reported that recombinant HCV envelope proteins can alter the function of NK cells in vitro [88, 89]. However, this result was not reproducible using infectious HCV particles [90] unless the particles were immobilized on a solid support [91]. The latter observation underscores the need for caution in interpretation of studies based on single recombinant HCV proteins; it is not certain whether NK cells encounter significant levels of immobilized HCV particles in vivo. Recombinant purified NS5A was reported to inhibit NK cell activation by activating IL10 and, indirectly, TGFb production [92]; however, it is not known whether such a mechanism operates in vivo. Furthermore, NK cells are activated during acute HCV infection regardless of the outcome of infection [93, 94].

3.4.2

Dendritic Cells

There is some controversy regarding the effects of HCV infection on DC function (reviewed in [95, 96]). Differences in purification or maturation protocols may account for some differences between studies. Two functionally distinct DC populations must be considered: Plasmacytoid dendritic cells (pDCs) are a significant source of type I (a/b) IFNs. Viral infection is detected through the endosomal TLRs 7 and 9, and this detection is followed rapidly by tremendous up-regulation of type I IFN expression [97]. While HCV itself

3

Immune Control of HCV Infection

is a poor inducer of IFNs by pDCs [98, 99], HCVinfected hepatoma strongly induce IFNa [45]. Type I IFNs can activate antiviral programs, summarized above, in other cells. They promote increased expression of major histocompatibility complex antigen ligands for NK and CD8+ T cells. Type I IFNs also stimulate another DC subset, the conventional or myeloid DCs, to produce IL12 – an essential factor for the maturation of Th1 cells [100]. In addition, type I IFNs directly promote NK cell activation and the proliferation of antigen-specific effector CD8+ T cells [101]. pDCs can directly control HCV replication in infected liver-derived cells by secreting IFNs [45]. Some authors have reported that HCV partially blocks pDC function [99, 102], perhaps by down-modulating IRF-7 [99]. However, HCV patients do not demonstrate a major increase in susceptibility to other viral diseases, arguing against a significant degree of pDC impairment [96]. Myeloid dendritic cells (mDCs) develop from monocyte precursors. These cells are potent antigen presenters [103] and are major producers of IL12 and l IFNs [97, 104]. In the liver, mDCs steer the adaptive immune response through antigen presentation and the production of soluble mediators [105]. The mediators include cytokines such as the anti-inflammatory IL10 [106], and IL12, which promotes differentiation of T and NK cells primed for IFNg production [100]. Another important DC-derived mediator is the tryptophan catabolic enzyme, indoleamine 2, 3-dioxygenase, which blocks proliferation of T cells as well as many pathogens [103, 107]. While some investigators have reported reduced immunostimulatory function in DCs during chronic HCV infection [108–110], others have found that these cells are phenotypically and functionally normal [111–115]. We would expect nonspecific impairment in mDC function to have a devastating impact on immune function, yet there is little evidence for global immune dysfunction in HCV patients. Indeed, mDCs isolated from HCV patient livers had enhanced antigen presentation ability, and specifically secreted less of the inhibitory cytokine IL10 than was the case in mDCs from non-infected livers [116].

3.4.3

Does HCV Infect DCs?

Some groups, finding HCV genetic material associated with DC subsets, have proposed that infection of DCs could lead to impaired function [110, 117]. When any

25

quantitation is reported, the level of HCV RNA associated with DCs is far below one copy per cell, indicating that infected DCs must be rare if they exist [95, 114]. In some reports, but not others, expression or uptake of HCV gene products hindered DC functions [95, 118–120]. It is not clear how HCV would infect DCs, which do not express all of the entry factors required for HCV infection of hepatocytes [98]. Laboratory strains of HCV do not replicate in either mDCs or pDCs [98, 102]. The fact that most HCV patients do not suffer from global immunological dysfunction argues against a general defect in antigenpresenting or cytokine-production functions of patient DCs. However, it is possible that infection or impairment is limited to local populations of DCs in the liver or in the draining lymph nodes.

3.5

Adaptive Immunity and HCV

Since the development of methods to screen blood and blood products for HCV in the early 1990s, most new HCV infections have occurred away from medical care. Thus, our knowledge of immunological mechanisms that clear acute HCV infection comes from chimpanzee studies and from specific populations with ready access: healthcare workers who have suffered needle-stick injuries, the rather unusual individuals who develop symptoms of acute hepatitis, and people at risk of exposure who are followed prospectively. In addition, some retrospective studies have examined patients infected in single-source outbreaks.

3.5.1

Cell-Mediated Immune Responses to HCV

Although innate antiviral responses are observed early after HCV infection, resolution of infection is dependent on adaptive immunity. Spontaneous clearance of HCV infection is correlated with T cell responses that target multiple HCV epitopes early in infection (reviewed in [6, 55]). Both CD4+ and CD8+ T cells target diverse sets of epitopes in patients who effectively control HCV [121–123]. In contrast, infections are more likely to become persistent when the acute immune response is narrowly focused [124]. It is widely assumed that broadly focused, sustained immune responses to HCV benefit the patient by increasing the number of HCV epitopes that must be

26

L.B. Dustin

changed by mutation in order to escape immune recognition. However, this hypothesis is not yet formally proven. An alternative interpretation is that early control of infection prevents T cell exhaustion and permits the maintenance of a broadly focused HCV-specific immune response. Furthermore, at least transient HCV-specific T cell activation can be detected even as HCV establishes persistent infection. In most patients, cell-mediated immunity fails to eradicate HCV infection. Even after spontaneous clearance of HCV, re-infections by homologous and heterologous HCV strains are possible [125]. Protective immunity reduces the duration and the level of viremia in repeated infection [126–128], but sterilizing immunity has been difficult to prove. Protection from recurrent HCV infection is dependent on both CD4+ [129] and CD8+ [130] memory T cells.

3.5.2

T Cell Responses in Acute Infection

Acute HCV infection – approximately the first 6 months after onset of viremia – is usually asymptomatic. Symptomatic acute infection occurs in <20% of patients and is associated with a higher rate of spontaneous clearance [131]. During acute HCV infection, the serum viral load increases exponentially for weeks without evidence of liver injury. HCV-specific T cells are often detectable in the blood around 4–8 weeks after the onset of infection [132–134]. A significant HCV-specific T cell response is detected in the blood during acute infection and corresponds to evidence of transient liver injury [132, 135]. Spontaneous control of HCV is associated with a robust and sustained T cell response targeting multiple HCV epitopes at once, and with intrahepatic IFNg production [21, 122, 132, 133, 136, 137]. HCV-specific CD8+ T cells in the blood and liver can produce IFNg and exhibit CTL activity [122, 136]. IFNg production is essential to control of viremia [133], while cytolytic activity may be dispensable [138]. HCV-specific CD4+ T cells are more readily detected during acute infection (regardless of the outcome of infection) than in chronic infection [139, 140]. Early CD4+ T cell responses predict at least transient control of acute HCV infection, while the loss of these responses predicts recurrence of viremia and development of persistent infection [141–143]. While many HCV-specific T cells have a “stunned” phenotype (even in hosts that go

on to resolve infection) [122, 132], they recover an activated and functional phenotype during and after spontaneous resolution of HCV infection [132, 133]. In contrast, persisting infection is associated with further loss of functional T cells [140].

3.5.3

Recruiting Immune Cells to the Liver

Intrahepatic immune responses can mediate both viral clearance and tissue damage. Thus, immune-mediated pathology must be tightly controlled. NK and T cell recruitment to the infected liver is influenced by chemokines including MIP-1a, CXCL9 (MIG), and CXCL10 [80, 144, 145]. MIP-1a recruits NK cells, an early source of IFNg; CXCL-9 and CXCL10, which can be induced by IFNg, are thought to recruit T cells into the liver parenchyma. An important role for MIP1a in HCV clearance is suggested by the increased intrahepatic MIP-1a RNA expression in chimpanzees with resolving HCV infection compared to those whose infection persists [22]. An important role for CXCL10 in HCV infection is suggested by the observation that HCV patients with high plasma levels of CXCL10 are likely to fail IFN-based antiviral therapy [71, 146, 147]. The elevated CXCL10 in HCV patient plasma may actually be an antagonist blocking recruitment of HCV-specific T cells into the infected liver [70, 148].

3.5.4

T Cell Responses in Chronic Infection

While broadly-targeted anti-HCV T cell responses are readily detected in the peripheral blood of patients who have spontaneously controlled HCV infection [121, 149], HCV-specific T cell responses in chronically infected patients are typically limited in scope and function. The number of epitopes targeted declines as HCV infection persists [134, 141], and a loss of CD4+ T cell responses predicts recurrence of viremia and establishment of chronic infection [139, 140, 143]. HCV-specific CD8+ T cells may be abundant, accounting for 1–2% or more of total blood or intrahepatic CD8+ T cells in chronically infected patients [150–152]. Even if they are abundant, HCV-specific CD8+ T cells in the blood and liver are functionally deficient as shown by reduced IFNg production,

3

Immune Control of HCV Infection

proliferation, and cytolytic activity [151, 153–155]. Their cell surface phenotype is suggestive of incomplete differentiation to memory or effector cells [156]. Phenotypically normal HCV-specific CD8+ T cells may recognize historical epitopes – that is, viral sequences that have been lost through mutation [157].

3.5.5

Deficient CD4+ T Cell Help Leads to CD8+ T Cell Exhaustion

The functionally deficient CD8+ T cells seen in acute and chronic HCV infection resemble the “exhausted” CD8+ T cells reported in various mouse models of persistent infection (reviewed in [158, 159]). These T cells lose the ability to produce key cytokines in a stepwise fashion (IL2, TNFa, and finally IFNg). Such exhausted T cells are observed under conditions of high, persistent antigen load [160]. CD4+ T cell help is critical for maintenance of CD8+ T cell responses during chronic infection [161]. Defective CD4+ T cell help is already evident in acute HCV patients, when many HCV-specific CD8+ T cells show evidence of cytokine deprivation [135]. When CD4+ helper T cells are impaired or absent – as observed in chronic HCV infection [139, 162] – CD8+ T cell exhaustion may be an inevitable consequence [158, 163]. Activated helper T cells promote dendritic cell maturation, and recognition of antigen on the same antigen presenting cell by CD4+ and CD8+ T cells is a key feature of antigenspecific T cell help [159, 164]. Thus, the failure of CD4+ HCV-specific T cells may doom CD8+ T cells by limiting their opportunities for priming by fully activated, HCV antigen-loaded DCs.

3.5.6

Role of PD-1

A number of groups have reported that HCV-specific CD8+ T cells express PD-1, a marker associated with exhausted T cells [165–167]. The inhibitory ligands of PD-1, PD-L1 and PD-L2, are highly expressed in the liver during several disease processes including, but not limited to, HCV infection [168]. In a mouse model of persistent infection, PD-1 blockade reversed T cell exhaustion and permitted resolution of infection [169]. This observation led to the hope that blocking PD-1 would restore T cell function in vivo in HCV patients, and some in vitro results have been encouraging

27

[166, 170]. Importantly, however, PD-1 expression is observed on HCV-specific T cells during resolving infection as well [171, 172]. Furthermore, mice genetically deficient in PD-1 are vulnerable to lethal immunemediated pathology [169]. PD-1 expression, and T cell exhaustion in general, may contribute to homeostatic mechanisms that reduce immune-mediated tissue damage by limiting T cell activation. It may also be possible to revive exhausted HCV-specific T cells by targeting multiple cell surface markers [173]. Given the consensus that much of the liver damage in HCV patients is immunologically mediated, approaches aimed at enhancing T cell activation will require rigorous safety testing.

3.5.7

Escape Mutations

As discussed earlier, HCV’s error prone RNAdependent RNA polymerase permits rapid sequence changes. These mutations provide the raw material for escape from immune recognition in each host. The emergence of CD8+ T cell escape variants correlates with loss of immunological control and progression to chronic infection [124, 174, 175], while escape variants are less frequently observed in self-limiting HCV infections (reviewed in [176]). The emergence of epitope escape variants may represent a balance between escape from immunological recognition on the one hand [175, 177–179] and viral fitness costs on the other [179, 180]. HLA class I-restricted epitopes undergo patterns of mutation and epitope escape that correlate with the host’s HLA-A and HLA-B type, suggesting that CD8+ T cells apply strong selective pressure [177–179]. In contrast, epitopes targeted by CD4+ T cells may not be subjected to strong selective pressure [181]. In the absence of strong immune selection, viral sequences may remain unchanged, or they may revert to the consensus sequence for their genotype [175, 177, 180]. Epitope escape mutations occur early in infection. Once escape is achieved, there is little selective pressure for continued evolution of these sequences [176, 182], since new T cell specificities do not arise after the acute infection period [134]. Mutational escape, T cell exhaustion, and poor recognition may each contribute to development of persistent HCV infection. Thus, mutation of a few HCV epitopes, or even just one epitope, may be observed in chronically infected hosts with diverse T cell repertoires [124, 175, 183, 184]. In a prospective

28

L.B. Dustin

study of six acutely infected patients, only epitopes recognized with high avidity early in infection underwent mutational escape as infection persisted [185]. Other epitopes, recognized by larger populations of CD8+ T cells with poor effector function or lower avidity, did not change over time [185]. Of interest, T cells specific for HCV epitopes that had not undergone mutational escape from recognition expressed high levels of PD-1 [167, 186] and other molecules associated with the exhausted state [167]. While HCV evolves under immunological pressure, the cellular immune response remains focused on viral sequences encountered early in infection. In contrast, antibody neutralization data suggest that the humoral immune response is more flexible in HCV infection.

3.5.8

Regulatory T Cells (Treg)

There is no consensus regarding the roles played by Treg cells in HCV clearance and persistence. While some groups have reported that these cells are increased in number or function in HCV patients [187–190], there does not seem to be any correlation between their abundance and the outcome of acute HCV infection [191]. Furthermore, Treg cells are also increased in frequency after resolution of HCV infection [189]. Tregs may protect the liver from damage mediated by effector T cells, and seem to be subject to regulation by PD-1 just as effector T cells are [192, 193].

3.5.9

LSEC can promote a vigorous immune response [75, 105, 197]. CD8+ T cells stimulated on LSECs acquire expression of PD-1 and fail to differentiate to cytotoxic effectors. Antigen presentation by LSEC promotes differentiation of a regulatory T cell subset that lacks expression of the markers IL25 and FoxP3. T cells can interact with hepatocytes from within the sinusoid by means of T cell and hepatocyte microvilli that protrude through fenestrated LSECs [196]. Antigen presentation by hepatocytes can promote tolerance rather than immunity [198]. In the HCV-infected liver, the majority of HCV-specific CD8+ T cells appear prone to apoptosis [135]. There may be important advantages to blunting immune responses in the liver. HCV is not directly cytopathic to infected cells, and it is believed that much of the damage observed in HCV infection occurs secondary to the immune response rather than the virus.

Immune Tolerance in the Liver

Perhaps because of its exposure to a barrage of antigens from foods and gut microflora, the liver is rich in tolerogenic antigen-presenting cells [105]. The liver microenvironment also favors the development of tolerogenic, rather than stimulatory, DCs [194, 195]. The anatomy of the hepatic sinusoid, its sluggish blood flow and narrow spaces, all promote T cell contact with liver sinusoidal endothelial cells (LSECs) and the underlying hepatocytes [196]. LSECs express a variety of lymphocyte adhesion molecules, class I and class II major histocompatibility complex molecules, and costimulators, but also inhibitory molecules, including PD-L1. Antigen presentation by LSECs typically results in T cell tolerance, although virus-infected

3.6

Humoral Immune Responses to HCV

Although humans [199] and chimpanzees [200] can clear HCV infection without mounting a detectable humoral immune response to HCV, it is clear that antibodies (Abs) can contribute to protection from HCV infection and to the control of established infection. Seroconversion is slow after HCV infection [201]; Ab responses are often not detected until after a peak in liver enzymes indicates the onset of immune-mediated damage in the liver [22, 132, 202]. Ab responses to HCV envelope proteins do not correlate with the outcome of infection in experimentally infected chimpanzees [22, 136, 203]. While Ab responses are dispensable for clearance of acute infection [21, 136, 200, 204], a robust neutralizing Ab response during acute infection is associated with spontaneous clearance [204]. HCVspecific Abs may wane after spontaneous recovery [204, 205].

3.6.1

Persistence of Infection Despite High Levels of HCV-Specific Neutralizing Abs

Neutralizing Abs (nAbs) are defined based on their ability to block the entry of either HCV or pseudoparticles bearing HCV envelope glycoproteins into

3

Immune Control of HCV Infection

appropriate target cells. These Abs recognize epitopes on the viral envelope, including but not limited to the hypervariable regions (HVR1 and HVR2) of the E2 glycoprotein [206, 207]. The appearance of HCVspecific nAbs is significantly delayed during acute infection [200, 201]. Broadly reactive nAbs are readily detected in the serum of persistently infected patients [200, 207, 208]. The presence of high-titer nAbs in patients who remain chronically infected demonstrates that such antibodies do not mediate sterilizing immunity. In addition to epitope mutational escape, HCV may bypass antibody neutralization by spreading from cell to cell without exposure to the extracellular environment [209]. Studies of acutely and chronically infected HCV patients have demonstrated that the antibody response exerts continuous evolutionary pressure on viral envelope proteins and particularly on the HVR segments of E2 [210, 211]. New viral envelope glycoprotein sequences arise repeatedly as the humoral immune response targets those variants already encountered. The continued emergence of new antibody specificities contrasts sharply with the static T cell response in chronic infection, as described earlier in this chapter. The importance of nAbs is demonstrated by clinical experience. Before the advent of serological screening for HCV, immunoglobulin preparations such as GammaGard, made from pooled human donor sera, did not transmit HCV infection. However, when manufacturers began excluding donor serum containing HCV-specific Abs, Gammagard preparations lacking HCV-specific Ab but containing infectious virus were distributed and some recipients developed HCV infection [212, 213]. In other studies, it has been shown that pre-treatment with anti-HCV serum or monoclonal antibodies can, in some cases, reduce transmission of HCV in experimental animals [214–216]. While most studies have thus far emphasized the ability of HCV-specific Abs to block viral infection in vitro, the roles of Abs in vivo may be far more complex. It has been argued that the majority of binding sites must be coated with Ab in order for neutralization of infection to occur [217]. This may be readily achieved using HCV pseudoparticles, with their poor incorporation of HCV envelope glycoproteins [218]. Neutralization epitopes on authentic HCV are expected to be more densely packed and may also be masked due to associated lipoproteins. Sub-neutralizing quantities of Ab, or Ab that binds but does not neutralize

29

infection, may still decrease infection by fixing complement, opsonizing particles for phagocytosis, and enhancing clearance and antigen presentation.

3.7

Antiviral Treatment and Immune Responses

At this writing, therapy for chronic HCV depends on pegylated IFNa combined with the nucleoside analog ribavirin [9]. Depending on the infecting genotype, patients are treated for up to 48 weeks, and nonresponders or relapsers may receive additional courses of therapy [9, 219, 220]. The goal of treatment is a sustained virologic response (SVR). A patient who has achieved SVR has no detectable HCV RNA in serum when tested 6 months after completion of therapy. Depending on the patient population and the infecting genotype, SVR rates range from well below 50% to >80% of those who complete treatment. With the addition of novel STAT-C (specifically-targeted antiviral therapy for HCV) drugs currently in clinical trials [10, 11], there is hope many more patients can achieve SVR with shorter treatment regimens. However, HCV has already demonstrated the ability to evolve resistance to several STAT-C drugs. Thus, STAT-C drugs will likely be used mainly in combination with IFNa-based treatment. While a review of established and novel antiviral therapies is beyond the scope of this chapter, some antiviral therapies may impact innate and adaptive immunity to HCV. In IFNa-based therapeutic regimens, viral loads decline in two distinct phases [16, 17, 221]. The first phase lasts approximately 24 h and is attributed to a decline in virus production by infected cells – a direct effect of IFNa and innate antiviral mechanisms [17, 221]. A slower, second phase decline occurs over weeks to months and is attributed to the death of infected cells [16, 17, 221]. The role of adaptive immunity in this second-phase decline is controversial (reviewed in [5, 6]). Responsiveness to IFNa-based HCV therapies is affected by polymorphisms in the IL28B locus (reviewed in [53]), suggesting that the l IFNs play a significant role in IFNa-stimulated antiviral responses. STAT-C drugs directed at HCV’s NS3/4A protease may, in addition to blocking processing of the HCV polyprotein, restore some innate responses by impairing NS3/4A-mediated cleavage of IPS-1 and TRIF.

30 Acknowledgments The author’s research is supported by the National Institutes of Health (AI60561).

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L.B. Dustin 166. Golden-Mason L, Palmer B, Klarquist J et al (2007) Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction. J Virol 81:9249–9258 167. Bengsch B, Seigel B, Ruhl M et al (2010) Coexpression of PD-1, 2B4, CD160 and KLRG1 on exhausted HCV-specific CD8+ T cells is linked to antigen recognition and T cell differentiation. PLoS Pathog 6:e1000947 168. Kassel R, Cruise MW, Iezzoni JC et al (2009) Chronically inflamed livers up-regulate expression of inhibitory B7 family members. Hepatology 50:1625–1637 169. Barber DL, Wherry EJ, Masopust D et al (2006) Restoring function in exhausted CD8 T cells during chronic viral infection. Nature 439:682–687 170. Nakamoto N, Kaplan DE, Coleclough J et al (2008) Functional restoration of HCV-specific CD8 T cells by PD-1 blockade is defined by PD-1 expression and compartmentalization. Gastroenterology 134:1927–1937. e1922 171. Bowen DG, Shoukry NH, Grakoui A et al (2008) Variable patterns of programmed death-1 expression on fully functional memory T cells after spontaneous resolution of hepatitis C virus infection. J Virol 82:5109–5114 172. Kasprowicz V, Schulze Zur Wiesch J, Kuntzen T et al (2008) High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome. J Virol 82: 3154–3160 173. McMahan RH, Golden-Mason L, Nishimura MI et al (2010) Tim-3 expression on PD-1+ HCV-specific human CTLs is associated with viral persistence, and its blockade restores hepatocyte-directed in vitro cytotoxicity. J Clin Invest 120:4546–4557 174. Erickson AL, Kimura Y, Igarashi S et al (2001) The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes. Immunity 15:883–895 175. Cox AL, Mosbruger T, Mao Q et al (2005) Cellular immune selection with hepatitis C virus persistence in humans. J Exp Med 201:1741–1752 176. Bowen DG, Walker CM (2005) Mutational escape from CD8+ T cell immunity: HCV evolution, from chimpanzees to man. J Exp Med 201:1709–1714 177. Timm J, Lauer GM, Kavanagh DG et al (2004) CD8 epitope escape and reversion in acute HCV infection. J Exp Med 200:1593–1604 178. Ray SC, Fanning L, Wang XH et al (2005) Divergent and convergent evolution after a common-source outbreak of hepatitis C virus. J Exp Med 201:1753–1759 179. Neumann-Haefelin C, McKiernan S, Ward S et al (2006) Dominant influence of an HLA-B27 restricted CD8+ T cell response in mediating HCV clearance and evolution. Hepatology 43:563–572 180. Uebelhoer L, Han JH, Callendret B et al (2008) Stable cytotoxic T cell escape mutation in hepatitis C virus is linked to maintenance of viral fitness. PLoS Pathog 4: e1000143 181. Fuller MJ, Shoukry NH, Gushima T et al (2010) Selectiondriven immune escape is not a significant factor in the failure of CD4 T cell responses in persistent hepatitis C virus infection. Hepatology 51:378–387

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182. Chang KM, Rehermann B, McHutchison JG et al (1997) Immunological significance of cytotoxic T lymphocyte epitope variants in patients chronically infected by the hepatitis C virus. J Clin Invest 100:2376–2385 183. Meyer-Olson D, Shoukry NH, Brady KW et al (2004) Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape. J Exp Med 200:307–319 184. Komatsu H, Lauer G, Pybus OG et al (2006) Do antiviral CD8+ T cells select hepatitis C virus escape mutants? Analysis in diverse epitopes targeted by human intrahepatic CD8+ T lymphocytes. J Viral Hepat 13:121–130 185. Urbani S, Amadei B, Cariani E et al (2005) The impairment of CD8 responses limits the selection of escape mutations in acute hepatitis C virus infection. J Immunol 175:7519–7529 186. Rutebemberwa A, Ray SC, Astemborski J et al (2008) High-programmed death-1 levels on hepatitis C virus-specific T cells during acute infection are associated with viral persistence and require preservation of cognate antigen during chronic infection. J Immunol 181:8215–8225 187. Sugimoto K, Ikeda F, Stadanlick J et al (2003) Suppression of HCV-specific T cells without differential hierarchy demonstrated ex vivo in persistent HCV infection. Hepatology 38:1437–1448 188. Boettler T, Spangenberg HC, Neumann-Haefelin C et al (2005) T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. J Virol 79:7860–7867 189. Manigold T, Shin EC, Mizukoshi E et al (2006) Foxp3+CD4+CD25+ T cells control virus-specific memory T cells in chimpanzees that recovered from hepatitis C. Blood 107:4424–4432 190. Ward SM, Fox BC, Brown PJ et al (2007) Quantification and localisation of FOXP3+ T lymphocytes and relation to hepatic inflammation during chronic HCV infection. J Hepatol 47:316–324 191. Heeg MH, Ulsenheimer A, Gruner NH et al (2009) FOXP3 expression in hepatitis C virus-specific CD4+ T cells during acute hepatitis C. Gastroenterology 137(1280–1288): e1281–e1286 192. Franceschini D, Paroli M, Francavilla V et al (2009) PD-L1 negatively regulates CD4+CD25+Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV. J Clin Invest 119:551–564 193. Radziewicz H, Dunham RM, Grakoui A (2009) PD-1 tempers Tregs in chronic HCV infection. J Clin Invest 119:450–453 194. Schildberg FA, Hegenbarth SI, Schumak B et al (2008) Liver sinusoidal endothelial cells veto CD8 T cell activation by antigen-presenting dendritic cells. Eur J Immunol 38:957–967 195. Cabillic F, Rougier N, Basset C et al (2006) Hepatic environment elicits monocyte differentiation into a dendritic cell subset directing Th2 response. J Hepatol 44:552–559 196. Warren A, Le Couteur DG, Fraser R et al (2006) T lymphocytes interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells. Hepatology 44: 1182–1190 197. Limmer A, Ohl J, Kurts C et al (2000) Efficient presentation of exogenous antigen by liver endothelial cells to

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4

B Cell Activation: General to HCV-Specific Considerations Vito Racanelli and Claudia Brunetti

4.1

Human B Cell Response in Physiology

B cell differentiation into antibody-secreting cells (ASC) provides the basis of the humoral adaptive immune system. All stages of this process require strict homeostatic controls in order to keep the cell pool size constant and to avoid the emergence of aberrant or self-reactive B cell populations. Indeed, naïve and memory B cells are governed by independent homeostatic mechanisms and different activation requirements [1–3]. In the context of a T cell-dependent (TD) B cell response, naïve B cells proliferate and differentiate into memory B cells and long-lived plasma cells only after the integration of three signals, namely: (i) B cell receptor (BCR) triggering by the antigen; (ii) CD4+ T cell help via CD40, following cognate interaction; and (iii) activation of Toll-like receptors (TLRs), whose expression is induced after BCR cross-linking [4]. By contrast, memory B cells can be effectively triggered in the absence of BCR engagement via polyclonal stimuli, such as bystander T cell help, provided in a non-cognate fashion by CD4+ T cells activated by a third-party antigen, and microbial products acting on TLRs, which are constitutively expressed by these cells [4]. In the context of thymus-independent (TI) B cell responses, naïve B cells exhibit the same differential requirements as memory B cells, since both BCR cross-linking and TLR stimulation are required. V. Racanelli (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_4, © Springer-Verlag Italia 2012

However, this response might also require surrogate T cell help, such as that provided by C4BP or BAFF [4]. Intriguingly, TLRs agonists are required both in the TD and TI responses, irrespective of the nature of T cell help, suggesting a pivotal role in B cell compartment activation. Naïve B cells do not express TLRs constitutively and cannot respond directly to microbial products; however, their capacity to rapidly up-regulate TLRs upon BCR stimulation endows them with high specificity, in that only naïve B cells stimulated by an antigen will focus on innate signals. In contrast, memory B cells constitutively express TLRs and can readily respond to a variety of environmental stimuli [5]. It has been speculated that TLR activation plays a distinct role in B cell response to viruses. The dual BCR-TLR engagement model of B cell activation perfectly fits with virus recognition by B cells, as viral particles expose an abundance of foreign antigens and contain non-self nucleic acids, a combination that should result in effective BCR and TLR triggering, followed by B cell activation and differentiation [6].

4.2

Human B Cell Response in Pathology

Several infectious agents including Epstein-Barr virus (EBV), Human Immunodeficiency Virus (HIV) and Helicobacter pylori have been reported to play an etiological role in B cell lymphoproliferative disorders. Direct infection of B lymphocytes, generalized deregulation of the immune response, and chronic activation of specific cell-surface receptors are the main pathogenetic mechanisms by which these viruses act. Abnormal antibody production (hypergammaglobulinemia) and 37

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HCV proteins and RNA

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Immunecomplexed HCV

Self lgG HCV antigen

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TLR7

Fig. 4.1 Possible mechanisms of B cell activation during chronic HCV infection. (a) After HCV entry and replication in B cells, viral proteins could exert their oncogenic potential by altering essential cell activation pathways. Consequently, infected B cells might lose proliferation control. (b) The ability of HCV-E2 to bind CD81 could determine a strong and sustained polyclonal stimulation of the B-cell compartment. (c) Dual engagement of CD81 and specific BCR by HCV-E2 could

effectively activate B cells, leading to their clonal expansion. (d) Molecular mimicry between an HCV antigen and a self-IgG could explain the selection of RF-producing B cell clones. (e) Immune-complexed HCV could effectively activate specific B cells through BCR. After immune complex internalization and HCV disruption in the endosomal/lysosomal compartment, viral RNA could reach TLR7 and start molecular pathways leading to B cell aberrant activation

B cell lymphoproliferative diseases, namely mixed cryoglobulinemia (MC) and B cell non-Hodgkin’s lymphoma (B-NHL) [5, 7, 8], are frequently observed during the chronic course of HCV infection. Several models have been proposed to explain how HCV, a predominantly hepatotropic virus, induces dysfunction of the B cell compartment.

the pathogenesis of HCV-related MC, which is characterized by a generalized B cell compartment expansion, even if the emergence of a specific rheumatoid-factorpositive (RF+) B cell clone suggests that HCV tropism is restricted to a B cell subset. According to this model, treatment of MC patients with rituximab should reduce HCV replication, but the increased serum HCV RNA levels often detected following its administration argues against the hypothesis of a B cell reservoir of infection [9]. Detectable levels of both HCV RNA and proteins [10, 11] have been reported in association with B cells and other blood cell subsets of infected individuals. In some reports [11], although not in others [12], HCV RNA was associated with peripheral blood

4.3

Direct Infection of B Cells

A number of studies hypothesized that direct infection of B cells leads to their malignant transformation (Fig. 4.1a). HCV lymphotropism may be involved in

4

B Cell Activation: General to HCV-Specific Considerations

39

mononuclear cells (PBMCs) even after antiviral therapy. Also, blood cells have been successfully infected in vitro [11, 13, 14]. However, no HCV replication was detected when PBMCs were treated with the HCV replicase inhibitor 2’C-methyl adenosine (2’-CMA), which allows viral replication to be discriminated from viral persistence [15]. In addition, the absence of important HCV entry factors, including scavenger receptor BI (SR-BI) and the tight-junction protein claudin-1 (CLDN1), on B cells impairs their proneness to infection [15]. Cumulative evidence suggests that viral RNA is bound or taken-up by cells without undergoing a complete infectious cycle. For instance, CD81, a known HCV entry factor, is expressed on B cells, thus mediating HCV capture from the environment. Surface anti-HCV antibodies may enable HCV-specific B cells to bind HCV, while Fc receptors, complement receptors, or RF on B cell membranes might be engaged by circulating virus-antibody complexes. Thus, viral particles may be detected in association with B cells, but HCV rarely infects them.

to be directed against E2 [20], suggesting specific BCR stimulation by HCV-E2. Moreover, the BCR cloned from an HCV-associated NHL was shown to specifically bind E2, although the RF activity of this E2 specific antibody has not been demonstrated [21]. Therefore, a modified model for HCV-E2 mediated B cell stimulation has been proposed. According to this model, the simultaneous engagement of BCR and CD81 by E2 would effectively reduce the B cell stimulation threshold, leading to extensive proliferation of E2-specific B cell clones [21] (Fig. 4.1c).

4.4

Polyclonal Stimulation of B Cells by HCV Antigens

The second portion of the HCV envelope glycoprotein E2 binds with high affinity to CD81, a tetraspanin expressed on several cell types [16]. Since on B cells CD81 is a component of the B cell co-receptor signaling complex [17], it has been proposed that CD81 engagement by E2 lowers the activation threshold of B cells, thus facilitating their proliferation and differentiation [18] (Fig. 4.1b). In agreement with this model, recombinant E2 protein, combined with crosslinking anti-CD81 monoclonal antibodies, polyclonally stimulates B cells in vitro [18]. However, the non-physiologically high concentration of E2 protein used in this study makes understanding of the events that occur in vivo difficult. It has also been claimed that E2/CD81 interaction triggers activation-induced (cytidine) deaminase (AID) expression and double-stranded breaks, resulting in stochastic immunoglobulin (Ig) hypermutation [19], but this model does not explain the emergence of clonal RF+ B cells and the presence of poorly hypermutated self-reacting antibodies, both features of MC. Intriguingly, an efficient HCVneutralizing monoclonal antibody, cloned from an asymptomatic HCV-infected patient, was demonstrated

4.5

Chronic Antigenic Stimulation by Specific HCV Proteins

The alternative scenario proposed for microbial species-associated B cell aberrations is chronic antigenic stimulation. This model has emerged with the description of several lymphomas developing in the context of chronic antigen-dependent immune stimulation, such as gastric mucosa-associated lymphoid tissue (MALT) positive for Helicobacter pylori [22], but it can also be transposed to other B cell abnormalities. According to this model, the pathogen is neither intrinsically transforming nor oncogenic, but its ability to persist in the host offers a chronic source of antigens to specific B cell clones [23]. Depending on the length of infection, additional oncogenic events may occur, leading the B cell proliferation to become independent of antigen stimulation [24]. According to this multistep mechanism of lymphomagenesis, t(14;18) and bcl-2 overexpression in lymphoid cells have been proposed to be the leading events for the pathogenesis of HCV-related lymphoproliferative disorders [25, 26]. Nonetheless, experimental data are still controversial [27], and t(14;18)-positive cells have also been detected in healthy individuals [28]. Of note, HCV-associated MC is characterized by monoclonal or oligoclonal expansion of specific B cell subsets both in the liver [29] and in the peripheral blood [30]. It can therefore be envisaged that an antigen-driven process is involved in B cell growth and clonal evolution. Molecular analysis of the IgH VDJ region in expanded B cell clones supports this hypothesis, showing restricted VH gene usage shared by the anti-HCV antibody response, e.g., directed against E2 (HCV/E2) [31–33]. Moreover, antibodies to E2 encode the VH/VL gene pair VH1-69 and VK3-23, which is also

40

utilized in the synthesis of WA cross-idiotype monoclonal RF in patients with MC [20, 34]. These findings suggest that RF secretion in part occurs as an antibody response to HCV antigens, such as E2. Molecular mimicry, a phenomenon in which microbial pathogens share structural similarities with self motifs, is an immune-evasion strategy possibly adopted by HCV in order to persist. As a secondary effect, molecular mimicry could confer distinctive characteristics to certain HCV antigens, making them more prone to evoke a strong autoimmune response. It has indeed been reported that the structural and antigenic homologies between the N-terminal region of the HCV-E2 protein and the human Ig variable domains (IgV) are responsible for the strong association between HCV and detection of RF [35]. Alternatively, it has been demonstrated that in MC patients the IgM RF cross-reacts with IgG Fc and HCV NS3, due to the molecular mimicry between these two proteins [36] (Fig. 4.1d). Though fascinating, these data account only for a minority of HCV MC patients and they are not sufficient to explain the general mechanisms underlying the pathogenesis of the disease. Recently, a new model of chronic antigenic stimulation has been proposed, in which the antigen can also stimulate pattern recognition receptors (PRR) receptors in B cells. According to this hypothesis, during chronic HCV infection, immune-complexed HCV stimulates the expansion of self-reactive B cells, acting through the dual ligation of BCR and TLR7 by IgG and HCV RNA, respectively [9] (Fig. 4.1e). In explorations of the pathogenetic mechanisms driving the self-reactive B cell clone expansion, the human leukocyte antigen (HLA) system has generated increasing interest. The DR5/DQ3 HLA combination [37] and a higher frequency of the DR11 HLA phenotype [38] have been proposed as predictive factors associated with an increased risk for HCV-related MC development. Although the mechanisms by which HLA polymorphysms might predispose to autoimmunity are still unknown, it has been proposed that continuous presentation of HCV-derived epitopes, through a limited set of HLA molecules, result, on the one hand, in low/ineffective viral clearance and, on the other, in positive selection of self-reactive B cell clones, sustained by T-helper lymphocytes [39]. In conclusion, studies of the human B cell response, both at steady state and during chronic HCV infection,

V. Racanelli and C. Brunetti

could help in unravelling HCV/B cell interactions in addition to providing relevant information with the potential to be promptly translated into new therapies both for autoimmune and infectious diseases.

References 1. Lanzavecchia A (1983) One out of five peripheral blood B lymphocytes is activated to high-rate Ig production by human alloreactive T cell clones. Eur J Immunol 13(10):820–824 2. Bernasconi NL, Traggiai E, Lanzavecchia A (2002) Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298(5601):2199–2202 3. Bernasconi NL, Onai N, Lanzavecchia A (2003) A role for toll-like receptors in acquired immunity: up-regulation of TLR9 by BCR triggering in naive B cells and constitutive expression in memory B cells. Blood 101(11):4500–4504 4. Ruprecht CR, Lanzavecchia A (2006) Toll-like receptor stimulation as a third signal required for activation of human naive B cells. Eur J Immunol 36(4):810–816 5. Lanzavecchia A, Bernasconi N, Traggiai E et al (2006) Understanding and making use of human memory B cells. Immunol Rev 211:303–309 6. Lanzavecchia A, Sallusto F (2007) Toll-like receptors and innate immunity in B-cell activation and antibody responses. Curr Opin Immunol 19(3):268–274 7. El-Serag HB, Hampel H, Yeh C et al (2002) Extrahepatic manifestations of hepatitis C among United States male veterans. Hepatology 36(6):1439–1445 8. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297(18):2010–2017 9. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76(8):818–824 10. Zehender G, Meroni L, De Maddalena C et al (1997) Detection of hepatitis C virus RNA in CD19 peripheral blood mononuclear cells of chronically infected patients. J Infect Dis 176(5):1209–1214 11. Navas MC, Fuchs A, Schvoerer E et al (2002) Dendritic cell susceptibility to hepatitis C virus genotype 1 infection. J Med Virol 67(2):152–161 12. Bernardin F, Tobler L, Walsh I et al (2008) Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia. Hepatology 47(5):1446–1452 13. Sung VM, Shimodaira S, Doughty AL et al (2003) Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol 77(3):2134–2146 14. Kondo Y, Sung VM, Machida K et al (2007) Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines. Virology 361(1):161–173 15. Marukian S, Jones CT, Andrus L et al (2008) Cell cultureproduced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 48(6):1843–1850 16. Pileri P, Uematsu Y, Campagnoli S et al (1998) Binding of hepatitis C virus to CD81. Science 282(5390):938–941

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17. Bradbury LE, Kansas GS, Levy S et al (1992) The CD19/ CD21 signal transducing complex of human B lymphocytes includes the target of antiproliferative antibody-1 and Leu13 molecules. J Immunol 149(9):2841–2850 18. Rosa D, Saletti G, De Gregorio E et al (2005) Activation of naive B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 102(51):18544–18549 19. Machida K, Cheng KT, Pavio N et al (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79(13):8079–8089 20. Keck ZY, Xia J, Cai Z et al (2007) Immunogenic and functional organization of hepatitis C virus (HCV) glycoprotein E2 on infectious HCV virions. J Virol 81(2):1043–1047 21. Quinn ER, Chan CH, Hadlock KG et al (2001) The B-cell receptor of a hepatitis C virus (HCV)-associated non-Hodgkin lymphoma binds the viral E2 envelope protein, implicating HCV in lymphomagenesis. Blood 98(13):3745–3749 22. Cavalli F, Isaacson PG, Gascoyne RD et al (2001) MALT lymphomas. Hematology Am Soc Hematol Educ Program: 241–258 23. Suarez F, Lortholary O, Hermine O et al (2006) Infectionassociated lymphomas derived from marginal zone B cells: a model of antigen-driven lymphoproliferation. Blood 107(8):3034–3044 24. Mayo MJ (2003) Extrahepatic manifestations of hepatitis C infection. Am J Med Sci 325(3):135–148 25. Zignego AL, Giannelli F, Marrocchi ME et al (2000) T(14;18) translocation in chronic hepatitis C virus infection. Hepatology 31(2):474–479 26. Zuckerman E, Zuckerman T, Sahar D et al (2001) bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection. Br J Haematol 112(2):364–369 27. Sansonno D, Tucci FA, De Re V et al (2005) HCV-associated B cell clonalities in the liver do not carry the t(14;18) chromosomal translocation. Hepatology 42:1019–1027 28. Roulland S, Lebailly P, Lecluse Y et al (2006) Long-term clonal persistence and evolution of t(14;18)-bearing B cells in healthy individuals. Leukemia 20(1):158–162 29. Racanelli V, Sansonno D, Piccoli C et al (2001) Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients. J Immunol 167(1):21–29

30. Charles ED, Green RM, Marukian S et al (2008) Clonal expansion of immunoglobulin M+CD27+ B cells in HCV-associated mixed cryoglobulinemia. Blood 111(3):1344–1356 31. Carbonari M, Caprini E, Tedesco T et al (2005) Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis. J Immunol 174(10):6532–6539 32. Chan CH, Hadlock KG, Foung SK et al (2001) V(H)1-69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen. Blood 97(4):1023–1026 33. De Re V, De Vita S, Marzotto A et al (2000) Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 96(10):3578–3584 34. Machida K, Kondo Y, Huang JY et al (2008) Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein. J Virol 82(13):6711–6720 35. Hu YW, Rocheleau L, Larke B et al (2005) Immunoglobulin mimicry by hepatitis C virus envelope protein E2. Virology 332(2):538–549 36. De Re V, Sansonno D, Simula MP et al (2006) HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 20(6):1145–1154 37. De Re V, Caggiari L, Monti G et al (2010) HLA DR-DQ combination associated with the increased risk of developing human HCV positive non-Hodgkin’s lymphoma is related to the type II mixed cryoglobulinemia. Tissue Antigens 75(2): 127–135 38. Cacoub P, Renou C, Kerr G et al (2001) Influence of HLA-DR phenotype on the risk of hepatitis C virusassociated mixed cryoglobulinemia. Arthritis Rheum 44(9): 2118–2124 39. De Re V, Caggiari L, De Vita S et al (2007) Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus. Dig Liver Dis 39 (Suppl 1):S65–S71

5

Organ-Specific Autoimmunity in HCV-Positive Patients Corrado Betterle and Fabio Presotto

5.1

Introduction

Hepatitis C virus (HCV) is a RNA virus that chronically infects more the 170 million individuals around the world. In the majority of cases, the acute infection develops undiagnosed such that HCV becomes a persistent infection that gives rise to chronic hepatitis, fibrosis, and even cirrhosis in about 20% of affected patients. The related severe complications and death are due to decompensated cirrhosis, end-stage liver disease, and hepatocellular carcinoma [1]. In the course of the disease, according to various studies, 40–80% of HCV-infected patients may develop at least one extrahepatic manifestation, which can be the first or only clinical sign of chronic infection with the virus [2]. Increasing attention is currently being focused on the pathogenic role of chronic HCV infection in triggering autoantibody production, autoimmune disease, and lymphoproliferative disorders [3].

5.2

Extrahepatic Manifestations

5.2.1

HCV-Associated Mixed Cryoglobulinemia

Cryoglobulins are antibody complexes that precipitate when serum is cooled and dissolve on rewarming [4]. Mixed cryoglobulinemia (MC), or type II

cryoglobulinemia, is the disease most commonly associated with chronic HCV infection. Cryoprecipitates usually contain large amounts of HCV antigens and/ or antibodies against HCV, and they can precipitate in the walls of small and medium-sized vessels, leading to activation of the complement cascade and manifestations of systemic vasculitis. The classical MC syndrome clinically presents as a triad of purpura, weakness, and arthralgias. The skin, kidney, nerves, and joints are the most frequent target organs affected by cryoglobulins. Although more than 50% of patients with chronic HCV infection have circulating serum cryoglobulins (cryoglobulinemia), the majority do not develop clinical signs or symptoms or need specific treatment [5, 6]. MC is extensively discussed in other chapters of this volume.

5.2.2

The most frequent and clinically important extrahepatic manifestations of HCV-related chronic infection involving organ-specific autoimmunity are thyroid disorders and diabetes mellitus. In the majority of cases, autoimmune endocrine disorders develop after treatment with interferons (IFNs) [7–9].

5.2.3 C. Betterle (*) Unit of Endocrinology, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_5, © Springer-Verlag Italia 2012

HCV-Related Organ-Specific Autoimmune Disorders

Pathogenesis of Extrahepatic Autoimmune Manifestations

The mechanisms that link HCV infection with autoimmunity are unknown. Moreover, it is unimportant to establish whether the presence of extrahepatic autoim43

44

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mune manifestations (EHAMs) is a direct effect of HCV infection or the consequence of its treatment. In fact, antiviral therapy with IFN-a has been accused of triggering autoimmunity [10]. Both B cell and T cell lines of the immune system are thought to be involved in the generation of autoimmunity during chronic HCV infection. A non-specific activation of the immune system triggered by HCV infection seems to be responsible for the production of autoantibodies directed against extrahepatic antigens, such as the non-organ-specific autoantibodies (NOSA), but molecular mimicry also appears to be involved in their appearance [4, 11]. The specific binding of the HCV-related E2 protein to the CD81 molecule of B cells prompted the hypothesis of a role for HCV in stimulating a chronic polyclonal B cell response to viral antigens that facilitates the onset of a lymphoproliferative dysregulation [12]. The binding of HCV to B cells may also support viral persistence and drive the immune response towards a Th2-associated profile with an increased humoral response and autoantibody production [13]. As for the cell-mediated immune response, it has been reported that CD81 receptors are expressed on various target organ cells able to bind the HCV envelope glycoprotein E2. This binding may induce several signaling cascades and cause the release of both cytotoxic and proinflammatory mediators, such as tumor necrosis factor or interleukin (IL)-8, which in turn can lead to the bystander killing of neighboring (uninfected) cells and activation of an autoimmune response [14].

5.3

Autoimmune Thyroid Diseases

Two main and distinct entities of autoimmune thyroid disease (AITD) are recognized: chronic thyroiditis (CT) and Graves’ disease (GD) [15, 16]. CT is probably the most common autoimmune disease in the world, presenting with or without goiter. CT with goiter can be associated with frank hypothyroidism, subclinical hypothyroidism, or normal thyroid function. The non-goitrous type includes two main subtypes, i.e. primary myxedema and symptomless autoimmune thyroiditis. Early genetic studies in white people showed that HLA-B8 and HLA-DR3 haplotypes were associated with symptomless autoimmune thyroiditis and HLA-DR5 with goitrous autoimmune thyroiditis, thus suggesting that the two disorders

have different genetic backgrounds [15]. CT is also associated with polymorphisms of the gene encoding cytotoxic T-lymphocyte antigen 4 (CTLA-4) [16, 17]. Thyroglobulin and/or thyroid peroxidase autoantibodies are a common finding when the different variants of CT are diagnosed, being detectable in 80–99% of cases. The reported prevalence of CT varies considerably, depending on the diagnostic criteria employed and the age and characteristics of the patient considered (e.g., gender, genetic differences, geographical origin, and iodine intake) [15, 18]. In autopsy studies performed in the UK and the USA, CT was identified in 40–45% of women and 20% of men if any degree of focal thyroiditis was considered, while its prevalence dropped to 5–15% in women and 1–5% in men when only cases of severe thyroiditis were taken into account [15]. The prevalence of subclinical hypothyroidism is estimated to be 1–10%, and the highest age- and sexspecific rate is seen in women over 60 years of age, in whom it approaches 20% [18]. In community surveys to screen the apparently normal population, 10–13% of women and 2–3% of men tested positive for thyroid autoantibodies [15, 19]. The prevalence of thyroid autoantibodies increases with age, becoming as high as 33% in women age ³ 70 years, but it otherwise peaks in the fifth and sixth decades of life. The presence of circulating thyroid autoantibodies is related to lymphocytic infiltration in the thyroid gland [20]. Graves’ disease is an AITD classically presenting with hyperthyroidism (clinical or subclinical), with or without goiter, ophthalmopathy, or dermopathy. The clinical manifestations of GD are strictly related to its severity, the duration of hyperthyroidism, and the patient’s age [16]. In patients with hyperthyroidism, high serum levels of thyroid peroxidase autoantibodies (detectable in 75% of patients) and/ or thyroid-stimulating autoantibodies (detectable in 80–95% of patients), and/or the demonstration by radionuclide thyroid scan of a diffuse increased uptake are evidence of GD. Thyroid-stimulating autoantibodies cause both thyroid hyperfunction and thyroid follicle hypertrophy/hyperplasia, resulting in a characteristic diffuse goiter. There is a well-established association between GD and certain class II HLA alleles, which varies among different racial groups. In whites, HLA-DR3 and HLA-DQA1*0501 are positively associated with GD, whereas HLA-DRB1*0701 protects against the disease [16]. GD is also associated with CTLA-4 polymorphisms in several racial groups

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Table 5.1 Frequency of thyroid autoimmunity in patients with chronic HCV infection before interferon therapy and controls Author Tran Pateron Watanabe Boadas Carella Preziati Roti Loviselli Metcalfe Floreani Fernandez-Soto Ganne-Carrie Betterle Carella Antonelli Zusinaite Gehring All studies

Reference [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [7] [33] [9] [34] [35]

Year 1993 1993 1994 1995 1995 1995 1996 1997 1997 1998 1998 2000 2000 2002 2004 2004 2006

Thyroid autoantibodies Patients (n) (%) 72 12.5 66 13.5 109 9.2 96 5.2 75 10.7 78 35.9 32 9.4 86 9.3 111 4.5 47 12.7 134 20.0 97 13.4 70 5.7 147 10.2 630 21.0 90 2.2 123 1.5 2,066 Range 1.5–21

Controls (n) 60a nt nt 96 nt nt nt 1147 99 nt 41 97 100 nt 657 nt nt 2,297

Thyroid autoantibodies (%) 1.5 nt nt 12.0 nt nt nt 17.0 11.1 nt 5.0 3.1 3.0 nt 11.2 nt nt Range 1.5–17

P value 0.02 – – ns – – – ns ns – 0.02 0.02 ns – 0.001 – ns

nt not tested, ns not significant Patients with chronic HBV infection

a

[16]. The annual incidence of GD is around 0.14 per 1,000 [18], with the highest risk of onset between the ages of 40 and 60 years. GD is five to ten times more common in women than in men and is unusual in children [16].

5.3.1

Autoimmune Thyroid Diseases and HCV Before Interferon Therapy

The etiology of AITD remains unknown. Genetic predisposition is important, but non-genetic factors also play a major part. HCV infection seems to be one of the non-genetic factors involved. Many studies have evaluated the prevalence of thyroid autoantibodies (TA) or AITD in patients with chronic HCV infection, with conflicting and inconclusive results. Table 5.1 summarizes the studies performed, showing the frequency of AITD in patients and controls. An association between HCV infection and AITD was initially suggested by Tran [21], who studied 72 patients with chronic HCV infection (43 men and 29 women) and found TA in nine cases (12%), all women (31%). Two of these nine women also had hypothyroidism. Only 1.5% of 60 patients with chronic HBV infection (used as controls) had low titers of TA.

Further studies on these patients are listed in Table 5.1. Considering the cited publications as a whole, in a cohort of 2,066 patients with chronic HCV infection TA were detected in 1.5–21% of cases as opposed to 1.5–17% in controls. Only four studies reported a significant association between HCV infection and TA [9, 21, 31, 32]. However, five studies found no such association [7, 24, 28, 29], and another eight studies provided no data on matched controls [22, 23, 25–27, 30, 33, 34] (Table 5.1). Disparities in the prevalence of TA may be related to the different methods used to detect TA, the female/male ratio, or the ages of the patients examined. The majority of the studies also failed to provide data on matched controls. To ascertain whether a correlation exists between HCV infection and AITD, other studies investigated the frequency of HCV infection in patients with AITD. In 1993, Quaranta et al. studied 147 patients with AITD and identified HCV infection in 10 (6.8%), a frequency significantly higher than in controls (0.7%) [36]. By contrast, another study found that none of 30 patients with CT had HCV infection [37], and another no significant association between HCV genotypes and the development of TA [34]. We investigated the prevalence of HCV infection and TA in a general population of 697 adult Italians and found that 10.1% were

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C. Betterle and F. Presotto

Table 5.2 Occurrence of thyroid antibodies after interferon therapy in patients with chronic HCV infection

Author Baudin Watanabe Imagawa Carella Preziati Marazuela Roti Carella Gehring All studies

Reference [40] [23] [41] [25] [26] [42] [27] [43] [35]

Year 1993 1994 1995 1995 1995 1996 1996 2001 2006

Occurrence of thyroid Patients (n) antibodies (%) 68 5.9 109 1.9 58 8.9 75 34.6 78 40.0 144 4.9 32 12.5 114 31.6 123 15.5 801 Range 1.9–40

positive for HCV and 6.1% for TA, but no significant association emerged between those testing positive for HCV and for TA [38]; therefore, these findings failed to demonstrate any clear relationship between chronic HCV infection and AITD.

relative risk than men of developing thyroid disease; (f) while on IFN therapy, several patients develop thyroid dysfunction in the absence of TA, suggesting a direct toxic effect of IFN on the thyrocytes (so-called destructive thyroiditis); (g) in most cases of destructive thyroiditis, thyroid dysfunction is mainly subclinical and may regress spontaneously [44–46]. In conclusion, while receiving IFN therapy, patients with chronic HCV infection may develop either AITD (CT or GD), or even TA with no gland dysfunction, or non-AITD conditions (destructive thyroiditis). All patients with chronic HCV infection should therefore be screened for thyroid diseases by means of baseline TSH and TA assays before starting IFN therapy, and then every 2–3 months during treatment. If TSH levels are normal but TA are positive, the patient risks developing thyroid dysfunction. If thyroid dysfunction sets in and TA are negative, it may be advisable to test for TSH receptor antibodies and perform thyroid ultrasound [45].

5.4 5.3.2

Autoimmune Thyroid Diseases and HCV During or After Interferon Therapy

In 1992, Marcellin et al. first reported on two patients with chronic HCV hepatitis who developed hypothyroidism while on IFN therapy [39]. Many other authors subsequently confirmed this observation (Table 5.2). Judging from these studies, the prevalence of IFNinduced thyroid autoimmunity ranges from 1.9% to 40%, possibly depending on the dosage and duration of the medical therapy, the patients’ characteristics, and the definition of thyroid dysfunction. An analysis of the main data collected after IFN therapy suggests that: (a) patients already positive for TA may experience an increase in their antibody titers and develop a clinical or subclinical thyroid dysfunction; (b) initially negative patients acquire TA in 1.9–40% of cases, and a proportion of them will develop clinical or subclinical thyroid dysfunction; (c) serum thyroid-stimulating hormone (TSH) and anti-thyroperoxidase antibody (TPO) status before IFN treatment predict progression to AITD; (d) thyroid autoimmunity is reversible in some cases, but permanent in others, although the percentage of the latter cannot be predicted from the available data; (e) women have a two-fold higher

Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (DM) is a chronic autoimmune disorder with variable degrees of insulin deficiency resulting from an immune-mediated destruction of the pancreatic beta cells [47]. The disease is characterized by T lymphocyte infiltration of the pancreatic islets, with the disappearance of the beta cells. A genetic predisposition to type 1 DM has been mapped to the HLA region on chromosome 6, but environmental factors have also been implicated in the pathogenesis of this disorder. Islet cell autoantibodies (ICA), insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies (GADA), and antibodies to the second islet antigen (IA-2A/ICA512) are the main serological markers of DM. One or more of these autoantibodies are detectable when diabetes is diagnosed in 90% of the cases, and they often appear even before the clinical onset of the disease [47].

5.4.1

Type 1 Diabetes Mellitus and HCV Chronic Infection Before Interferon Therapy

Epidemiological studies have shown a relationship between HCV infection and DM, suggesting that this disease is another extrahepatic manifestation. However,

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Table 5.3 Autoantibodies to endocrine pancreas in patients with HCV chronic hepatitis before and after interferon (IFN) therapy

Authors Imagawa Di Cesare Imagawa Hieronimus Floreani Betterle Piquer Wesche Wasmuth All studies

Year 1995 1996 1996 1997 1998 2000 2001 2001 2001

Reference [41] [52] [53] [54] [30] [7] [55] [56] [57]

Patients (n) 58 48 40 47 47 70 277 75 56 718

Pancreatic autoantibodies before IFN (%) 0 0 0 2 4.2 2.8 1.4 4.0 0 Range 0–4.2

Pancreatic autoantibodies during IFN (%) 0 0 2.5 nt 12.7 nt 0 10.6 5.3 Range 0–12.7

Development of diabetes mellitus (%) 0 0 0 nr 2 nr 0 0 0 0–2

nt not tested, nr not reported

these studies found a greater association of HCV infection with type 2 diabetes, not type 1 [48–51]. The lack of association between HCV chronic infection and pancreatic autoimmunity before IFN therapy has also been demonstrated in many studies (Table 5.3). A meta-analysis was performed on 718 patients with chronic HCV infection. Pancreatic autoantibodies were detected in 0–4.2% before IFN therapy, a frequency that did not differ significantly from the situation in controls [58, 59] and suggesting that HCV infection is not linked to pancreatic autoimmunity.

5.4.2

Type 1 Diabetes Mellitus and Chronic HCV Infection During or After Interferon Therapy

The situation is different in patients with chronic HCV hepatitis treated with IFN. The first case of autoimmune diabetes occurring after IFNa treatment was described in 1992, in a male patient with chronic HCV infection found to have pancreatic autoantibodies in a serum sample collected before IFNa treatment. This prompted the speculation that IFNa triggers the onset of overt type 1 diabetes in patients with islet cell antibodies [60]. A study performed in Italy on more than 11,000 patients with chronic HCV infection treated with IFNa reported ten new cases of DM (0.08%), nine of them requiring permanent insulin therapy [61]. Another study, performed in Japan on 667 HCVpositive patients with chronic hepatitis, reported five new cases of type 1 diabetes (0.7%) after IFN therapy

[62]. In both studies, the prevalence of diabetes was higher than among controls. In the Japanese study, the majority of the cases of hyperglycemia regressed, albeit incompletely, after therapy was withdrawn. These studies did not consider the presence of pancreatic autoantibodies, however, which could have discriminated between patients with and without the autoimmune form of DM. From 1995 to 2001, other reports were published on the prevalence of autoantibodies to the endocrine pancreas after IFNa treatment (Table 5.3). These studies showed that, while on IFNa therapy, patients already positive for such autoantibodies had an increase in their antibody titers, and that up to 12.7% of patients initially negative acquired pancreatic antibodies de novo and then developed autoimmune type 1 diabetes [58, 59]. Following the first description, in 1992, of a case of autoimmune DM induced by IFN, by 2004 another 31 patients had reportedly developed type 1 DM during, or soon after, IFN therapy, as reviewed by Fabris et al. [58] and Davendra et al. [59]. An analysis of these cases showed that: (a) there were 25 males and 6 females, with a mean age at the onset of DM of 46.7 years (range 23–66 years); (b) 25 patients were treated with IFN for chronic HCV hepatitis, 3 for HBV hepatitis, and 3 for cancer; (c) nine patients were treated with IFNa and ribavirin, 1 with IFNa and IL-2, 1 with both IFNa and IFNb, 1 with IFNb, and the remainder with IFNa; the total dose of IFN before DM developed was 65–1350 U; (d) nine of the 25 cases evaluated had a family history of DM (type 1 in 3 cases, type 2 in 6); (e) the latency period from starting

48

C. Betterle and F. Presotto

IFN therapy to DM varied from 10 days to 4 years; (f) one or more pancreatic antibodies were positive in 9 of 18 patients tested before any IFN treatment, but in 23 of 30 (77%) evaluated at the onset of DM; (g) in 16 of 18 (89%) patients evaluated, an HLA conferring a susceptibility to type 1 DM was detected; (h) in the majority of patients, the clinical onset of DM was acute and the disease persisted even after IFN therapy was withdrawn, but the diabetes did regress spontaneously in some cases [58, 59]. Other cases of patients developing autoimmune DM after IFN therapy have since been reported [63–67]. The considerable number of patients developing IFN-related DM has been recently pointed out in a literature review investigating the Japanese population from 1992 to 2009 [68]. Out of the 143 collected cases, 104 were type 1 DM and 39 were nonautoimmune type-2-like DM. Patients with IFN-related type 1 DM had a HLA type similar to Japanese type 1 diabetic patients, and a high positive rate of GAD antibodies [68] These data confirm our initial hypothesis, that IFNa triggers the onset of overt type 1 diabetes in patients with pancreatic autoantibodies, or modifies the natural history of the disease in genetically predisposed individuals [59]. Patients with chronic HCV hepatitis who are candidates for IFN therapy are therefore considered at risk of autoimmune DM. Accordingly, they should be screened for pancreatic autoimmunity and glycemia before and during IFN therapy in order to identify DM or the risk of it developing [58].

5.5

Autoimmune Gastritis

Autoimmune gastritis is a disease affecting the body and fundus of the stomach, with lymphocytic infiltration and autoantibodies to parietal cells (PCA). Patients with chronic HCV infection test positive for PCA in 1.6–5% of cases, and 5% have high serum gastrin levels. After IFNa treatment, however, 13% of patients become PCA-positive and 16% develop hypergastrinemia [69]. In 22 patients, PCA and hypergastrinemia developed during IFN treatment; endoscopic investigation revealed chronic atrophic gastritis in 59%. Gastric autoimmunity correlated closely with the presence of thyroid autoimmunity (thyrogastric syndrome) [69]. Cases of pernicious anemia developing in patients with antibodies to intrinsic factor have also been described after IFNa therapy [70].

5.6

Celiac Disease

Autoantibodies against tissue transglutaminase or endomysium, which are recognized as serological markers of celiac disease, have been found in 1.3–2.0% of patients with HCV-positive chronic hepatitis, compared with 0.16–0.4% of controls [71, 72]. During IFNa treatment, six of seven patients (86%) with transglutaminase autoantibodies developed moderate-severe symptoms of celiac disease, which improved after therapy was withdrawn [71]. Based on these findings, transglutaminase autoantibodies should be assayed if gastrointestinal disorders occur during IFNa therapy.

5.7

Mechanisms of Interferon-Related Damage to Thyrocytes and Pancreatic Beta Cells

Although it remains to be determined exactly how IFN triggers or exacerbate autoimmune diseases such as CT, GD, autoimmune DM, autoimmune gastritis, or celiac disease, according to experimental data summarized by Mandac et al. [45], IFNa can: (a) increase the expression of class I HLA antigens and induce the expression of class II HLA on thyrocytes; this aberrant expression is then associated with cytotoxic T lymphocyte activation by a Th1-oriented response; (b) stimulate, in vitro, thyrocyte transcription of different genes encoding cytokines and adhesion molecules and significantly increase the expression of ICAM-1 and B7 molecules; this increased expression of adhesion molecules favors the presentation of thyroid autoantigens; (c) increase the activity of lymphocytes, macrophages, natural killer cells, neutrophils, and monocytes; (d) increase the production of cytokines such as IL-6 (IL-6 receptors on thyrocytes reduce TSH-induced iodine uptake and the release of thyroid hormones); and (e) reduce T regulatory lymphocyte function. A possible pathogenic mechanism behind autoimmune thyroiditis is therefore that IFNa polarizes the immune reaction towards a Th1 response, which in turn induces the release of IFNg and IL-2, both of which are potent proinflammatory cytokines [45]. IFNa may also be implicated in the onset or acceleration of autoimmune processes against pancreatic beta cells, thus triggering the onset of type 1 DM. Experimental data summarized by Devendra et al. [59]

5

Organ-Specific Autoimmunity in HCV-Positive Patients

indicate that: (a) high levels of serum IFNa are detectable in the majority of patients at the onset of type 1 diabetes; (b) the beta cells involved in insulitis produce IFNa; (c) high levels of IFNa are detectable in enterovirus-associated type 1 diabetes; (d) Coxsackie B4 virus stimulates IFNa production by beta cells in vitro; (e) transgenic mice carrying beta cells that produce IFNa develop autoimmune diabetes with insulitis, and IFNa inhibition with monoclonal antibodies protects these animals against the occurrence of diabetes; (f) IFNa expression is associated with the increased expression of HLA class I molecules on pancreatic beta cells.

5.8

Non-Organ-Specific Autoimmune Diseases or Systemic Autoimmune Diseases

Although nearly 40% of unselected patients with HCV have at least one extrahepatic manifestation, the prevalence of patients fulfilling the criteria for nonorgan-specific autoimmune diseases (NOSAD) is much lower (2–6%). In the HISPAMEC Registry (HispanoAmerican Study Group of Autoimmune Manifestations Associated with Hepatitis C Virus), the most commonly associated NOSAD was Sjögren’s syndrome, which accounted for 47.5% of all NOSADs, followed by rheumatoid arthritis (14.7%), systemic lupus erythematosus (12.6%), polyarteritis nodosa (7.6%), antiphospholipid syndrome (5.8%), and inflammatory myopathies (3.8%) [73]. The strong association between HCV infection and Sjögren’s syndrome is probably related to the sialotropism of HCV [74]. This syndrome is the NOSAD with the highest prevalence of chronic HCV infection, which was found in 151 (18%) of 858 of these patients who were tested for the virus [73]. Antinuclear antibodies, in addition to rheumatoid factor, anticardiolipin antibodies, and smooth muscle, liver/kidney, and microsomal antibodies, are detectable in 40–65% of patients with HCV infection [38, 75, 76]. The characteristic lymphotropism of HCV is probably behind the increased production of autoantibodies. The prevalence and titer of these autoantibodies remain substantially unchanged after IFNa therapy [71]. In a cohort of 963 treatment-naïve HCV patients tested for anti-nuclear and anti-smooth muscle antibodies, 172 (17.9%) had at least one autoantibody: anti-smooth muscle in 104 patients (10.8%), anti-nuclear in 54 (5.6%), and both in 14 (1.5%).

49

5.9

Conclusions

Hepatitis C virus infection appears to be primarily, and in most patients almost exclusively, confined to the liver, but there is a wide variety of extrahepatic manifestations that seem to be associated with the infection, especially autoimmune disorders. A common hypothesis is that non-hepatic diseases are caused by the widespread tropisms of HCV, and particularly lymphotropism, which may explain the production of autoantibodies and/or the activation of self-reactive lymphocytes. In genetically susceptible individuals at least, this may give rise to clinically overt autoimmune disorders. The link between chronic HCV infection and NOSADs has been clearly documented, whereas the links between chronic HCV infection and AITD or type 1 diabetes mellitus are less clear, but IFN treatment may have a crucial role in triggering latent autoimmune reactions in these patients and in inducing the development of the related clinical diseases.

References 1. Lauer GM, Walker BD (2001) Hepatitis C virus infection. N Engl J Med 345:41–52 2. Cacoub P, Renou C, Rosenthal E et al (2000) Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. Groupe d’Etude et de Recherche en Médecine Interne et Maladies Infectieuses sur le Virus de l’Hepatite C. Medicine (Baltimore) 79:47–56 3. Zignego AL, Piluso A, Giannini C (2008) HBV and HCV chronic infection: autoimmune manifestations and lymphoproliferation. Autoimmun Rev 8:107–111 4. Ferri S, Muratori L, Quarneti C et al (2008) HCV and autoimmunity. Curr Pharm Des 14:1678–1685 5. Sene D, Ghillani-Dalbin P, Thibault V et al (2006) Longterm course of mixed cryoglobulinemia in patients infected with hepatitis C virus. J Rheumatol 31:2199–2206 6. Saadoun D, Landau DA, Calabrese LH, Cacoub PP (2007) Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation. Rheumatology (Oxford) 46:1234–1242 7. Betterle C, Fabris P, Zanchetta R et al (2000) Autoimmunity against pancreatic islets and other tissue before and after interferon-a therapy in patients with hepatitis C virus chronic infection. Diabetes Care 23:1177–1181 8. Bini EJ, Mehandru S (2004) Incidence of thyroid dysfunction during interferon alpha-2b and ribavirin therapy in men with chronic hepatitis C: a prospective cohort study. Arch Intern Med 164:2371–2376 9. Antonelli A, Ferri C, Pampana A et al (2004) Thyroid disorders in chronic hepatitis C. Am J Med 117:10–13 10. Baccala R, Kono DH, Theofilopoulos AN (2005) Interferons as pathogenic effectors in autoimmunity. Immunol Rev 204:9–26

50 11. Gregorio GV, Choudhuri K, Ma Y et al (2003) Mimicry between the hepatitis C virus polyprotein and antigenic targets of nuclear and smooth muscle antibodies in chronic hepatitis C virus infection. Clin Exp Immunol 133:404–413 12. Pileri P, Uematsu Y, Campagnoli S et al (1998) Binding of hepatitis C virus to CD81. Science 282:938–941 13. Deng J, Dekruyff RH, Freeman GJ et al (2002) Critical role of CD81 in cognate T-B cell interactions leading to Th2 responses. Int Immunol 14:513–523 14. Akeno N, Blackard JT, Tomer Y (2008) HCV E2 protein binds directly to thyroid cells and induces IL-8 production: a new mechanism for HCV induced thyroid autoimmunity. J Autoimmun 31:339–344 15. Dayan CM, Daniels GH (1996) Chronic autoimmune thyroiditis. N Engl J Med 335:99–107 16. Weetman AP (2000) Graves’ disease. N Engl J Med 343: 1236–1248 17. Vaidya B, Pearce S (2004) The emerging role of the CTLA-4 gene in autoimmune endocrinopathies. Eur J Endocrinol 150:619–626 18. Cooper DS (2002) Subclinical hypothyroidism. N Engl J Med 345:260–265 19. Betterle C, Callegari GP, Presotto F et al (1987) Thyroid autoantibodies: a good marker for the study of symptomless autoimmune thyroiditis. Acta Endocrinol (Copenh) 114: 321–327 20. Yoshida H, Amino N, Yagawa K et al (1978) Association of serum anti-thyroid antibodies with lymphocytic infiltration of the thyroid gland: studies on seventy autopsied cases. J Clin Endocrinol Metab 46:858–862 21. Tran A, Quaranta JF, Benzaken S et al (1993) High prevalence of thyroid autoantibodies in a prospective series of patients with chronic hepatitis C before interferon therapy. Hepatology 18:253–257 22. Pateron D, Hartmann DJ, Jouanolle DV, Beaugrand M (1993) Latent autoimmune thyroid disease in patients with chronic HCV hepatitis. J Hepatol 17:417–419 23. Watanabe U, Hashimoto E, Hisamitsu T et al (1994) The risk factor for development of thyroid disease during interferonalpha therapy for chronic hepatitis C. Am J Gastroenterol 89: 399–403 24. Boadas J, Rodriguez-Espinosa J, Enriquez J et al (1995) Prevalence of thyroid autoantibodies is not increased in blood donors with hepatitis C virus infection. J Hepatol 22:611–615 25. Carella C, Amato G, Biondi B et al (1995) Longitudinal study of antibodies against thyroid in patients undergoing interferon-a therapy for HCV chronic hepatitis. Horm Res 44:110–114 26. Preziati D, La Rosa L, Covini G et al (1995) Autoimmunity and thyroid function in patients with chronic active hepatitis treated with recombinant interferon alpha-2. Eur J Endocrinol 132:587–593 27. Roti E, Minelli R, Giuberti T et al (1996) Multiple changes in thyroid function in patients with chronic active HCV hepatitis treated with recombinant interferon-alpha. Am J Med 101:482–487 28. Loviselli A, Oppo A, Velluzzi F et al (1999) Independent expression of serological markers of thyroid autoimmunity and hepatitis virus C infection in the general population: results of a community-based study in north-western Sardinia. J Endocrinol Invest 22:660–665

C. Betterle and F. Presotto 29. Metcalfe RA, Ball G, Kudesia G, Weetman AP (1997) Failure to find an association between hepatitis C virus and thyroid autoimmunity. Thyroid 22:660–665 30. Floreani A, Chiaramonte M, Greggio NA et al (1998) Organspecific autoimmunity and genetic predisposition in interferon-treated HCV-related chronic hepatitis patients. Ital J Gastroenterol Hepatol 30:71–76 31. Fernandez-Soto L, Gonzales A, Escobar-Jimenez F et al (1998) Increased risk of autoimmune thyroid disease in hepatitis C vs hepatitis B before, during and after discontinuing interferon therapy. Arch Intern Med 158:1445–1448 32. Carella C, Mazziotti G, Morisco F et al (2002) The addition of ribavirin to interferon-alpha therapy with hepatitis C virus-related chronic hepatitis does not modify the thyroid autoantibody pattern but increases the risk of developing hypothyroidism. Eur J Endocrinol 146:743–749 33. Ganne-Carrie N, Medini A, Coderc E et al (2000) Latent autoimmune thyroiditis in untreated patients with HCV chronic hepatitis: a case-control study. J Autoimmun 14:189–193 34. Zusinaite E, Metskula K, Salupere R (2005) Autoantibodies and hepatitis C virus genotypes in chronic hepatitis C patients in Estonia. World J Gastroenterol 11:488–494 35. Gehring S, Kullmer U, Koeppelmann S et al (2006) Prevalence of autoantibodies and the risk of autoimmune thyroid disease in children with chronic hepatitis C virus infection treated with interferon-a. World J Gastroenterol 12:5787–5792 36. Quaranta JR, Tran A, Regnier D et al (1993) High prevalence of antibodies to hepatitis C virus (HCV) in patients with anti-thyroid autoantibodies. J Hepatol 18:136–138 37. Marcellin P, Pouteau M, Benhamou JP (1995) Hepatitis C virus infection, alpha interferon therapy and thyroid dysfunction. J Hepatol 22:364–369 38. Floreani A, Betterle C, Carderi I, Arsita-Research Group et al (2006) Is hepatitis C virus a risk factor for thyroid autoimmunity? J Viral Hepat 13:272–277 39. Marcellin P, Pouteau M, Renard P et al (1992) Sustained hypothyroidism induced by recombinant alpha interferon in patients with chronic hepatitis C. Gut 33:855–856 40. Baudin E, Marcellin P, Pouteau M et al (1993) Reversibility of thyroid dysfunction induced by recombinant alpha interferon in chronic hepatitis C. Clin Endocrinol 39:657–661 41. Imagawa A, Itoh N, Hanafusa T et al (1995) Autoimmune endocrine disease induced by recombinant interferon-alpha therapy for chronic active hepatitis. J Clin Endocrinol Metab 80:922–926 42. Marazuela M, Garcia-Buey L, Gonzalez-Fernandez B et al (1996) Thyroid autoimmune disorders in patients with chronic hepatitis C before and during interferon-a therapy. Clin Endocrinol 44:635–642 43. Carella C, Mazziotti G, Morisco F et al (2001) Long-term outcome of interferon-alpha-induced thyroid autoimmunity and prognostic influence of thyroid autoantibody pattern at the end of treatment. J Clin Endocrinol Metab 86:1925–1929 44. Oppenheim Y, Ban Y, Tomer Y (2004) Interferon induced autoimmune thyroid disease (AITD): a model for human autoimmunity. Autoimmun Rev 3:388–393 45. Mandac JC, Chaudhry S, Sherman KE, Tomer Y (2006) The clinical and physiological spectrum of interferon-a induced thyroiditis: toward a new classification. Hepatology 43: 661–672

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46. Costelloe SJ, Wassef N, Schulz J et al (2010) Thyroid dysfunction in a UK hepatitis C population treated with interferon-alpha and ribavirin combination therapy. Clin Endocrinol (Oxf) 73:249–256 47. Atkinson MA, Eisenbarth GS (2001) Type 1 diabetes: new perspectives on disease pathogenesis and treatment. Lancet 358:221–229 48. Simo R, Hernandez C, Genesca J, Jardi R, Mesa J (1996) High prevalence of hepatitis C virus infection in diabetic patients. Diabetes Care 19:998–1000 49. Nizar N, Zein NN (1998) Hepatitis C and diabetes mellitus: an ongoing controversy. Am J Gastroenterol 93:2320–2322 50. Everarth JA (2001) Confluences of epidemics: does hepatitis C cause type 2 diabetes? Hepatology 33:762–763 51. Mehta SH, Brancati FL, Sulkowski MS, Strathdee SA, Szklo M, Thomas DL (2000) Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the united states. Ann Intern Med 133:592–599 52. Di Cesare E, Previti M, Russo F et al (1996) Interferonalpha therapy may induce insulin autoantibodies development in patients with chronic viral hepatitis. Dig Dis Sci 41: 1672–1677 53. Imagawa A, Itoh N, Hanafusa T et al (1996) Antibodies to glutamic acid decarboxylase induced by interferon-alpha therapy for chronic viral hepatitis. Diabetologia 39:126 54. Hiéronimus S, Fredenrich A, Tran A et al (1997) Antibodies to GAD in chronic hepatitis C patients. Diabetes Care 20: 1044 55. Piquer S, Hernandez C, Enriquez J et al (2001) Islet cell and thyroid antibody in patients with hepatitis C virus infection: effect of treatment with interferon. J Lab Clin Med 137: 38–42 56. Wesche B, Jaechel E, Trautwein C et al (2001) Induction of autoantibodies to adrenal cortex and pancreatic islet cells by interferon alpha therapy for chronic hepatitis C. Gut 48: 378–383 57. Wasmuth HB, Stolte C, Geier A, Gartung C, Matern S (2001) Induction of multiple autoantibodies to islet cell antigens during treatment with interferon alpha for chronic hepatitis C. Gut 49:596–597 58. Fabris P, Floreani A, Tositti G et al (2003) Review article: type 1 diabetes mellitus in patients with chronic hepatitis C before and after interferon therapy. Aliment Pharmacol Ther 18:549–558 59. Devendra D, Eisenbarth GS (2004) Interferon alpha – a potential link in the pathogenesis of viral-induced type 1 diabetes and autoimmunity. Clin Immunol 111:225–233 60. Fabris P, Betterle C, Floreani A et al (1992) Development of type 1 diabetes mellitus during alpha-interferon therapy for chronic HCV hepatitis. Lancet 340:548 61. Fattovich G, Giustina G, Favarato S, Ruol A (1996) A survey of adverse events in 11,241 patients with chronic viral hepatitis treated with alpha interferon. J Hepatol 24:38–47 62. Okanoue T, Sakamoto S, Itoh Y et al (1996) Side effects of high-dose interferon therapy for chronic hepatitis. J Hepatol 25:283–291

51 63. Cozzolongo R, Betterle C, Fabris P et al (2006) Onset of type 1 diabetes mellitus during peginterferon alpha-2b plus ribavirin treatment for chronic hepatitis C. Eur J Gastroenterol Hepatol 18:689–692 64. Soultati A, Dourakis S, Alexopoulou A et al (2007) Simultaneous development of diabetic ketoacidosis and hashitoxicosis in a patient treated with pegylated interferon-alpha for chronic hepatitis C. World J Gastroenterol 13:1292–1294 65. Tanaka J, Sugimoto K, Shiraki K et al (2008) Type 1 diabetes mellitus provoked by peginterferon a-2b plus ribavirin treatment for chronic hepatitis C. Intern Med 47:747–749 66. Ogihara T, Katagiri H, Yamada T et al (2009) Peginterferon (PEG-IFN) plus ribavirin combination therapy, but neither interferon nor PGE-IFN alone, induced type 1 diabetes in a patient with chronic hepatitis C. Intern Med 48: 1387–1390 67. Yamazaki M, Sato A, Takeda T, Komatsu M (2010) Distinct clinical courses in type 1 diabetes mellitus induced by peginterferon-alpha treatment for chronic hepatitis C. Intern Med 49:403–407 68. Muraishi K, Sasaki Y, Kato T et al (2011) Classification and characterization of interferon-related diabetes mellitus in Japan. Hepatol Res 41:184–188 69. Fabbri C, Jaboli F, Giovanelli S et al (2003) Gastric autoimmune disorders in patients with chronic hepatitis C before, during and after interferon-alpha therapy. World J Gastroenterol 9:1487–1490 70. Andrès E, Loukili NH, Ben Abdelghani M, Noel E (2004) Pernicious anemia associated with interferon-alpha therapy and chronic hepatitis C infection. Clin Gastroenterol 38: 382–383 71. Durante-Mangoni E, Iardino P, Resse M et al (2004) Silent celiac disease in chronic hepatitis C – impact of interferon treatment on the disease onset and clinical outcome. J Clin Gastroenterol 38:901–905 72. Ruggeri C, La Masa AT, Rudi S et al (2008) Celiac disease and non-organ-specific autoantibodies in patients with chronic hepatitis C virus infection. Dig Dis Sci 53:2151–2155 73. Ramos-Casals M, Muñoz S, Medina F et al (2009) HISPAMEC Study Group. Systemic autoimmune diseases in patients with hepatitis C virus infection: characterization of 1020 cases (The HISPAMEC Registry). J Rheumatol 36: 1442–1448 74. Arrieta JJ, Rodriguez-Inigo E, Ortiz-Movilla N et al (2001) In situ detection of hepatitis C virus RNA in salivary glands. Am J Pathol 158:259–264 75. Agnello V, De Rosa FG (2004) Extra-hepatic disease manifestations of HCV infection: some current issues. J Hepatol 40:341–352 76. Williams MJ, Lawson A, Neal KR, on behalf of the Trent HCV Group et al (2009) Autoantibodies in chronic hepatitis C virus infection and their association with disease profile. J Viral Hepat 16:325–333

Part II Cellular Compartments of HCV Infection (and Replication)

6

HCV and Blood Cells: How Can We Distinguish Infection from Association? Lynn B. Dustin and Charles M. Rice

6.1

Introduction

While the primary target of hepatitis C virus (HCV) infection is the liver, the frequent occurrence of extrahepatic manifestations such as mixed cryoglobulinemia (MC) has led investigators to examine the possibility that HCV replicates in other cells and tissues. A number of groups have reported association of HCV RNA with blood cells in patients with ongoing or even resolved HCV infection. These reports raise the possibility that HCV infection of peripheral blood mononuclear cells (PBMCs) affects their function, contributing to the pathogenesis of MC, B cell nonHodgkin lymphoma, or ineffective anti-HCV immune responses. It may be tempting to conclude that MC is a consequence of B cell infection by HCV. However, there are reasons to be skeptical [1, 2]. B cells, monocytes, and dendritic cells are all specialized to capture exogenous material for antigen presentation. Patients with chronic HCV infection, and certainly those with MC, have abundant viral material circulating in the form of immune complexes—a form likely to enhance capture by these cell types. Claims of HCV infection in lymphocytes should be subjected to the same rigorous scrutiny that has been applied to studies of HCV infection in hepatocytes. There is no reason a priori to assume that HCV replication proceeds by different pathways in lymphocytes than in liver cells. Thus, we expect that in any

L.B. Dustin (*) Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY, USA e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_6, © Springer-Verlag Italia 2012

susceptible cell HCV replication would involve a measurable increase in viral RNA over time, a measurable increase in HCV protein levels over time, and, importantly, a demonstration that the increased RNA and protein levels are sensitive to specific antiviral drugs. If these criteria are not met, then the study fails to distinguish HCV infection from simple association of HCV RNA and/or proteins. Studies lacking these features do not meet the criteria that have been adopted as proof of HCV infection in liver cells.

6.2

HCV Genome and Protein Products

The genome of HCV is a single, positive-sense RNA approximately 9,600 bases in length [3]. HCV RNA is highly structured, with stem-loop and pseudoknot structures that play essential roles in RNA translation and replication. An internal ribosomal entry site, near the 5¢ end of the genome, directs the ribosome to translate HCV RNA. When a susceptible cell is infected, HCV RNA is translated as a single polyprotein of about 3,000 amino acids. The polyprotein is cleaved during and after translation by host and virus-encoded proteases, yielding the structural proteins core, E1, E2, and p7, and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B. The viral particle is believed to include HCV RNA in complex with core protein, surrounded by a lipid bilayer decorated with the envelope glycoproteins E1 and E2. In vivo, much of the extracellular HCV RNA is associated with lipoprotein particles. The NS proteins are present in the infected cell, where they modulate cellular functions and contribute to viral replication and assembly. Cells cannot be said to be productively infected simply because they 55

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are associated with HCV RNA and proteins. Capture or association of viral material, including debris from infected cells, by B cells or other cell types could allow the detection of HCV proteins and RNA even if these cells are not sites of active viral replication.

6.3

ranged from a high of less than one HCV genome per 30 B cells to a low of one HCV genome per 100,000 PBMCs. An exception is the study in [12]. Since a cell cannot be infected by a fraction of an HCV genome, these results strongly suggest that the number of infected cells cannot be high.

Which Cell Types Are Infected? 6.5

The association of HCV RNA with B cells has been reported by a number of groups [4–13]. One group reported that a B lymphoma cell line produced infectious HCV in culture [14]. In some of these studies HCV RNA was quantified but many others have used semi-quantitative assays, or simply reported that the reaction was positive. Other groups have found that T cells, monocytes, neutrophils, or dendritic cells contain HCV RNA and/or proteins [5, 6, 13, 15–18]. Of note, a recent report demonstrated the presence of HCV RNA and a NS protein exclusively in blood cells expressing the phagocytic Fc receptor, FcgRIIIA or CD16 [19]. This observation is consistent with the possibility that viral material is taken up from the blood in the form of immune complexes, or from sites of infection by phagocytosis of dying infected cells.

6.4

How Much HCV RNA Is There in Lymphocytes?

Published reports and our own data indicate that the level of HCV RNA associated with PBMCs in patients’ blood samples is actually very small. In most studies that have provided quantitative measurements of HCV RNA, the number of HCV genomes has been far below one copy per cell. As summarized in Table 6.1, reported levels of HCV RNA in B cells and in PBMCs have

Is It Really Replicating?

While some groups have shown that HCV RNA increases in B cells following activation in vitro, these studies have not included rigorous quantitation. In some, the amount of HCV protein demonstrated by immunofluorescence seems not to correlate with the level of viral RNA, suggesting that staining artifacts or uptake of material from infected tissues contributed to the observed signal. Others, using quantitative methods, found that residual HCV RNA declines rapidly when patient B cells are cultured in vitro with or without mitogens [2, 20].

6.6

Challenges in the Detection and Quantitation of HCV RNA Replication

The genetic material of HCV is a single positive-sense RNA. In susceptible cells, HCV RNA is translated to produce replicase enzymes, which use genomic RNA as a template for the production of a replicative intermediate, i.e., negative sense or minus strand RNA. This strand is then used as a template for the synthesis of more positive sense genomes. Measurement of the minus strand is challenging. In infected liver-derived cell lines, the minus strand is present at approximately tenfold lower levels than the genomic plus strand RNA.

Table 6.1 Measurements of HCV RNA levels in B cells and PBMCsa PBMCs 1.5 × 10−4a (median) Not reported

B cells Notes 2.6 × 10−3 (median) < 2.5 × 10−4 to 4.9 × 10−3 (range) Replicative intermediate detected only in liver except for one case in dendritic cells and one case in plasma 1 × 10−4 to 0.01 (range) 1 × 10−5 to 1 × 10−3 (range) Calculations assume one mg RNA/106 cells 0.033 (mean) Replicative intermediate detected only in liver 2.5 × 10−3 (mean) cells “Sustained virologic responders” 1.7 × 10−4 to 5.6 × 10−4 (range) Not reported 2.2 × 10−2 (mean) Calculations assume one mg RNA/106 cells a

Copies per cell

Reference [4] [6]

[13] [20] [21] [22]

6

HCV and Blood Cells: How Can We Distinguish Infection from Association?

Given the excess levels of plus strand relative to minus strand RNA, it is quite possible that the minus strand exists largely as a duplex with the plus strand, or may form a duplex when infected cells are lysed. Therefore, such duplexes should be rigorously denatured—or data obtained to prove that this is not necessary—before the minus strand is isolated. In addition, the HCV genome is extensively structured [23–25]. Most of the 5¢ noncoding region typically targeted for RT-PCR amplification is involved in stem-loop structures [25] that may be prone to self-priming, and the 3¢ non-translated region forms a stem-loop structure that acts as a primer in vitro. Thus, it is advisable to target less structured regions of the genome. Artifacts such as false priming and mispriming can result in detection of the plus strand under conditions thought to allow detection of only the minus strand [26]. Finally, levels of HCV RNA associated with blood cells are very low, and therefore the number of PCR cycles needed to detect either RNA strand is very high. In real-time PCR, background signals increase with each PCR cycle. We need to critically consider the possibility that PCR signals may be influenced by contamination, background noise, or falsepositive reactions. Controls that should be shown in each experiment include reactions without reverse transcriptase, no-template reactions, and wrong-strand controls. A standard curve should also be provided to document assay sensitivity—and specificity—using the exact conditions being tested (that is, in the presence of B cell RNA).

6.7

Measurement of HCV Protein Expression

Productively infected cells should express both structural and NS proteins. In order to demonstrate HCV protein expression, investigators must document the specificity of all antibodies used. Some published studies have depended on immunostaining with a single monoclonal antibody (mAb) to support the hypothesis that HCV replication occurs in lymphoid cells. However, certain commercially available mAbs produce peculiar immunostaining patterns suggesting protein localization in cellular locations—such as the nucleus—that do not correspond to known sites of HCV replication. In cells actively engaged in HCV replication, NS proteins are concentrated in punctate cytoplasmic structures [27–30]. Studies using flow cytometry would be expected to demonstrate a distinct

57

subpopulation of NS protein-expressing cells, since the number of HCV RNA copies is less than one per cell and therefore not all cells are expected to be infected. Data indicating a fluorescence shift of the entire lymphocyte population are inconsistent with the presence of far less than one HCV genome per cell. Studies reporting de novo HCV infection of B cells or PBMCs should document quantitation of HCV NS proteins over time, demonstrating increased levels of these proteins during the course of the experiment. More than one mAb should be used to establish the presence of HCV NS proteins.

6.8

Do Antiviral Drugs Affect the Level of HCV RNA and Protein?

In infected hepatoma cells, HCV replication is sensitive to interferons (IFNs) and to a number of antiviral drugs. Type I IFNs block HCV infection in cell culture [31] and are the backbone of the only currently approved therapeutic regimen for HCV infection [32, 33]. B cells and other blood cells express the type I IFN receptor and respond to IFN stimulation; therefore, it seems reasonable to expect that HCV replication in these cells is equally sensitive to IFNs, but this remains to be tested. Several small molecules targeting specific HCV enzyme activities are now in development [33, 34]. These drugs have demonstrated effectiveness against HCV replication and infection in vitro and in human HCV patients. Initial studies of the HCVcc (cell culture derived HCV) system used sensitivity to potent, specific antiviral drugs active against HCV as one criterion for bona fide HCV replication [31]. The use of these drugs in cell culture can help investigators distinguish HCV RNA persistence from HCV RNA replication [2]. In hepatoma cells as well as primary hepatocytes infected with HCVcc, HCV RNA and protein levels increase during the course of infection. Drugs that target HCV’s RNA-dependent RNA polymerase or NS3-4A protease block such increases in HCV RNA and protein. We have tested the same antiviral drugs on HCV RNA and protein levels in B cells, T cells, monocytes, macrophages, dendritic cells, and unfractionated PBMCs cultured with HCVcc. These cells, unlike hepatoma cells or primary hepatocytes, showed no increase in HCV RNA or protein levels over time. Even though HCV RNA persisted in these cells for varying lengths of time, there was no evidence

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B cell

APO-E/B HCV HCV (LVP)

HSPG SR-BI

CD81

CLDN1

LDL-R

OCLN

Hepatocyte

Fig. 6.1 HCV entry factors and mechanisms. Infection of target cells by HCV or HCV lipoviral particles (LVP) is a multistep process [35, 36]. Viral particles are first captured by heparin sulfate proteoglycans (HSPG), scavenger receptors (SR-BI) and/or the low-density lipoprotein receptor (LDL-R). This capture step may permit HCV binding to CD81. It is hypothesized that CD81-bound virus is actively transferred to intercellular tight junctions [37]. HCV entry is dependent on the presence of the

tight junction proteins CLDN1 and OCLN. It is not clear whether these proteins play roles in endocytosis, fusion, or other steps in infection. HCV is taken up by clathrin-dependent endocytosis. After acidification of the endocytic vesicle, the envelope glycoproteins E1 and E2 mediate fusion of the viral envelope with the vesicle membrane. This step releases HCV RNA into the cytoplasm. B cells express CD81 and a splice variant of SR-BI, but do not express appreciable levels of CLDN1 or OCLN

for any increase in HCV RNA levels during culture. Furthermore, such persistence was completely unaffected by antiviral drugs [2].

(Fig. 6.1). Not all of these proteins are expressed by B cells or by other cells in the blood. To study HCV entry, researchers use systems including HCVcc and HCV pseudoparticles (HCVpp) [3]. HCVpp bear HCV envelope proteins but deliver a reporter gene rather than the HCV genome; thus, it is even possible to study HCV entry using cells that may not permit a complete infectious cycle. HCVpp bearing the envelope proteins E1 and E2 from a large variety of HCV isolates have been prepared and extensively studied; the entry requirements of different HCV

6.9

How Can HCV Enter Blood Cells?

Investigators reporting B cell infection must evaluate how the virus was able to enter these cells. A number of proteins are now known to play essential roles in HCV glycoprotein-mediated entry into liver cells

6

HCV and Blood Cells: How Can We Distinguish Infection from Association?

genotypes are known to be very similar. As for HCVpp, it is now possible to prepare HCVcc chimeric viruses using structural-protein sequences from a variety of different HCV genotypes [38]. HCVcc can recapitulate the entire HCV infectious cycle in hepatoma cells, primary human hepatocytes, mice bearing human hepatocyte grafts, and chimpanzees. Studies using these systems and others have shown that CD81 expression, while necessary, is not sufficient for HCV infection. Therefore, although CD81 is widely expressed by blood cells (and by nearly every cell type), it cannot by itself mediate their infection with HCV. Susceptible cells may capture viral particles by means of heparin sulfated proteoglycans, the scavenger receptor SR-BI, and/or the low-density lipoprotein (LDL) receptor (reviewed in [35, 36]). Such capture may facilitate E2 interaction with CD81. It is now evident that HCV entry to susceptible cells is dependent on the tight junction proteins claudin-1 (CLDN1) [39] and occludin (OCLN) [40] in addition to SR-BI and CD81. Expression of all four of these factors together (SR-BI, CD81, CLDN1, and OCLN) conferred on mouse cells the ability to permit HCVpp entry [40]. B cells express CD81 and a splice variant of SR-BI, but do not express appreciable levels of the other entry factors [2]; (Marukian and Dustin, unpublished data). Of note, a subpopulation of B cells expressing the lectin DC-SIGN may capture and internalize HCV, but rather than becoming infected these B cells may release the captured virus to susceptible liver cells [41]. While it has been reported that CLDN6 and CLDN9 may also support HCV entry [42], these are also not expressed at high levels in B cells [2]. We and others have been unable to demonstrate B cell infection by HCVcc or HCVpp that bear envelopes representing a variety of isolates [2, 43, 44]. A B cell line engineered to express high levels of several entry factors remained resistant to infection with HCVcc or HCVpp [2].

6.10

Do Blood Cells Provide the Necessary Cofactors for Productive HCV Infection?

In liver cells, emerging evidence shows that HCV makes use of cellular machinery during RNA translation, replication, and virus assembly. HCV replication is dramatically enhanced by the liver-specific

59

microRNA miR-122, which binds to a pair of seed sites in the 5¢ non-translated region of the HCV genome [45–47]. Indeed, HCV does not replicate well in cells deficient in microRNA processing pathways [46]. HCV-infected chimpanzees treated with a miR122 antagonist demonstrate a dramatic reduction in serum HCV levels [48]. Despite its abundance in liver cells, miR-122 levels are limiting even in susceptible liver cell lines [49]. Also, miR-122 expression has not been reported in B cells. Mounting evidence indicates that production of infectious HCV virions is intimately tied in with the assembly of low and very-low-density lipoproteins (LDL and VLDL), particularly the latter [50]. HCV viral particles in the blood are associated with LDL and VLDL, and are termed “lipoviral particles” or LVP [50–53]. HCV isolated from the liver has a similar low density, and is associated with apolipoproteins B and E [53]. LDL and VLDL particles are produced only in the liver [54]. HCV is replicated and assembled in subcellular compartments associated with lipid droplets [30] and lipoprotein assembly [55, 56]. Inhibition of VLDL assembly—whether by drugs inhibiting microsomal transfer protein [55, 57] or by siRNA targeting of apolipoprotein E [58, 59], apolipoprotein B [55], or enzymes involved in VLDL biosynthesis [60]—strongly blocks HCV virus production in cell culture. Lower-density, lipoprotein-associated HCV is significantly more infectious than higher-density HCV lacking associated lipoproteins [30, 61]. There is, as yet, no published evidence that HCV-infected blood cells can provide the lipoprotein biosynthetic pathway components apparently required and used by the virus in hepatocytes.

6.11

Is There Evidence for B Cell-Specific HCV Quasispecies?

While there are reports of differential association of HCV RNA sequences with different compartments, researchers have not yet identified any sequences that are consistently associated with B cells as opposed to other cell types [8, 62]. B cell-associated HCV sequences differ from one report to another. Furthermore, it may be difficult to accurately characterize HCV quasispecies uniquely associated with B cells because the amounts of viral RNA that can be recovered from patient B cells are typically very low—and include peripherally associated

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viral RNA from the serum. HCV RNA diversity is analyzed, by necessity, by reverse transcription (RT) and PCR amplification followed by cloning and sequencing; less than perfect efficiency at the beginning of this process will result in the loss of some templates and therefore of some sequence variants. Low template copy numbers in the material under study, i.e. B cells, thus present a risk of under-sampling artifacts leading to incorrect conclusions of limited HCV sequence diversity in this compartment. At this time, the field does not enforce a standard for demonstrating that adequate sampling has taken place. Sequencing multiple clones from a single RT-PCR experiment does not address the undersampling problem. To reduce the likelihood of an undersampling artifact, it is important to analyze more than one RNA sample, verify the integrity of the RNA, and perform the entire RT-PCR amplification process more than once for each sample.

6.12

Summary

It is widely accepted that HCV RNA and even some viral proteins can be associated with B cells in HCV patients with and without MC. The level of HCV RNA, when measurements are reported, is very low. The frequency of infected cells, if any, must be correspondingly low. It is not yet clear how the possible infection of a small subset of B cells could lead to the functional changes associated with MC. Scientists and clinicians studying the potential role of B cell infection by HCV in the pathogenesis of MC would do well to apply state-of-the-art virological tools to the study of this important area. Quantitative assays must be used to document changes in HCV RNA and protein over time. Controls for the specificity of both RNA and protein measurements must be presented. In order to distinguish replicating virus from viral material that is adsorbed to the cell or is taken up by phagocytosis or endocytosis, it will be useful to determine the effects of specific antiviral drugs on the levels and rate of change of viral RNA and protein. If these assays are carried out and strong evidence is found to support the conclusion that B cells are sites of productive HCV infection, many new questions arise. We must understand how the virus is able to enter cells, given that B cells do not express the full complement of known HCV entry factors. We must also understand how HCV replicates and produces infectious viral particles in the

setting of a lymphoid cell, which lacks many of the features required for efficient HCV replication in liver cells. These studies will be important in order to understand how HCV infection of blood cells could possibly contribute to MC and the other immunological abnormalities seen in HCV patients.

References 1. Lanford RE, Chavez D, Chisari FV et al (1995) Lack of detection of negative-strand hepatitis C virus RNA in peripheral blood mononuclear cells and other extrahepatic tissues by the highly strand-specific rTth reverse transcriptase PCR. J Virol 69:8079–8083 2. Marukian S, Jones CT, Andrus L et al (2008) Cell cultureproduced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 48:1843–1850 3. Tellinghuisen TL, Evans MJ, von Hahn T et al (2007) Studying hepatitis C virus: making the best of a bad virus. J Virol 81:8853–8867 4. Zehender G, Meroni L, De Maddalena C et al (1997) Detection of hepatitis C virus RNA in CD19 peripheral blood mononuclear cells of chronically infected patients. J Infect Dis 176:1209–1214 5. Lerat H, Rumin S, Habersetzer F et al (1998) In vivo tropism of hepatitis C virus genomic sequences in hematopoietic cells: influence of viral load, viral genotype, and cell phenotype. Blood 91:3841–3849 6. Mellor J, Haydon G, Blair C et al (1998) Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/ GB virus C in peripheral blood mononuclear cells. J Gen Virol 79(Pt 4):705–714 7. Fornasieri A, Bernasconi P, Ribero ML et al (2000) Hepatitis C virus (HCV) in lymphocyte subsets and in B lymphocytes expressing rheumatoid factor cross-reacting idiotype in type II mixed cryoglobulinaemia. Clin Exp Immunol 122:400–403 8. Ducoulombier D, Roque-Afonso AM, Di Liberto G et al (2004) Frequent compartmentalization of hepatitis C virus variants in circulating B cells and monocytes. Hepatology 39:817–825 9. Baré P, Massud I, Parodi C et al (2005) Continuous release of hepatitis C virus (HCV) by peripheral blood mononuclear cells and B-lymphoblastoid cell-line cultures derived from HCV-infected patients. J Gen Virol 86:1717–1727 10. Pham TN, Macparland SA, Coffin CS et al (2005) Mitogeninduced upregulation of hepatitis C virus expression in human lymphoid cells. J Gen Virol 86:657–666 11. Pal S, Sullivan DG, Kim S et al (2006) Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo: implications of HCV lymphotropism. Gastroenterology 130:1107–1116 12. Sansonno D, Tucci FA, Lauletta G et al (2007) Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia. Clin Exp Immunol 147:241–248 13. Pham TN, King D, Macparland SA et al (2008) Hepatitis C virus replicates in the same immune cell subsets in chronic hepatitis C and occult infection. Gastroenterology 134:812–822

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14. Sung VM, Shimodaira S, Doughty AL et al (2003) Establishment of B-cell lymphoma cell lines persistently infected with hepatitis C virus in vivo and in vitro: the apoptotic effects of virus infection. J Virol 77:2134–2146 15. Pham TN, MacParland SA, Mulrooney PM et al (2004) Hepatitis C virus persistence after spontaneous or treatmentinduced resolution of hepatitis C. J Virol 78:5867–5874 16. Goutagny N, Fatmi A, De Ledinghen V et al (2003) Evidence of viral replication in circulating dendritic cells during hepatitis C virus infection. J Infect Dis 187:1951–1958 17. Laporte J, Bain C, Maurel P et al (2003) Differential distribution and internal translation efficiency of hepatitis C virus quasispecies present in dendritic and liver cells. Blood 101:52–57 18. Rodrigue-Gervais IG, Jouan L, Beaule G et al (2007) Poly(I:C) and lipopolysaccharide innate sensing functions of circulating human myeloid dendritic cells are affected in vivo in hepatitis C virus-infected patients. J Virol 81:5537–5546 19. Coquillard G, Patterson BK (2009) Determination of hepatitis C virus-infected, monocyte lineage reservoirs in individuals with or without HIV coinfection. J Infect Dis 200: 947–954 20. Boisvert J, He XS, Cheung R et al (2001) Quantitative analysis of hepatitis C virus in peripheral blood and liver: replication detected only in liver. J Infect Dis 184:827–835 21. Radkowski M, Gallegos-Orozco JF, Jablonska J et al (2005) Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C. Hepatology 41:106–114 22. Inokuchi M, Ito T, Uchikoshi M et al (2009) Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C. J Med Virol 81:619–627 23. Tuplin A, Wood J, Evans DJ et al (2002) Thermodynamic and phylogenetic prediction of RNA secondary structures in the coding region of hepatitis C virus. RNA 8:824–884 24. You S, Rice CM (2008) 3¢ RNA elements in hepatitis C virus replication: kissing partners and long poly(U). J Virol 82: 184–195 25. Lukavsky PJ (2009) Structure and function of HCV IRES domains. Virus Res 139:166–171 26. Lerat H, Berby F, Trabaud MA et al (1996) Specific detection of hepatitis C virus minus strand RNA in hematopoietic cells. J Clin Invest 97:845–851 27. Gosert R, Egger D, Lohmann V et al (2003) Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons. J Virol 77:5487–5492 28. Rouillé Y, Helle F, Delgrange D et al (2006) Subcellular localization of hepatitis C virus structural proteins in a cell culture system that efficiently replicates the virus. J Virol 80:2832–2841 29. Targett-Adams P, Boulant S, McLauchlan J (2008) Visualization of double-stranded RNA in cells supporting hepatitis C virus RNA replication. J Virol 82:2182–2195 30. Miyanari Y, Atsuzawa K, Usuda N et al (2007) The lipid droplet is an important organelle for hepatitis C virus production. Nat Cell Biol 9:1089–1097 31. Lindenbach BD, Evans MJ, Syder AJ et al (2005) Complete replication of hepatitis C virus in cell culture. Science 309: 623–626 32. Heathcote EJ (2007) Antiviral therapy: chronic hepatitis C. J Viral Hepat 14(Suppl 1):82–88

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33. Zeuzem S (2008) Interferon-based therapy for chronic hepatitis C: current and future perspectives. Nat Clin Pract Gastroenterol Hepatol 5:610–622 34. Manns MP, Foster GR, Rockstroh JK et al (2007) The way forward in HCV treatment – finding the right path. Nat Rev Drug Discov 6:991–1000 35. Burlone ME, Budkowska A (2009) Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol 90:1055–1070 36. Bartosch B, Cosset FL (2006) Cell entry of hepatitis C virus. Virology 348:1–12 37. Brazzoli M, Bianchi A, Filippini S et al (2008) CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes. J Virol 82:8316–8329 38. Gottwein JM, Scheel TK, Jensen TB et al (2009) Development and characterization of hepatitis C virus genotype 1–7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 49: 364–377 39. Evans MJ, von Hahn T, Tscherne DM et al (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446:801–805 40. Ploss A, Evans MJ, Gaysinskaya VA et al (2009) Human occludin is a hepatitis C virus entry factor required for infection of mouse cells. Nature 457:882–886 41. Stamataki Z, Shannon-Lowe C, Shaw J et al (2009) Hepatitis C virus association with peripheral blood B lymphocytes potentiates viral infection of liver-derived hepatoma cells. Blood 113: 585–593 42. Zheng A, Yuan F, Li Y et al (2007) Claudin-6 and claudin-9 function as additional coreceptors for hepatitis C virus. J Virol 81:12465–12471 43. McKeating JA, Zhang LQ, Logvinoff C et al (2004) Diverse hepatitis C virus glycoproteins mediate viral infection in a CD81 dependent manner. J Virol 78:8496–8505 44. Bartosch B, Dubuisson J, Cosset F-L (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642 45. Jopling CL, Yi M, Lancaster AM et al (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309:1577–1581 46. Randall G, Panis M, Cooper JD et al (2007) Cellular cofactors affecting hepatitis C virus infection and replication. Proc Natl Acad Sci USA 104:12884–12889 47. Chang J, Guo JT, Jiang D et al (2008) Liver-specific microRNA miR-122 enhances the replication of hepatitis C virus in nonhepatic cells. J Virol 82:8215–8223 48. Lanford RE, Hildebrandt-Eriksen ES, Petri A et al (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327(5962): 198–201, Epub 2009 Dec 3 49. Jopling CL, Norman KL, Sarnow P (2006) Positive and negative modulation of viral and cellular mRNAs by liverspecific microRNA miR-122. Cold Spring Harb Symp Quant Biol 71:369–376 50. Syed G, Amako Y, Siddiqui A (2010) Hepatitis C virus hijacks host lipid metabolism. Trends Endocrinol Metab 21(1):33–40, Epub 2009 Oct 23 51. Andre P, Komurian-Pradel F, Deforges S et al (2002) Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J Virol 76:6919–6928

62 52. Nielsen SU, Bassendine MF, Burt AD et al (2006) Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients. J Virol 80:2418–2428 53. Nielsen SU, Bassendine MF, Martin C et al (2008) Characterization of hepatitis C RNA-containing particles from human liver by density and size. J Gen Virol 89:2507–2517 54. Gibbons GF, Wiggins D, Brown AM et al (2004) Synthesis and function of hepatic very-low-density lipoprotein. Biochem Soc Trans 32:59–64 55. Huang H, Sun F, Owen DM et al (2007) Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins. Proc Natl Acad Sci USA 104:5848–5853 56. Benga WJ, Krieger SE, Dimitrova M et al (2010) Apolipoprotein E interacts with hepatitis C virus nonstructural protein 5A and determines assembly of infectious particles. Hepatology 51:43–53 57. Gastaminza P, Cheng G, Wieland S et al (2008) Cellular determinants of hepatitis C virus assembly, maturation, degradation, and secretion. J Virol 82:2120–2129

L.B. Dustin and C.M. Rice 58. Chang KS, Jiang J, Cai Z et al (2007) Human apolipoprotein E is required for infectivity and production of hepatitis C virus in cell culture. J Virol 81:13783–13793 59. Jiang J, Luo G (2009) Apolipoprotein E but not B is required for the formation of infectious hepatitis C virus particles. J Virol 83:12680–12691 60. Yao H, Ye J (2008) Long chain acyl-CoA synthetase 3-mediated phosphatidylcholine synthesis is required for assembly of very low density lipoproteins in human hepatoma Huh7 cells. J Biol Chem 283:849–854 61. Lindenbach BD, Meuleman P, Ploss A et al (2006) Cell culture-grown hepatitis C virus is infectious in vivo and can be recultured in vitro. Proc Natl Acad Sci USA 103: 3805–3809 62. Zehender G, De Maddalena C, Bernini F et al (2005) Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome. J Virol 79:9145–9156

7

Mechanisms of Cell Entry of Hepatitis C Virus Franco Dammacco and Vito Racanelli

7.1

Introduction

Hepatitis C virus (HCV) is a positive-strand RNA belonging to the Flaviviridae family. Its identification required long and painstaking research, involving the use of a number of molecular and serological methods [1], which finally led to the recognition of this third (in addition to HAV and HBV) etiological agent of infectious hepatitis. The existence of HCV had long been suspected and it was even provisionally named as hepatitis nonA-nonB virus, which emphasized its nosographic differentiation from the other two viruses [2]. Final confirmation of the structural uniqueness of HCV came in 1989, with the determination of its complete amino acid sequence [3]. In spite of the early and often remarkable production of neutralizing antibodies in the acute phase of HCV infection, the large majority of patients are unable to clear the virus and become chronically infected. One of the most intriguing and fascinating aspects of the molecular biology of HCV is the complex and so far largely unexplained strategy it has developed to escape host immune response and gain entry into target cells. Indeed, the HCV internalization pathway may be considered the forefront of viral-entry research. Deeper insight into the precise mechanisms whereby HCV evades (avoids) antibody-mediated neutralization and spreads from cell to cell would not only contribute to the comprehension of infection chronicity, but would also provide invaluable help in F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_7, © Springer-Verlag Italia 2012

devising novel therapeutic compounds targeting entry. Here, we summarize the main advances that have been accomplished in this field in the last decade.

7.2

HCV: The Hepatic Environment and Model Systems of Cell Entry

The molecular features of HCV have been described in several papers, including recent reviews [4, 5]. It is an enveloped virus that bears two surface glycoproteins, E1 and E2, and shows a primary tropism for human liver cells, with a less obvious and still controversial lymphotropism. A peculiar feature of hepatocytes is their polarized structure, in that basolateral and apical poles, through tight-junction separation, face the blood and the bile, respectively. Circulating HCV, linked to lipoproteins, wanders in the liver microenvironment and, when it comes into contact with the basolateral membranes of liver cells, interacts via entry molecules. In 1998, it was shown that protein E2 binds human CD81, a tetraspanin expressed on hepatocytes, B lymphocytes, and other cell types [6]. More precisely, the ability to bind E2 was ascribed to the major extracellular loop of CD81. It soon became evident that HCV cell entry is a much more complex and multi-step process, whose clarification has been largely hampered by the lack of suitable animal models and the limitations of in vitro systems based on the reaction of HCV particles with cell cultures. An important contribution to the advancement of knowledge in this field came from the establishment of new methods in molecular virology and, in terms of HCV research, a replicon system [7] with applications to basic as well as clinical research [8]. 63

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Several model systems, discussed in an excellent review [9], have been employed for the study of HCV cell entry. They include: (a) plasma-derived HCV, an older procedure [10] largely abandoned because of its many limitations [11]; (b) recombinant E2 glycoprotein, which allowed identification of the already-mentioned tetraspanin CD81 and of the human scavenger receptor class B (SR-BI) [12]; (c) HCV-like particles (HCV-LPs [13]), whose main limitation is their retention in intracellular vesicles rather than their secretion; (d) HCV pseudoparticles (HCVpp [14]), namely, the production of lentiviral particles able to incorporate HCV glycoproteins into the lipid envelope, although such pseudoparticles are not associated with lipoproteins; (e) cell-culture-produced HCV (HCVcc [15, 16]), an in vitro model that has marked a true milestone as it allows the reproduction of both early and late steps of the viral replication cycle and supports the production of virus particles with infectious properties both in vitro and in vivo; (f) a recently described smallanimal model, namely, the tree shrew or tupaia (Tupaia belangeri), which resembles a squirrel in its external appearance and habits. Primary tupaia hepatocytes have been shown to support the complete HCV infection cycle and are endowed with all of the receptors or co-receptors required for HCV entry; thus, it promises to become an important animal model in HCV-related research [17].

7.3

Receptors and Co-receptors for HCV Cell Entry

Following the identification of tetraspanin CD81 [6], several additional cell-surface molecules were demonstrated to play an important role as virus receptors on host cells. We briefly describe their properties, but refer the reader to specific reviews [9, 11, 18, 19] for a more detailed analysis of this probably still incomplete set of entry molecules.

7.3.1

Tetraspanin CD81

As a non-glycosylated protein, this tetraspanin is expressed on many cell types, acts as an HCV receptor molecule, and binds the virus surface glycoprotein E2 [6]. Its role is strongly suggested by studies showing

that anti-CD81 monoclonal antibodies are able to inhibit the infectivity of HCVpp and HCVcc in vitro [14, 16]. Growing evidence indicates that CD81 is a post-binding entry molecule, with the absence in liver cells of a natural inhibitor of CD81, EWI-2wint, probably accounting for the selective hepatotropism of HCV [20]. Of interest, CD81 also seems capable of modulating the adaptive immune response through its interaction with HCV on T and B lymphocytes. If a polyclonal B cell activation ensues, extrahepatic manifestations (including mixed cryoglobulinemia) of chronic HCV infection may occur [21].

7.3.2

Scavenger Receptor Class B (SR-BI)

Like tetraspanin CD81, SR-BI is a glycoprotein that also acts as a post-binding receptor. It is largely expressed by hepatocytes but to a lesser extent also by many other mammalian cells [22]. At least two mRNA splice variants are produced by the SR-BI gene, SR-BI and SR-BII, which differ in their carboxy-terminal ends [23]. SR-BI binds to high-, low- and very-lowdensity lipoproteins, but chemically modified lipoproteins can also be bound. There seems to be a close cooperation between SR-BI and CD81 in the process of HCV cell entry [22] in that SR-BI acts as a major cholesterol provider and controls the organization of CD81 at the level of plasma membrane. SR-BI is also able to bind serum amyloid A (SAA) protein, an acutephase reactant induced by a number of viral and bacterial infections [24]. SAA reduces HCV infectivity in cultured cells when added during HCV infection, but not after viral entry. Finally, interferon-a seems to exert its antiviral effects through a cell surface decrease in the expression of SR-BI, resulting in reduced HCV attachment and entry into liver cells [25].

7.3.3

Tight-Junction Proteins

These include four different components, but only claudins and occludins will be mentioned here as they play a crucial role in the cell-to-cell adhesion mechanism and in the separation of the apical from the basolateral membrane. Claudin-1 is expressed by all epithelial tissues and especially liver cells. Although it is not known whether

7

Mechanisms of Cell Entry of Hepatitis C Virus

claudin-1 directly interacts with HCV, it is probably involved in a later step of infection, after the virus binds to CD81 and SR-BI [26]. It has been shown that HCV infection of Huh-7 hepatoma cells down-regulates the expression of claudin-1 (and occludin as well), thus preventing superinfection [27]. Claudin-6 and claudin-9, which are additional members of the claudin family, are also involved in HCV entry but their roles in this process are still poorly defined. Occludin is a trans-membrane member of the tightjunction complex and is structurally related to the claudins. It is equally essential in HCV cell entry and in the activation of a productive HCV infection. Experimental data indicate that occludin, by direct interaction with glycoprotein E2, is capable of enhancing viral entry through the tight junctions of liver cells. In addition, based on the observed down-regulation of claudin-1 and occludin expression following HCV infection, this reduction of tight-junction proteins not only prevents, as already stated, superinfection of infected cells by HCVpp, but also affects the polarity of liver cells [27].

7.3.4

Glycosaminoglycans

These polysaccharides are largely expressed on the cell membrane and are able to bind many types of viruses, including HCV. They act as initial, low-affinity receptors in the early step of viral attachment, well before the virus is bound to higher-affinity receptors. Among glycosaminoglycans, the highly sulfated heparan sulfate seems to be particularly active. Lipoprotein lipase possibly mediates HCV cell entry by a mechanism resembling hepatic clearance of triglyceride-rich lipoproteins from the blood, thus favoring non-productive viral uptake [28].

7.3.5

Lectins

Two membrane proteins belonging to the C-type lectin family, DC-SIGN (dendritic-cell-specific intercellular adhesion molecule-3 grabbing non-integrin) and its homolog L-SIGN, have been found to take part in the binding, internalization, and clearance of several viruses, including HCV. Both are able to bind glycoprotein sE2 as well as natural HCV. DC-SIGN has

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been demonstrated on Kupffer cells, dendritic cells, and B cells, whereas L-SIGN is largely expressed on endothelial cells of liver sinusoids. Thus, both lectins likely act as capture receptors that, by binding and transmitting the virus to permissive cells, effectively start the infectious process [29].

7.4

Steps of Cell Entry

Through a multi-step, intricate, and finely regulated interplay between viral receptors and lipoprotein components, the virus is able to escape host immune responses [30]. Despite the large body of evidence on the mechanism(s) of cell entry, elucidated following advances in molecular virology, such as HCVpp and HCVcc [14–16], the precise sequence of events, the role played by each receptor or co-receptor, the cellular signaling network, and the overall coordination underlying “one virus/many receptors” still present pitfalls and knowledge gaps. It also seems reasonable to assume that the different circulating forms of HCV activate different pathways of cell entry such that infection proceeds by different modalities [9]. As shown in Fig. 7.1, largely inspired by models proposed by other authors [9, 11], the starting step of binding and internalization of ApoB-associated HCV requires the interplay of virus-associated VLDL and SR-BI. LDL receptors and glycosaminoglycans are possibly involved in this early phase, thus emphasizing the crucial role of lipoproteins, and in particular LDL receptors, in virus cell entry, as has been proposed since 1999 [31]. In the next step, the virus binds to the SR-BI/ CD81 complex, which results in its subsequent transfer to the tight-junction proteins claudin-1 and occludin. Following clathrin-mediated endocytosis, the virus enters the cell from the tight junction and undergoes a process of fusion that involves viral envelope glycoproteins and the endosomal membrane. In turn, this allows release of the viral RNA genome into the cytosol.

7.5

HCV Entry Inhibitors as Novel Therapeutic Agents

Precise recognition of the biomolecular mechanisms underlying cellular entry of HCV has obvious therapeutic implications, through the identification of

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F. Dammacco and V. Racanelli

novel therapeutic compounds targeting entry. At the moment, this approach includes inhibitors of glycosylation that target envelope glycans or cyanovirin-N, a lectin able to inhibit binding of the virus to cell-surface receptors [32]. The same therapeutic potential should be achievable using antibodies capable of downregulating the cell-surface expression of HCV receptors or co-receptors. Using a human liver-uPA-SCID mice system, a new chimeric mouse model permissive for HBV and HCV infection [33], it has been shown

a

that prophylactic treatment with anti-CD81 antibodies completely protected human liver-uPA-SCID mice from a subsequent challenge with HCV consensus strains of different genotypes, whereas the administration of anti-CD81 antibodies after viral challenge had no effect [34]. This observation suggests that it should be possible to prevent allograft reinfection after orthotopic liver transplantation in chronically infected HCV patients [35]. The same model is also being used in the development of new anti-viral compounds.

VLDL, LDL, ox LDL ↓ HDL ↑

LPL ↓↑ ?

SAA? ↓ ?

Claudin-1 SR-BI

Occludin

CD81

Clathrincoated pit

GAGs LDLR Early endosome H+

Fusion and genome release

Fig. 7.1 (a) A hypothetical model of cell entry of natural ApoB-associated HCV. The model implies that envelope glycoproteins directly interact with co-receptors. The SR-BI/CD81 complex binds the virus, which is then transferred to tight junctions where it interacts with claudin-1 and occludin. In the following step, HCV enters the cell by clathrin-dependent

endocytosis, followed by fusion of the viral envelope glycoproteins with the membrane of an early endosome. This results in the release of viral nucleocapsid into the cytoplasm. (b) Schematic representation of the localization of HCV entry molecules in the liver epithelium

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Mechanisms of Cell Entry of Hepatitis C Virus

b

CD81 DC-SIGN/L-SIGN

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SR-BI

HCV

Claudin-1

Sinusoidal space

Blood (basolateral side)

Endothelium Space of Disse

Tight junction Hepatocytes Lumen of bile canaliculus (apical side)

Fig. 7.1 (continued)

References 1. Houghton M (2009) The long and winding road leading to the identification of the hepatitis C virus. J Hepatol 51:939–948 2. Alter MJ (1989) Non-A, non-B hepatitis: sorting through a diagnosis of exclusion. Ann Intern Med 110:583–585 3. Choo QL, Kuo G, Weiner AJ et al (1989) Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362 4. Fraser CS, Doudna J (2007) Structural and mechanistic insights into hepatitis C viral translation initiation. Nat Rev Microbiol 5:29–38 5. Poenisch M, Bartenschlager R (2010) New insights into structure and replication of the hepatitis C virus and clinical implications. Semin Liver Dis 30:333–347 6. Pileri P, Uematsu Y, Campagnoli S et al (1998) Binding of hepatitis C virus to CD81. Science 282:938–941 7. Lohmann V, Körner F, Koch J et al (1999) Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285:110–113 8. Bartenschlager R (2005) The hepatitis C virus replicon system: from basic research to clinical application. J Hepatol 43:210–216

9. Burlone ME, Budkowska A (2009) Hepatitis C virus cell entry: role of lipoproteins and cellular receptors. J Gen Virol 90:1055–1070 10. Shimizu YK, Iwamoto A, Hijikata M et al (1992) Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line. Proc Natl Acad Sci USA 89:5477–5481 11. von Hahn T, Rice CM (2008) Hepatitis C virus entry. J Biol Chem 283:3689–3693 12. Scarselli E, Ansuini H, Cerino R et al (2002) The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus. EMBO J 21:5017–5025 13. Barth H, Liang TJ, Baumert TF (2006) Hepatitis C virus entry: molecular biology and clinical implications. Hepatology 44:527–535 14. Bartosch B, Dubuisson J, Cosset FL (2003) Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J Exp Med 197:633–642 15. Lindenbach BD, Evans MJ, Syder AJ et al (2005) Complete replication of hepatitis C virus in cell culture. Science 309: 623–626 16. Wakita T, Pietschmann T, Kato T et al (2005) Production of infectious hepatitis C virus in tissue culture from a cloned viral genome. Nat Med 11:791–796

68 17. Tong Y, Zhu Y, Xia X et al (2011) Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection. J Virol 85:2793–2802 18. Brass V, Moradpour D, Blum HE (2006) Molecular virology of hepatitis C virus (HCV): 2006 update. Int J Med Sci 3: 29–34 19. Zeisel MB, Fofana I, Fafi-Kremer S et al (2011) Hepatitis C virus entry into hepatocytes: molecular mechanisms and targets for antiviral therapies. J Hepatol 54:566–576 20. Rocha-Perugini V, Montpellier C, Delgrange D et al (2008) The CD81 partner EWI-2wint inhibits hepatitis C virus entry. PLoS One 3:e1866 21. Machida K, Cheng KT, Pavio N et al (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79:8079–8089 22. Zeisel MB, Koutsoudakis G, Schnober EK et al (2007) Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81. Hepatology 46:1722–1731 23. Grove J, Huby T, Stamataki Z et al (2007) Scavenger receptor BI and BII expression levels modulate hepatitis C virus infectivity. J Virol 81:3162–3169 24. Cai Z, Cai L, Jiang J et al (2007) Human serum amyloid A protein inhibits hepatitis C virus entry into cells. J Virol 81:6128–6133 25. Murao K, Imachi H, Yu X et al (2008) Interferon alpha decreases expression of human scavenger receptor class BI, a possible HCV receptor in hepatocytes. Gut 57:664–667 26. Evans MJ, von Hahn T, Tscherne DM et al (2007) Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry. Nature 446:801–805

F. Dammacco and V. Racanelli 27. Liu S, Yang W, Shen L et al (2009) Tight junction proteins claudin-1 and occludin control hepatitis C virus entry and are downregulated during infection to prevent superinfection. J Virol 83:2011–2014 28. Andréo U, Maillard P, Kalinina O et al (2007) Lipoprotein lipase mediates hepatitis C virus (HCV) cell entry and inhibits HCV infection. Cell Microbiol 9:2445–2456 29. Gardner JP, Durso RJ, Arrigale RR et al (2003) L-SIGN (CD 209 L) is a liver-specific capture receptor for hepatitis C virus. Proc Natl Acad Sci USA 100:4498–4503 30. Perrault M, Pécheur EI (2009) The hepatitis C virus and its hepatic environment: a toxic but finely tuned partnership. Biochem J 423:303–314 31. Agnello V, Abel G, Elfahal M et al (1999) Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA 96: 12766–12771 32. Steinmann E, Whitfield T, Kallis S et al (2007) Antiviral effects of amantadine and iminosugar derivatives against hepatitis C virus. Hepatology 46:330–338 33. Meuleman P, Leroux-Roels G (2008) The human liver-uPASCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV. Antiviral Res 80:231–238 34. Meuleman P, Hesselgesser J, Paulson M et al (2008) Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo. Hepatology 48:1761–1768 35. Fafi-Kremer S, Fofana I, Soulier E et al (2010) Viral entry and escape from antibody-mediated neutralization influence hepatitis C virus reinfection in liver transplantation. J Exp Med 207:2019–2031

8

HCV Infection of Hematopoietic and Immune Cell Subsets Tram N.Q. Pham and Tomasz I. Michalak

8.1

Introduction

Hepatitis C virus (HCV) is an important human pathogen that causes chronic hepatitis, with 170 million people infected with the virus world-wide. The 9600-base-pair genome of this single-stranded RNA virus encodes a number of structural (core, envelope) and non-structural (NS2–NS5) proteins that determine the morphology of the virus and are essential to its replication, respectively. Although hepatocytes are considered to be the primary targets of HCV, a large body of clinical and experimental evidence implies that this pathogen also invades and replicates in cells of other organs, particularly those of the immune system [1, 2]. It is conceivable that by setting up these extrahepatic sanctuaries of replication, HCV is able to establish persistence through evasion or modulation of host’s immune responses—a typical propensity of viruses capable of causing chronic infections [3]. In this respect, there are also studies that do not support the notion of HCV lymphotropism. The goal of this chapter is to provide an overview of our current understanding of the lymphoid cell compartment’s involvement in HCV infection in highly viremic patients with chronic hepatitis C (CHC) and in those with low-level, occult HCV infection (OCI). Regarding the latter, we summarize what is known to date on the occurrence of HCV in immune cell subsets of those individuals who either have or lack a past history of acute or chronic

T.I. Michalak (*) Molecular Virology and Hepatology Research Group, Faculty of Medicine, Health Sciences Center, Memorial University, St. John’s, NL, Canada e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_8, © Springer-Verlag Italia 2012

hepatitis C. Finally, the chapter reviews the data addressing the issue of HCV infection in cultured immune cells.

8.2

HCV Replication in Hematopoietic and Immune Cells of Patients with Acute or Chronic Hepatitis C

Over the past two decades, HCV lymphotropism has been the subject of numerous investigations. These works have reported the identification of HCV RNA positive and negative (replicative) strands as well as viral proteins in peripheral blood mononuclear cells (PBMCs) of patients with acute or chronic hepatitis C (AHC, CHC, respectively) [1, 4, 5]. Interestingly, the levels of the virus’ RNA replicative strand were shown to be significantly higher in the PBMCs of patients with CHC than in those of patients with AHC, suggesting that HCV infection of the immune system has a role in sustaining chronic infection [4, 5]. As alluded to earlier, by targeting the immune cells, the virus may likely acquire the ability to evade antiviral responses very early on, thereby establishing a stronghold within the infected host. Studies from different groups have addressed the question whether HCV displays a preferential tropism for specific PBMC subsets or infects different subtypes of circulating immune cells. Under certain conditions, HCV has been shown to compartmentalize much more frequently, if not exclusively, in unique cell subsets, such as CD19+ B cells [6–8]. However, in other studies, the detection of HCV RNA positive and negative strands was more widespread among the various immune cells, including CD4+ and CD8+ T cells, monocytes, dendritic cells and B cells; 69

70

although different cell subtypes might be infected to various degrees in different patients [9–12]. Clonal sequencing and single-stranded conformational polymorphism (SSCP) analyses of HCV variants residing in PBMCs revealed interesting features that argued against the possibility of either a carry-over from plasma-derived free HCV RNA or non-specific viral adsorption to the cell surface [10, 11, 13–16]. First, HCV variants from lymphoid cells were genetically related but highly distinct from those encountered in serum or liver. In certain cases, HCV quasi-species identifiable in lymphoid cells were detectable in serum but not in hepatic tissue, supporting their extrahepatic origin. Second, HCV quasi-species from one cellular compartment, for example CD8+ T cells, were statistically more genetically similar to one another than they were to variants found in other immune cell subsets, such as CD4+ T cells. In one relevant study, HCV RNA sequences in CD8+ T lymphocytes phylogenetically clustered with each other but not with sequences detected in CD4+ T cells or CD19+ B cells [11]. In terms of functionality, certain sequence polymorphisms within the internal ribosomal entry site (IRES) of the 5¢-untranslated region (5¢-UTR) of the HCV genome isolated from lymphoid cells were found to coincide with an IRES translational activity unlike that in other infected cell types which would support HCV replication in cells carrying those variations but not in cells that do not [17, 18]. Indeed, a very recent study showed that within liver-derived hepatoma Huh7 cells, HCV IRES variants originating from plasma displayed a significantly higher translational activity than those from HCV residing in B cells [19]. Conversely, IRES variants of the virus replicating in B cells were found to have a similarly low translational efficiency in B cell lines, such as Raji and Daudi, and in hepatoma Huh7 cells, suggesting not only their extrahepatic origin but also an overall low capacity of HCV replication in B cells [19]. The concept of HCV lymphotropism is consistent with the meaningfully greater occurrence of lymphoproliferative disorders, such as non-Hodgkin’s lymphoma (NHL) [20, 21] and type II mixed cryoglobulinemia [22], in patients with CHC, as discussed elsewhere in this book. Suffice it to mention that in patients with markers of NHL, HCV RNA has been found at significantly higher frequencies in B-cell NHL than in non-B-cell NHL [23, 24]. Along this line, HCV infection of B cells also appears to be predominant in patients with lymphoproliferative disorders

T.N.Q. Pham and T.I. Michalak

compared to those without [2, 9]. Furthermore, there is evidence implying that the progression of these diseases is associated with high HCV viremia and, conversely, that their remission is correlated with a reduction in viral load following antiviral treatment [2, 25]. Taken together, these findings point towards an etiologic role not only for HCV in general, but also for immune cell infection in particular in the pathogenesis of these disorders. It should be mentioned that in patients with CHC, the identification of replicating HCV in lymphoid cells is not limited to only those cells in the circulation. In fact, HCV genomes and/or proteins have also been found in lymph nodes (LN), bone marrow (BM), and even the brain [8, 26–28]. In LNs, replicating HCV genomes as well as HCV core and NS3 proteins have been identified within B-cell-rich lymphoid follicles of biopsy specimens from patients with end-stage CHC [8]. Remarkably, not only were B cells the primary site of HCV infection in this secondary lymphoid tissue, but clonal sequencing analyses also indirectly revealed that, in some patients, HCV residing in LN-derived B cells contributed up to 40% of the total level of viremia [8]. Similarly, HCV RNA sequences found in the cerebrospinal fluid of patients co-infected with human immunodeficiency virus (HIV) were shown to be more similar to those in PBMCs and LNs than to those in plasma, suggesting that cells of the monocyte/macrophage lineage carry HCV into the brain and that resident microglial cells independently maintain viral replication [26, 27]. Lastly, HCV RNA positive and negative strands, as well as HCV structural and nonstructural proteins, were readily detectable in CD34+ hematopoietic progenitor cells in the BM of patients with CHC [28], lending further support to the concept of extrahepatic HCV replication. Although in the same study there was no evidence that primary CD34+ cells from healthy individuals supported de novo HCV infection, CD34+ cells from CHC patients were shown to release HCV RNA into culture supernatants, linking the development of CD34+ cells to their susceptibility to HCV infection. In an elegant study reported more than 10 years ago by the Brechot group [29], PBMCs from patients with CHC were injected intraperitoneally into severe combined immunodeficiency mice (SCID), with the animals subsequently monitored for HCV RNA persistence in both serum and PBMC for several months. Intriguingly, the authors documented the presence of HCV RNA in both compartments and the identification

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of replicating HCV in circulating PBMCs. Furthermore, this was the case only in animals inoculated with HCV RNA-positive PBMCs and not in those injected with HCV RNA-positive sera, sheep PBMCs preincubated with HCV, or human fibroblasts preincubated with HCV [29].

further support for HCV lymphotropism as well as its existence independent of whether infection is symptomatic or silent. Furthermore, the cytokine expression profiles and HCV-specific T cell responses of circulating immune cells were investigated in individuals with clinical SVR and compared to those of patients with CHC or healthy controls. PBMCs from individuals with SVR: (1) exhibited a cytokine expression pattern clearly distinct from that of healthy individuals or patients with CHC; (2) mounted a stronger virus-specific proliferative response; and (3) produced a greater abundance of antiviral cytokines following exposure to recombinant HCV antigens than was the case with PBMCs from patients with CHC [40–43]. Of note, these responses were documented long after the achievement of SVR and coincided, in many cases, with HCV detection in PBMCs. This would imply that low-level persistent infection engaging lymphoid cells is unlikely to be an immunologically neutral event. In fact, OCI may even be beneficial to the host in keeping the virus under immunological control. Regarding whether HCV RNA detectable in secondary OCI reflects authentic, replication-competent virions or defective virus, it was documented that, at least in some affected individuals, the virus is capable of inducing de novo productive infection in primary T cells derived from healthy donors [44]. Specifically, primary T lymphocytes exposed to the virus were able to generate HCV RNA negative strands, produce viral NS5A protein, and release HCV virion particles into the culture supernatant, thus recapitulating the entire viral life cycle. As mentioned, other studies reported the failure to detect virus persisting as OCI after clinical resolution of hepatitis C [35–38]. The reasons behind these very different results and whether they can be reconciled have been addressed elsewhere [39]. However, in the following, we provide a succinct look at the most important arguments and offer possible explanations for the discrepancies.

8.3

HCV Persistence in Lymphoid Cells of Individuals with Clinically Resolved Hepatitis C

It was previously thought that resolution of hepatitis C, as evidenced by normalization of liver function tests and disappearance of serum HCV RNA identifiable by clinical laboratory assays for at least 6 months upon completion of antiviral therapy, was a reflection of a complete eradication of HCV. This state of clinically apparent HCV clearance was referred to as a sustained virological response (SVR). However, as early as 2004, several groups independently reported the detection of residual HCV infection, namely, occult HCV infection, in plasma, PBMCs, and/or liver tissue of individuals with clinical resolved hepatitis C. These results were obtained by testing the samples using highly sensitive nucleic acid amplification assays, either alone or combined with hybridization of the amplified signals with HCV-specific probes [30–33]. We refer to this form of HCV infection as secondary OCI [34]. Meanwhile, there were also studies that refuted the existence of this form of HCV infection [35–38]. To this end, although OCI remains relatively controversial, there are factors that may reconcile these discordant findings [39]. The most important elements are briefly summarized below. It is worthwhile noting that in studies documenting the existence of secondary OCI, viral loads reported were generally very low, ranging between 100 and 200 virus genome equivalents (vge)/mL plasma, and between 10 and 100 vge/mg total RNA in PBMCs and hepatic tissue [10, 31]. As in the case of CHC, different immune cell subsets were found to be infected with HCV and in certain situations the major, if not exclusive reservoir of replicating HCV resided within a particular immune cell subtype [2, 10]. Similar to investigations in patients with CHC, the use of clonal sequencing and highly sensitive SSCP enabled the identification of HCV variants unique to immune cells within the 5¢-UTR IRES or the hypervariable region of E2 envelope glycoprotein [10, 13, 15]. This provided

8.4

Occult HCV Infection in Immune Cells of Patients with No History of Hepatitis C

In parallel with the identification of secondary OCI, which normally occurs against a background of detectable antibodies against HCV (anti-HCV) and normal liver function tests, OCI has also been described in

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anti-HCV non-reactive patients with persistently elevated liver enzymes [45–47]. In contrast to the usual situation, the etiology of the anti-HCV non-reactive OCI is not clear; thus, we refer to this form as cryptogenic OCI [34, 39]. In such patients, Schmidt et al. [46] reported that HCV RNA was repeatedly detectable in the whole blood of up to 67% of infected individuals versus in about 30% in whom HCV RNA was only intermittently identifiable when plasma was screened instead, implying a potentially significant contribution of hematopoietic cells to the overall viral load. Similarly, Carreno’s group [45] described the presence of HCV RNA in PBMCs of nearly 40% of individuals who were negative for both anti-HCV antibody and serum HCV RNA by standard clinical testing. Of note, nearly 60% of patients in the Carreno study [45] were also found to be reactive for replicating HCV genomes in liver tissues. Interestingly, in cryptogenic OCI as in secondary OCI discussed above, HCV-specific CD4+ and CD8+ T cell responses were detected. These were postulated to be inversely correlated with the extent of hepatic OCI infection [48]. However, the opposite scenario was also reported, in which patients with cryptogenic liver disease were not found to carry detectable HCV RNA in PBMCs; but whether the patients investigated were positive for HCV RNA in the liver was not addressed [49]. Again, explanations for the apparently contradictory data might be similar to those applicable to secondary OCI, as considered below.

8.5

Reconciling the Inconsistencies in the Detection of Occult HCV Infection

As discussed in the previously cited publication [39], the contradictory results regarding the existence of OCI reported by different groups may be due to variations in: (1) the collection and preparation of plasma and liver samples, and in particular PBMCs; (2) the method used for RNA extraction and the amount of material used in the experiments; (3) the number of time points at which the samples were analyzed (i.e., singular or serial samples); (4) the amount of template RNA ultimately tested for HCV genome expression; and (5) the sensitivity of the HCV RNA detection assay used. Furthermore, as mentioned above, there is ample evidence from independent groups that HCV is

compartmentalized to different degrees in different immune cell subsets [6–8, 10–12]. Consequently, a negative finding of HCV RNA in PBMCs does not always translate as the absence of HCV infection in individual lymphoid cell subsets. Also, it seems that ex vivo stimulation of immune cells with mitogens enhances viral replication, allowing for more sensitive virus detection [30, 50], as demonstrated even when a single PBMC sample was tested. This rate of positivity could increase up to 15% when serial PBMC samples are examined.

8.6

In Vitro Propagation of Wild-Type HCV Isolates in Lymphoid Cells

Support for the inherent propensity of HCV to infect and propagate in cells of the immune system also comes from studies in which lymphocytic cells from established human B cell lines (e.g., Rajii and Daudi) and T cell lines (e.g., Molt-4 and Jurkat) were exposed to HCV either in plasma or derived from hematopoeitic cells of CHC patients with type II mixed cryoglobulinemia [51, 52]. In addition, other groups have demonstrated the ability of primary lymphoid cells from healthy individuals, including total PBMCs [53], T cells [54], purified CD45RA+ CD45RO− CD4+ T cells [51], and monocytes/macrophages [55, 56], to support productive HCV infection. It should be stated that the infection of various immune cell subsets with HCV, despite a relatively low efficiency, could still lead to modulation of the host’s immune responses by: (1) negatively affecting interferon (IFN)-g gamma signaling [51]; (2) inhibiting T cell proliferation while enhancing CD95 (Fas)mediated apoptosis [52]; and (3) triggering the release of inflammatory cytokines [56]. For the most part, researchers have employed several experimental approaches in attempts to show that wild-type HCV is authentically replicated in their respective lymphoid cell cultures. These involve documentation of: (1) the presence of HCV RNA replicative (negative) strands in infected cells; (2) detection of viral proteins within the cytoplasm of de novo infected cells; (3) the susceptibility of virally infected cells to antiviral treatments, including, but not limited to, recombinant IFN-a; (4) the ability of cell-free supernatant from infected cell cultures to transmit infection to virus-naive cells, (5) viral particles released from

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infected cells possessing structural and physical properties of complete HCV virions; (6) visualization of HCV-like virions released by cultured immune cells via immunoelectron microscopy, and (7) evolution of HCV variants over the course of de novo infection. In contrast to the overall message conveyed by the above studies, that HCV can replicate in cells of the immune system, a recent work by Marukian et al. [57] reported that no immune cell subsets within PBMCs were capable of supporting de novo HCV infection. In their study, HCV particles generated by the transfection of a laboratory-adapted HCV chimera J6/JFH construct into hepatoma Huh7 cell line were used as inoculum. This, in essence, was a fundamental difference from all previous studies, in which clinical HCV isolates obtained directly from plasma or from the infected cells of patients with CHC or OCI were used as the source of virus. Since the ability of the JFH-1 strain to establish HCV replication in hepatoma Huh7 cells but not in lymphoid cell lines had been previously confirmed [58], the inability of the J6/JFH chimera to infect peripheral immune cells was not surprising. However, it is generally acknowledged that the levels of HCV are lower in immune cells than in hepatocytes and that HCV replication in the former is much less robust than in JFH-1/Huh7 cells and related systems. As such, the methods commonly used to detect and quantify HCV replication in hepatoma cells might be insufficiently sensitive to identify viral replication in lymphoid cells. With respect to the postulated apparent lack on lymphoid cells of receptors shown to be required for HCV entry into Huh7 cells [57], it cannot be ruled out that HCV uses entirely different receptors to gain access to cells of the immune system. In this context, measles virus (MeV), another single-stranded RNA virus, normally uses a molecule known as signaling lymphocyte activation molecule (SLAM) [59], whose expression is restricted to hematopoietic cells, to gain entry to its primary targets, i.e., lymphocytes and macrophages. However, very recent data revealed that MeV apparently uses CD147 as a functional entry receptor on other, less-recognized target cells, such as epithelial cells, which coincidentally do not express SLAM [60]. In this scenario, the flexibility of using another entry receptor allows viruses (such as MeV) to expand their cell target repertoire and therefore to induce a more readily persistent infection. With respect to HCV, CD5, a glycoprotein belonging to the scavenger receptor cysteine-rich family and specifically

expressed on T and B cells, appears to be essential for HCV entry into primary T cells or Molt4 and Jurkat T cell lines, all of which are susceptible to infection with wild-type virus (Sarhan et al., manuscript submitted) [61]. Importantly, hepatoma Huh7, Huh7.5, and HepG2 cell lines do not display CD5 (Sarhan et al., manuscript submitted).

8.7

Concluding Remarks

For nearly two decades, the issue of HCV lymphotropism has been investigated. The body of data accumulated through studies using clinical samples or cell culture infection models has demonstrated the propensity of HCV to target cells of the immune system. Nonetheless, acceptance of the notion that HCV invades sites beyond the liver is by no means unanimous. That HCV does not infect or replicate in immune cells to the same degree as it does in hepatocytes together with the essentially asymptomatic nature of immune-system infection in the majority of cases is probably one of the most important reasons behind the lack of agreement. In addition, because of the basic properties of HCV propagation in immune cells, the sensitivity of HCV RNA detection assays is an important factor in determining whether or not HCV lymphotropism is observed. Certainly, further molecular characterization of the mechanisms underlying immune cell susceptibility to HCV and governing viral replication in different immune cell subtypes, along with recognition of the immunological consequences of HCV infection of immune cells would be prudent in broadening our understanding of the role of the lymphatic system in the pathogenesis and natural progression of HCV infection. Moreover, it will be important to determine whether elimination of the virus within the lymphoid cell compartment contributes to the overall success of current or future antiviral therapies for hepatitis C, and if so, then how this occurs.

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low translational efficiency in cultured hepatocytes. Gut 59: 934–942 Gisbert JP, Garcia-Buey L, Pajares JM, Moreno-Otero R (2003) Prevalence of hepatitis C virus infection in B-cell non-Hodgkin’s lymphoma: systematic review and metaanalysis. Gastroenterology 125:1723–1732 Matsuo K, Kusano A, Sugumar A et al (2004) Effect of hepatitis C virus infection on the risk of non-Hodgkin’s lymphoma: a meta-analysis of epidemiological studies. Cancer Sci 95:745–752 Agnello V, De Rosa FG (2004) Extrahepatic disease manifestations of HCV infection: some current issues. J Hepatol 40:341–352 Ferri C, Monti M, La Civita L et al (1993) Infection of peripheral blood mononuclear cells by hepatitis C virus in mixed cryoglobulinemia. Blood 82:3701–3704 Mizorogi F, Hiramoto J, Nozato A et al (2000) Hepatitis C virus infection in patients with B-cell non-Hodgkin’s lymphoma. Intern Med 39:112–117 Martyak LA, Yeganeh M, Saab S (2009) Hepatitis C and lymphoproliferative disorders: from mixed cryoglobulinemia to non-Hodgkin’s lymphoma. Clin Gastroenterol Hepatol 7:900–905 Bagaglio S, Cinque P, Racca S et al (2005) Hepatitis C virus populations in the plasma, peripheral blood mononuclear cells and cerebrospinal fluid of HIV/hepatitis C virus-coinfected patients. AIDS 19(Suppl 3):S151–S165 Radkowski M, Wilkinson J, Nowicki M et al (2002) Search for hepatitis C virus negative-strand RNA sequences and analysis of viral sequences in the central nervous system: evidence of replication. J Virol 76:600–608 Sansonno D, Lotesoriere C, Cornacchiulo V et al (1998) Hepatitis C virus infection involves CD34(+) hematopoietic progenitor cells in hepatitis C virus chronic carriers. Blood 92:3328–3337 Bronowicki JP, Loriot MA, Thiers V et al (1998) Hepatitis C virus persistence in human hematopoietic cells injected into SCID mice. Hepatology 28:211–218 Pham TN, MacParland SA, Mulrooney PM et al (2004) Hepatitis C virus persistence after spontaneous or treatmentinduced resolution of hepatitis C. J Virol 78:5867–5874 Radkowski M, Gallegos-Orozco JF, Jablonska J et al (2005) Persistence of hepatitis C virus in patients successfully treated for chronic hepatitis C. Hepatology 41:106–114 Ciancio A, Smedile A, Giordanino C (2006) Long-term follow-up of previous hepatitis C virus positive nonresponders to interferon monotherapy successfully retreated with combination therapy: are they really cured? Am J Gastroenterol 101:1811–1816 Gallegos-Orozco JF, Rakela J, Rosati MJ et al (2008) Persistence of hepatitis C virus in peripheral blood mononuclear cells of sustained viral responders to pegylated interferon and ribavirin therapy. Dig Dis Sci 53:2564–2568 Michalak TI, Pham TN (2009) Anti-HCV core antibody: a potential new marker of occult and otherwise serologically silent HCV infection. J Hepatol 50:244–246 Bernardin F, Tobler L, Walsh I et al (2008) Clearance of hepatitis C virus RNA from the peripheral blood mononuclear cells of blood donors who spontaneously or therapeutically control their plasma viremia. Hepatology 47: 1446–1452

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HCV Infection of Hematopoietic and Immune Cell Subsets

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36. George SL, Bacon BR, Brunt EM et al (2009) Clinical, virologic, histologic, and biochemical outcomes after successful HCV therapy: a 5-year follow-up of 150 patients. Hepatology 49:729–738 37. Marcellin P, Boyer N, Gervais A et al (1997) Long-term histologic improvement and loss of detectable intrahepatic HCV RNA in patients with chronic hepatitis C and sustained response to interferon-alpha therapy. Ann Intern Med 127:875–881 38. Maylin S, Martinot-Peignoux M, Ripault MP et al (2009) Sustained virological response is associated with clearance of hepatitis C virus RNA and a decrease in hepatitis C virus antibody. Liver Int 29:511–517 39. Pham TN, Coffin CS, Michalak TI (2010) Occult hepatitis C virus infection: what does it mean? Liver Int 30:502–511 40. Casanovas-Taltavull T, Ercilla MG et al (2004) Long-term immune response after liver transplantation in patients with spontaneous or post-treatment HCV-RNA clearance. Liver Transpl 10:584–594 41. Pillai V, Lee WM, Thiele DL, Karandikar NJ (2007) Clinical responders to antiviral therapy of chronic HCV infection show elevated antiviral CD4+ and CD8+ T-cell responses. J Viral Hepat 14:318–329 42. Quiroga JA, Llorente S, Castillo I et al (2006) Virus-specific T-cell responses associated with hepatitis C virus (HCV) persistence in the liver after apparent recovery from HCV infection. J Med Virol 78:1190–1197 43. Pham TNQ, Mercer SE, Michalak TI (2009) Chronic hepatitis C and persistent occult hepatitis C virus infection are characterized by distinct immune cell cytokine expression profiles. J Viral Hepat 16:547–556 44. MacParland SA, Pham TN, Guy CS, Michalak TI (2009) Hepatitis C virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro. Hepatology 49:1431–1441 45. Castillo I, Pardo M, Bartolome J et al (2004) Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver-function tests is unknown. J Infect Dis 189:7–14 46. Schmidt WN, Wu P, Cederna J et al (1997) Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests. J Infect Dis 176:27–33 47. Stapleton JT, Schmidt WN, Katz L (2004) Seronegative hepatitis C virus infection, not just RNA detection. J Infect Dis 190:651–652

48. Quiroga JA, Llorente S, Castillo I et al (2006) Cellular immune responses associated with occult hepatitis C virus infection of the liver. J Virol 80:10972–10979 49. Halfon P, Bourliere M, Ouzan D et al (2008) Occult hepatitis C virus infection revisited with ultrasensitive real-time PCR assay. J Clin Microbiol 46:2106–2108 50. Pham TN, MacParland SA, Coffin CS et al (2005) Mitogeninduced upregulation of hepatitis C virus expression in human lymphoid cells. J Gen Virol 86:657–666 51. Kondo Y, Sung VM, Machida K et al (2007) Hepatitis C virus infects T cells and affects interferon-gamma signaling in T cell lines. Virology 361:161–173 52. Kondo Y, Machida K, Liu HM et al (2009) Hepatitis C virus infection of T cells inhibits proliferation and enhances Fasmediated apoptosis by down-regulating the expression of CD44 splicing variant 6. J Infect Dis 199:726–736 53. Cribier B, Schmitt C, Bingen A et al (1995) In vitro infection of peripheral blood mononuclear cells by hepatitis C virus. J Gen Virol 76:2485–2491 54. MacParland SA, Pham TN, Gujar SA, Michalak TI (2006) De novo infection and propagation of wild-type Hepatitis C virus in human T lymphocytes in vitro. J Gen Virol 87: 3577–3586 55. Laskus T, Radkowski M, Jablonska J et al (2004) Human immunodeficiency virus facilitates infection/replication of hepatitis C virus in native human macrophages. Blood 103: 3854–3859 56. Radkowski M, Bednarska A, Horban A et al (2004) Infection of primary human macrophages with hepatitis C virus in vitro: induction of tumour necrosis factor-alpha and interleukin 8. J Gen Virol 85:47–59 57. Marukian S, Jones CT, Andrus L et al (2008) Cell cultureproduced hepatitis C virus does not infect peripheral blood mononuclear cells. Hepatology 48:1843–1850 58. Murakami K, Kimura T, Osaki M et al (2008) Virological characterization of the hepatitis C virus JFH-1 strain in lymphocytic cell lines. J Gen Virol 89:1587–1592 59. Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for measles virus. Nature 406:893–897 60. Watanabe A, Yoneda M, Ikeda F et al (2010) CD147/ EMMPRIN acts as a functional entry receptor for measles virus on epithelial cells. J Virol 84:4183–4193 61. Sarhan MA, Michalak TI (2009) CD5-Mediated susceptibility of human T lymphocytes to wild-type hepatitis C virus. Hepatology 50:350A

Part III Cryoglobulinemia and the Complement System

9

Cryoglobulinemia and Chronic HCV Infection: An Evolving Story Jürg A. Schifferli and Marten Trendelenburg

The observation of protein precipitation at low ­temperature is an old one. Heidelberger and Kendall, already in 1929, described the precipitation of immune complexes in the cold after an immune response to pneumococcal polysaccharides [1]. In 1933, Wintrobe and Buell described the same phenomenon in the plasma of a patient with multiple myeloma [2]. A more detailed analysis of different types of cryoglobulins, however, began later, after it became clear that some of the cryoprecipitates contained different types of immunoglobulins. Mixed cryoglobulins were first described in 1962, following the observation that the cold-­ precipitable proteins in the serum of a patient with renal tubular acidosis were complexes consisting of 7S gamma globulins and 19S rheumatoid factors (RF), with 7S and 19S indicating what would later be designated as monomeric IgG and IgM, respectively [3]. The IgM and IgG present in these complexes differed from monoclonal cryoproteins, which precipitate on their own in the cold. Indeed, each of the two components, IgG and IgM, was an “incomplete” cryoglobulin, referring to the ability to precipitate in the cold only when mixed with the other, thus explaining the terminology of “mixed cryoglobulin.” Since the IgM had rheumatoid factor (RF) activity, it was immediately considered that the reaction of IgM with its antigen (IgG) was the  necessary requirement for cryoprecipitation. The ­corresponding clinical syndrome of mixed cryoglobulinemia (palpable purpura,

J.A. Schifferli (*) Division of Internal Medicine, Department of Medicine, University Hospital Basel, Basel, Switzerland e-mail: [email protected] F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_9, © Springer-Verlag Italia 2012

arthralgias, and weakness) was first described by Meltzer and Franklin [4], who ­confirmed the mixed IgG-IgM composition of the cryoglobulins and found that the IgM-RF was polyclonal in some patients and monoclonal in others. These authors and their colleagues also provided the first evidence for a role of mixed cryoglobulins in the pathogenesis of renal lesions associated with the disease. Immunofluorescence studies demonstrated that both IgG and IgM were deposited intraluminally and along the basement membranes of glomeruli [5]. Neither the clinical nor the pathological features in 9 of the 11 originally described patients with mixed cryoglobulins were typical of any known disease entity, and the term essential mixed cryoglobulinemia (EMC) was therefore introduced. The immunochemical classification of cryoglobulins that is now widely used describes three types of cryoglobulins [6, 7]. Those in type I consist of a single monoclonal immunoglobulin and are predominantly associated with malignancies of the immune system. Types II and type III are mixed cryoglobulins. Whereas in type II the cryoglobulins consist of polyclonal IgG and monoclonal IgM RFs, in type III both the IgG and the RF are polyclonal. The RF seen in type II and type III cryoglobulinemia usually consists of IgM but other classes of immunoglobulins are possible as well. More recently, Tissot described the presence of a few dominant oligoclonal IgM RFs in the serum of some patients with mixed cryoglobulinemia, suggesting a transition phase from type III (polyclonal IgM RF) to type II (monoclonal IgM RF) [8]. It soon also became evident that many of the RFs of type II cryoglobulinemia were genetically related, since they expressed the common idiotype Wa, as if the events preceding the emergence of the RF had some common features [9]. 79

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As noted above, most of the type II and type III mixed cryoglobulinemias are classified as essential, reflecting the fact that they occur in the absence of any apparent underlying disease. Secondary mixed cryoglobulins, predominantly type III, are seen in infectious, autoimmune, and chronic liver diseases whereas the secondary type II mixed cryoglobulins having a monoclonal component are often found in association with malignancies of the immune system. The prominent role of chronic viral hepatitis in the pathogenesis of cryoglobulins was not obvious in initial reports [10], although Levo et al. [11] had suggested a major role for hepatitis B virus (HBV) in EMC based on their detection of hepatitis B antigen or antibody in 13 of 53 patients (25%) with the disease. However, 14% of their control groups, made up of systemic lupus erythematosus and rheumatoid arthritis patients were infected with HBV, which was a very high percentage. In addition, this association was not confirmed by others. In 1990, Pascual presented ­evidence of hepatitis C virus (HCV) infection in three out of ten patients with EMC based on the results of a ­first-generation assay for anti-HCV antibodies [12]. Subsequently and very rapidly, evidence for a strong association between HCV and EMC was provided by different groups using newer anti-HCV antibody assays [13–24]. The prevalence of HCV antibodies in the sera of patients with type II and type III mixed cryoglobulinemia ranged from 30% to 98%. It is worth emphasizing that in most of the initial studies on mixed cryoglobulinemia, the test results were obtained using different generations of enzymelinked immunosorbent and recombinant immunoblot assay (ELISAs and RIBAs, respectively), leading to conflicting results on the prevalence of anti-HCV antibodies [13, 14, 21–23, 25]. However, quantitative studies of HCV antibodies from dissociated cryoglobulins showed that these antibodies were concentrated in the cryoglobulin. The results suggested that in non-dissociated cryoglobulins the anti-HCV antibodies are blocked by antigen and therefore unavailable for detection. Over time, assays for the detection of anti-HCV antibodies became more sensitive such that nowadays there are few patients with cryoglobulinemia and HCV hepatitis who do not have detectable antibodies in the blood circulation. Moreover, the detection of HCV RNA is a more direct indication of infection. Advances in polymerase chain reaction (PCR) methodology have allowed this method to become a practical technique for detecting and measuring hepatitis C viremia. It is,

J.A. Schifferli and M. Trendelenburg

Fig. 9.1  Serum obtained from a patient with cryoglubulinemia type II and HCV. After 24 h at 4°C, a cryoprecipitate is clearly visible

however, worthwhile to emphasize that in some patients with mixed cryoglobulinemia (5% approximately) HCV is not detectable; instead, other viruses are thought to trigger the emergence of the polyclonal/ monoclonal RF, such as HBV or HIV. Alternatively, these patients may have a clinical syndrome that includes some autoimmune features. These isolated observations enhance the notion that while HCV is indeed an excellent trigger of mixed cryoglobulinemia, the cascade of events induced by the virus corresponds somehow to “a stereotyped response” of the immune system in certain individuals. Interestingly, evidence for a direct pathogenic effect of cryoglobulins is lacking although their pathogenicity appears to be obvious. Cryoprecipitation of immunoglobulins, as it is used for diagnostic purposes (Fig. 9.1), is primarily an in vitro phenomenon the relevance of which has not been clearly shown in vivo. The concentration of cryoglobulins was, from the start, shown not to correlate well with the clinical syndrome. In spite of this knowledge, it is intriguing to see that the measurements of cryoglobulins in many labs continues to be based on the cryocrit, which is performed very differently from one lab to another and regardless of the method used will fail to detect low levels of cryoglobulins. Efforts to standardize these measurements or to convert them into units of mg/l, as defined by protein determination of the precipitate, have not yet been successful and in part explain the large differences in reports of the prevalence of cryoglobulins in HCV infection. In contrast, the correlation of cryoglobulins with skin lesions is clinically well established in many patients, as illustrated by the local application

9  Cryoglobulinemia and Chronic HCV Infection: An Evolving Story

Fig. 9.2  Patient with HCV and a cryoglobulin type II. She applied ice cubes to her “burning” skin, with resulting necrosis

of ice cubes to the skin of patients suffering from type II cryoglobulinemia (Fig.  9.2). These observations strongly support the idea that the formation of cryoprecipitates is not only an in vitro phenomenon but is of importance in vivo as well. The physicochemical properties of cryoglobulins might play a major role here. Following a unique case of a cryoglobulin precipitating only in the presence of calcium, it was possible to analyze the mechanisms of temperature-dependent precipitation [26]. Cryoprecipitation was shown to be a two-step event: (1) the reaction between antibody (IgM) and antigen (i.e., the Fc portion of IgG) reaction, which is not significantly influenced by temperature and (2) an IgM- and temperature-dependent aggregation. These and other data indicate clearly that IgG-IgM complexes circulate in vivo. However, unlike immune complexes in which IgG is the antibody, they are not cleared but instead circulate with a half life of weeks, comparable to monomeric IgG [27]. In addition, although they activate complement, they do not fix enough C3b to allow clearance by the complement/ CR1 pathway [28, 29]. This “non-clearance” might lead to the accumulation of IgM (RF)-IgG complexes in blood vessels and their subsequent deposition when the temperature drops or “saturation” is achieved. Immune complex deposition would then lead to local inflammation mediated by complement activation, with local amplification of C3 deposition and the release of C5a. The latter is the most powerful chemotactic factor for polymorphonuclear leukocytes and is responsible for the initiation of local tissue damage [30]. Izui’s group suggested that the combination of RF and cryoprecipitability is responsible for the

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v­ asculitis seen in their mouse model of cryoglobulinemia. Interestingly, the temperature at which the mice are kept determines the presence of vasculitis and glomerulonephritis [31]. Whereas mice kept at room temperature develop glomerular depositions of cryoglobulins, glomerulonephritis is not seen in mice maintained in a warm environment (37°C). In line with the above-mentioned experimental findings, the most likely hypothesis explaining this result is that the large cryoglobulinemic aggregates formed in superficial blood vessels at temperatures below 37°C might not dissociate fast enough before arriving in the kidney or other organs. Despite the detection of HCV or proteins thereof in immune deposits, it remains unresolved whether their presence is directly involved in the local deposition of cryoglobulins in tissues such as the kidney and skin [32, 33]. Indeed, any antigen trapped in the cryoglobulin will be found at the site of immune aggregation. Furthermore, tissue deposition of type II cryoglobulins in Sjögren’s syndrome without HCV involvement occurs in the same organs and is indistinguishable from that of HCV-associated cryoglobulinemia. It is also worthwhile to think back about the treatment options that were at our disposal before we ­realized that HCV was the major cause of EMC. Immunosuppression and plasma exchange were efficient in many cases and, ironically, might correspond to the current use of rituximab [34], since both are aimed at inhibiting the synthesis of RF. Interestingly, interferon (IFN)-a was used in the treatment of cryoglobulinemia with some success by Bonomo in 1987, before HCV was identified as the cause of the disease [35]. Nowadays, IFN-a is the basis of anti-HCV therapy. It is also of interest that liver disease identical to that seen in many patients with mixed cryoglobulinemia was described in several reports long before 1990. The type of infiltration was reminiscent of the histology of “nonA-nonB” hepatitis, and in fact, as already mentioned, many authors had looked for viruses including HBV, Epstein-Barr virus, and cytomegalovirus even in the absence of convincing evidence for a viral origin to the pathology. The slow progression of many of these patients also suggested that EMC was more of a lowgrade lymphoma particularly, when histological images of even the kidney suggested tumor infiltration. This was the basis of the trial performed by Bonomo [35]. Most likely the future will teach us to recognize similar, apparently obvious relationships, since in many patients

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with HCV-related cryoglobulinemia the expansion of B cells across organs corresponds to the development of low-grade lymphoma. The history of essential cryoglobulinemia is fascinating since it demonstrates the slow progression of knowledge from one generation of research groups to the next, beginning with the initial emphasis on the immune reactants discovered to the realization that behind the deregulation of the immune system lay an infectious organism. Similar observations have been made for other viruses (Epstein-Barr virus) or bacteria (Helicobacter pylori), which have been shown to trigger a lymphoproliferation that starts as a benign process but becomes aggressive with time. The relation between HCV, the immune response, and induced deregulation are, however, far from being understood. While the present volume provides a synthesis of our current knowledge, there is much that remains to be understood: What are the steps between this specific viral trigger and the response of the human immune system? How can we better inhibit this response? Is the immune response genetically defined, and if so by which genes? Do other lymphoproliferative phenomena have similar triggers? Let’s hope that the readers of this book will be inspired to write the next chapters of the history of mixed HCV- and nonHCV-related cryoglobulinemia.

References 1. Heidelberger M, Kendall FE (1929) A quantitative study of the precipitin reaction between type III pneumococcus polysaccharide and purified homologous antibody. J Exp Med 50:809–823 2. Wintrobe MM, Buell MV (1933) Hypoproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 52: 156–165 3. Lospalluto J, Dorward B, Miller W Jr, Ziff M (1962) Cryoglobulinemia based on interaction between a gamma macroglobulin and 7s gamma globulin. Am J Med 32:142–147 4. Meltzer M, Franklin EC (1966) Cryoglobulinemia: a study of twentynine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 40:828–836 5. Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N (1966) Cryoglobulinemia: a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med 40:837–856 6. Brouet JC, Clauvel JP, Danon F et  al (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 57:775–788

J.A. Schifferli and M. Trendelenburg 7. Gorevic PD (1986) Mixed cryoglobulinemia: an update of recent clinical experience. In: Ponticelli C, Minetti L, D’Amico G (eds) Antiglobulins, cryoglobulins and glomerulonephritis. Martinus Nijhoff, Dordrecht 8. Tissot JD, Schifferli JA, Hochstrasser DF, Pasquali C, Spertini F, Clément F, Frutiger S, Paquet N, Hugues GJ, Schneider P (1994) Two-dimentional polyacrylamide gel electrophoresis analysis of cryoglobulins and identification of an IgM-associated peptide. J Immunol Methods 173: 63–75 9. Agnello V, Barnes JL (1986) Human rheumatoid factor crossidiotypes. I. WA and BLA are heat-labile conformational antigens requiring both heavy and light chains. J Exp Med 164:1809–1814 10. Brouet JC (1983) Les cryoglobulinémies. Presse Med 12: 2991–2996 11. Levo Y, Gorevic PD, Kassab HJ, Tobias H, Franklin EC (1977) Liver involvement in the syndrome of mixed cryoglobulinemia. Ann Intern Med 87:287–292 12. Pascual M, Perrin L, Giostra E, Schifferli JA (1990) Hepatitis C virus in patients with cryoglobulinemia type II (letter). J Infect Dis 162:569–570 13. Ferri C, Greco F, Longombardo G, Palla P, Moretti A, Marzo E, Fosella PV, Pasero G, Bombardieri S (1991) Antibodies to hepatitis C virus in patients with mixed cryoglobulinemia. Arthritis Rheum 34:1606–1610 14. Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II mixed cryoglobulinemia. N Engl J Med 327:1490–1495 15. Alter HE (1992) New kit on the block: evaluation of second generation assays for detection of antibody to the hepatitis C virus. Hepatology 15:350–353 16. Ferri C, Palla P, Greco F, Marzo E, Longombardo G, Moretti A (1991) Hepatitis C virus antibodies in mixed cryoglobulinemia (letter). Clin Exp Rheumatol 9:95–96 17. Bambara LM, Carletto A, Biasi D, Pacor ML, Caramaschi P (1991) Cryoglobulinemia and hepatitis C virus (HCV) infection (letter). Clin Exp Rheumatol 9:96–97 18. Durand JM, Lefevre P, Harle JR, Boucrat J, Vitvitski L, Soubeyrand J (1991) Cutaneous vasditis and cryoglobulinemia type II associated with hepatitis C virus infection (letter). Lancet 337:499–500 19. Casato M, Taliani G, Pucillo LP, Goffredo F, Lagana B, Bonomo L (1991) Cryoglobulinemia and hepatitis C virus (letter). Lancet 337:1047–1048 20. Arribas JR, Barbado FJ, Zapico R, Sendino A, Gonzalez I, Vazquez JJ (1991) Association between hepatitis C virus and mixed cryoglobulinemia. Rev Infect Dis 13:770–771 21. Disdier P, Harle JR, Weiller PJ (1991) Cryoglobulinemia and hepatitis C infection (letter). Lancet 338:1151–1152 22. Pechère-Bertschi A, Perrin L, de Saussure P, Widmann JJ, Giostra E, Schifferli JA (1992) Hepatitis C: a new etiology for cryoglobulinemia type II. Clin Exp Immunol 89: 419–422 23. Ferri C, Greco F, Longombando G, Palla P, Moretti A, Marzo E, Mazzoni A, Pasero G, Bombardieri S, Highfield P, Corbishley T (1991) Association between hepatitis C virus and mixed cryoglobulinemia. Clin Exp Rheumatol 9: 621–624

9  Cryoglobulinemia and Chronic HCV Infection: An Evolving Story 24. Misiani R, Bellavita P, Fenili D, Borelli G, Marchesi D, Massazza M, Vedramin G, Comotti B, Tanzi E, Scudeller G, Zanetti A (1992) Hepatitis C virus infection in patients with essential mixed cryoglobulinemia. Ann Intern Med 117: 573–577 25. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R (1990) Hepatitis C virus antibodies in chronic active hepatitis: pathogenetic factor or false-positive result? Lancet 335:754–757 26. Qi M, Steiger G, Schifferli JA (1992) A calcium dependent cryoglobulin IgM kappa/polyclonal IgG. J Immunol 149:2345–2351 27. Schifferli JA, Amos N, Pusey CD, Sissons JGP, Peters DK (1983) Metabolism of autologous and homologous IgG in patients with mixed essential cryoglobulinemia type II. Absence of fast elimination of IgG. Clin Exp Immunol 51: 305–316 28. Schifferli JA, Ng YC, Estreicher J, Walport MJ (1988) The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans: complementand erythrocyte CR1-dependent mechanisms. J Immunol 140:899–904 29. Madi N, Steiger G, Estreicher J, Schifferli JA (1991) Defective immune adherence and elimination of hepatitis B

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surface Ag/Ab complexes in patients with mixed essential cryoglobulinaemia type II. J Immunol 147:495–502 30. Schifferli JA, Steiger G, Polla L, Didierjean L, Saurat JH (1985) Activation of the alternative pathway of complement by skin immune deposits. J Invest Dermatol 85:407–411 31. Fulpius T, Berney T, Lemoine R et al (1994) Glomerulopathy induced by IgG3 anti-trinitrophenyl monoclonal cryoglobulins derived from non-autoimmune mice. Kidney Int 45:962–971 32. Sansonno D, Gesualdo L, Manno C, Schena FP, Dammacco F (1997) Hepatitis C virus-related proteins in kidney tissue from hepatitis C virus-infected patients with cryoglobulinemic membranoproliferative glomerulonephritis. Hepatology 25:1237–1244 33. Agnello V, Abel G (1997) Localization of hepatitis C virus in cutaneous vasculitic lesions in patients with type II cryoglobulinemia. Arthritis Rheum 40:2007–2015 34. Terrier B, Saadoun D, Sène D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 30:2531–2540 35. Bonomo L, Casato M, Afeltra A, Caccavo D (1987) Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 83:726–730

The Complement System in Cryoglobulinemia

10

Marten Trendelenburg

10.1 The Complement Cascade Complement is part of the innate immune system and underlies one of the main effector mechanisms of antibody-mediated immunity. It has three major physiologic activities: defending against pyogenic bacterial infection, bridging innate and adaptive immunity, and disposing of immune complexes and the products of inflammatory injury. There are three pathways of activation of the complement system: the classical, mannose-binding lectin, and alternative pathways (Fig. 10.1). These pathways leading to the cleavage of C3 are triggered enzyme cascades, analogous to the coagulation, fibrinolysis, and kinin pathways. The terminal complement pathway, resulting in the formation of the membrane-attack complex, is a unique system that builds up a lipophilic complex in cell membranes from several plasma proteins (C5b-9). Complement is a system comprising more than 30 proteins in plasma and on cell surfaces. Complement proteins in plasma are present in concentrations of > 3 g/l and they constitute approximately 15% of the globulin fraction. The nomenclature of complement follows the historical order of discovery and as a consequence might appear confusing. The first complement pathway that was discovered, the classical pathway, begins with the formation of immune complexes, e.g., when antibody binds to a cell surface, and ends with lysis of the cell. The proteins of this pathway

M. Trendelenburg  Clinic for Internal Medicine and Laboratory for Clinical Immunology, University Hospital Basel, Basel, Switzerland e-mail: [email protected]

are designated C1–C9. It was subsequently discovered that the numbering of the proteins did not quite correspond with the order of the reaction, since C1 is followed in succession by C4, C2, C3, and C5, with the numerical order being restored from C6 through C9. Proteins of the second pathway to be discovered, the alternative pathway, are called factors and are followed by a letter, such as factor B. The alternative pathway is spontaneously and constantly activated on biological surfaces in plasma and in most or all other body fluids. This spontaneous activation readily initiates amplification by the “amplification loop” and requires regulatory proteins protecting cells from complement attack. Therefore, complement proteins on cell membranes are not only receptors for activated complement proteins but also proteins that regulate complement. The third but evolutionarily perhaps the oldest pathway of complement activation is the mannose-binding lectin (MBL) pathway. As a multimeric complex, complement MBL is able to recognize carbohydrate pathogen-associated molecular patterns on a wide range of microorganisms, leading to complement activation in an antibody- and C1q-independent manner. Besides these three well-known pathways of complement activation, further pathways seem to exist, such as the direct cleavage of complement C5 by thrombin. Several complement proteins are cleaved during activation of the system, and the fragments are designated with lowercase suffixes; for example, C3 is cleaved into two fragments, C3a and C3b. With the exception of C2 for historical reasons, the large fragment is designated “b” and the small fragment “a.” The regulatory mechanisms of complement are finely balanced so that, on the one hand, the activation of complement is focused on the surface of invading microorganisms and, on the

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_10, © Springer-Verlag Italia 2012

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86 Fig. 10.1  Overview of the main components and activation pathways of complement

M. Trendelenburg Classical pathway - Immune complexes

Alternative pathway - Activating surfaces MBL pathway - carbohydrates (mannose)

C1 C4

MASP C2

C4 C2

C3b Factor B Factor D

C3 convertase C3a C5 convertase

Thrombin pathway - thrombin

C5a

C5b-9 Membrane-attack complex (MAC)

other hand, the deposition of complement on normal cells and tissues is limited. When the mechanisms that regulate this delicate balance go awry, the complement system may cause injury. Thus, complement may be friend or foe, depending on the circumstances. Under physiologic conditions, complement promotes the clearance of immune complexes, an important means of eliminating, e.g., antibody-coated bacteria. If, however, immune complexes cannot be eliminated, then complement becomes chronically activated and can incite inflammation. In addition, chronic infections can perpetuate the formation of immune complexes, which in hepatitis C infection and bacterial endocarditis cause relentless activation and consumption of complement [1–5].

10.2 Complement Consumption and Deposition in Cryoglobulinemia Low levels of complement suggesting ongoing complement activation and consumption are a well-known phenomenon in patients with cryoglobulinemia. It was first reported by Riethmüller et  al. in 1966 [6], who described seven patients with mixed cryoglobulins and very low levels of complement C2, suggesting activation and consumption of complement via the classical pathway. Further studies confirmed the predominant

consumption of components of the classical pathway [7–9]. In addition, it became apparent that some cryoglobulins are also able to directly activate the alternative pathway of complement [10]. However, whereas patients with cryoglobulinemia usually have low levels of C1, C4, and C2, concentrations of C3 and of the components of the terminal pathway often remain within the normal range, suggesting that activation of  complement beyond C3 is inefficient although not  absent [11, 12]. In a more recent analysis, cryoglobulins were found to also activate the MBL pathway of complement. All 16 cryoprecipitates of patients with mixed cryoglobulinemia were found to contain MBL [13]. In line with these data, it can frequently be observed that consumption of C4 is more pronounced than the relative consumption of C1q, suggesting that, in addition to activation of the classical pathway, complement activation by cryoglobulins occurs via the MBL pathway. The consumption of complement components is paralleled by the deposition of complement in affected tissues such as the kidney [13–15]. Whereas the predominant immunoreactants in mixed cryoglobulinemia are IgG and IgM, these deposits are usually accompanied by the glomerular deposition of MBL, C4 (C4d), C3, and, less frequently, C1q, with a distribution very similar to that of immunoglobulins. Complement deposition seems to be the consequence of both the local and ongoing activation of ­complement

10  The Complement System in Cryoglobulinemia

after trapping of pre-formed cryo-precipitates as well as of secondary deposition of complement components that are already present in the trapped complexes.

10.3 Interaction of Complement with Cryo-Immune Complexes Cryoglobulins can be considered as immune complexes consisting not only of immunoglobulins but also of trapped antigen, such as hepatitis C virus particles, and other serum components, such as C-reactive protein and complement [9, 16]. Complement participates in the elimination of immune complexes in many circumstances and is likely to be involved in the clearance of cryoglobulins as well. When immune complexes first form in the circulation, complement inhibits their aggregation because binding of components of the classical pathway as well as the covalent binding of C3b to the immune complexes modifies their biophysical properties [17]. This modification by the binding of complement facilitates the retention of immune complexes in solution, a process called “inhibition of immune precipitation.” In addition, complement has the capacity to solubilize immune precipitates that have already formed [18]. However, solubilization by complement is much less efficient, requiring a considerable amount of complement activation and thus bearing the risk of inflammatory tissue damage [19]. Once they are opsonized (C3b coated), immune complexes attach to cells bearing C3b receptors (complement receptor 1, CR1) in the circulation, in particular to erythrocytes, since in humans 85–90% of CR1 in the blood is located on these cells. This immune adherence binding reaction is a physiological system that allows immune complexes to be transported through the circulation to the fixed macrophages of the mononuclear phagocyte system, where they are safely eliminated. The deposition of circulating complement-fixing immune complexes in various organs such as the kidney may be considered as a failure of this transport system. This is apparent in complement-deficient and -depleted states as well as in non-complement-fixing immune complexes such as those of IgA. The formation of insoluble immune complexes (by definition, immune deposits found in human pathology are insoluble) produces complement activation and inflammation at the site of the immune aggregate. Type II

87

cryoglobulins were shown to activate complement in vitro but rapid clearance in vivo was not observed [20]. Most likely, on the one hand, the IgM rheumatoid factor (RF) covered the Fc fragments of IgG, thus altering their interference with Fc receptors. On the other hand, despite efficient complement activation, the cryo-immune complexes were not able to bind ­sufficient C3b and thus to be cleared via the erythrocyte-CR1 transport system [6, 21–23]. The insufficient binding of C3b perhaps explains the consumption of early components of the complement cascade that is usually not accompanied by similarly low C3 levels or the depletion of components of the terminal pathway, as outlined above. In addition, “non-clearance” might concur with the accumulation of IgM (RF)-IgG complexes in blood vessels that deposit when the temperature drops or “saturation” is achieved. Immune complex deposition then leads to local inflammation mediated by complement activation, with local amplification of C3 deposition and the release of C5a, which is the most powerful chemotactic factor for polymorphonuclear leukocytes and responsible for the initiation of local tissue damage [24].

10.4 Pathogenic Role of Complement Activation by Cryoglobulins In Vivo The abundant occurrence of IgG in cryoprecipitates and thus the large number of potentially available Fc fragments suggests that Fc gamma receptors play a major role in the pathogenic mechanisms of cryoglobulinemia. However, in  vivo experiments using mice that develop cryoglobulinemia could not confirm a major pathogenic role for Fc gamma receptors in this entity [25, 26]. These rather surprising results may be related to the fact that Fc fragments are no longer accessible after the binding of RFs, as occurs in mixed cryoglobulins. In contrast, there is some evidence that complement activation is a major factor explaining the inflammatory organ damage seen in cryoglobulinemia in  vivo. In our study, the role of complement was investigated in a mouse model of induced cryoglobulinemic glomerulonephritis. Depending on the genetic background of the mice, the cryoprecipitate consisted of a murine monoclonal IgG3 cryoglobulin only (type I cryoglobulinemia) or of murine monoclonal IgG3 RF–polyclonal IgG2a complexes (type II cryoglobulinemia). Several complement-deficient mice with

88

either genetic background were investigated and ­compared to strain-matched, wild-type controls. The ­survival of mice was not affected by complement deficiency but glomerular influx of neutrophils was ­significantly less in C3-, factor-B-, and C5-deficient mice than in wild-type and C1q-deficient mice. The influx of neutrophils did not correlate with the C3 deposition mediated by activation of the classical and alternative pathways, but did correlate with the amount of C6 deposited. In addition, deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Taken together, these results suggested that: (i) the generation of C5a was critical for the neutrophil influx observed in this model of cryoglobulinemia, and (ii) in spite of additional complement activation via the classical pathway, the alternative pathway played a prominent role in the cleavage of C5 [27]. However, data on the role of complement in ­cryoglobulinemic glomerulonephritis is conflicting. Transgenic mice that overexpress thymic stromal lymphopoietin (TSLP) and as a consequence develop mixed cryoglobulinemia with renal disease resembling human cryoglobulin-associated membranoproliferative glomerulonephritis. In these mice, no anti-inflammatory effect of the additional overexpression of the murine complement regulator Crry could be detected [28]. In contrast, factor B deficiency, which blocks the alternate pathway of complement, either alone or in addition to Crry overexpression did not alleviate, but instead ­aggravated the renal lesions in TSLP transgenic mice [29, 30]. However, it might be important to note that C1q staining in these mice was absent and that C3 deposition was much less than IgG and IgM deposition. Therefore, more studies are required to clarify the role of complement in cryoglobulinemia.

References 1. Fujita T (2002) Evolution of the lectin-complement pathway and its role in innate immunity. Nat Rev Immunol 2:346–353 2. Zipfel PF, Skerka C (2009) Complement regulators and inhibitory proteins. Nat Rev Immunol 9:729–740 3. Walport MJ (2001) Complement. First of two parts. N Engl J Med 344:1058–1066

M. Trendelenburg 4. Walport MJ (2001) Complement. Second of two parts. N Engl J Med 344:1140–1144 5. Huber-Lang M, Sarma JV, Zetoune FS et  al (2006) Generation of C5a in the absence of C3: a new complement activation pathway. Nat Med 12:682–687 6. Riethmüller G, Meltzer M, Franklin E, Miescher PA (1966) Serum complement levels in patients with mixed (IgM-IgG) cryoglobulinaemia. Clin Exp Immunol 1:337–339 7. Linscott WD, Kane JP (1975) The complement system in cryoglobulinaemia. Interaction with immunoglobulins and lipoproteins. Clin Exp Immunol 21:510–519 8. Tanimoto K, Cooper NR, Johnsons JS, Vaughan JH (1975) Complement fixation by rheumatoid factor. J Clin Invest 55:437–445 9. Wilson MR, Arroyave CM, Miles L, Tan EM (1977) Immune reactants in cryoproteins. Relationship to complement activation. Ann Rheum Dis 36:540–548 10. Poskitt TR, Poskitt PK (1979) Temperature dependent activation of the alternate complement pathway by an IgG cryoglobulin. Am J Hematol 7:147–154 11. Tarantino A, Anelli A, Costantino A et  al (1978) Serum complement pattern in essential mixed cryoglobulinaemia. Clin Exp Immunol 32:77–85 12. Greenstein JD, Peake PW, Charlesworth JA (1996) The metabolism of C9 in normal sbjects and in patients with autoimmune disease. Clin Exp Immunol 104:160–166 13. Ohsawa I, Ohi H, Tamano M et al (2001) Cryoprecipitate of patients with cryoglobulinemic glomerulonephritis contains molecules of the lectin complement pathway. Clin Immunol 101:59–66 14. D’Amico G, Colasanti G, Ferrario F, Sinico RA (1989) Renal involvement in essential mixed cryoglobulinemia. Kidney Int 35:1004–1014 15. Beddhu S, Bastacky S, Johnson JP (2002) The clinical and morphologic spectrum of renal cryoglobulinemia. Medicine (Baltimore) 81:398–409 16. Weiner SM, Prasauskas V, Lebrecht D et  al (2001) Occurrence of C-reactive protein in cryoglobulins. Clin Exp Immunol 125:316–322 17. Schifferli JA, Taylor RP (1989) Physiological and pathological aspects of circulating immune complexes. Kidney Int 35:993–1003 18. Miller GW, Nussenzweig V (1975) A new complement function: solubilization of antigen-antibody aggregates. Proc Natl Acad Sci USA 72:418–422 19. Takahshi M, Tack BF, Nussenzweig V (1977) Requirements for the solubilization of immune aggregates by complement; assembly of a factor B-dependent C3-convertase on the immune complexes. J Exp Med 145:86–100 20. Schifferli JA, Amos N, Pusey CD et al (1983) Metabolism of autologous and homologous IgG in patients with mixed essential cryoglobulinemia type II. Absence of fast elimination of IgG. Clin Exp Immunol 51:305–316 21. Ng YC, Peters DK, Walport MJ (1988) Monoclonal rheumatoid factor-IgG immune complexes: poor fixation of opsonic C4 and C3 despite efficient complement activation. Arthritis Rheum 31:99–107 22. Schifferli JA, Ng YC, Estreicher J, Walport MJ (1988) The clearance of tetanus toxoid- anti tetanus toxoid immune complexes from the circulation of humans: ­complement- and

10  The Complement System in Cryoglobulinemia erythrocyte CR1-dependent mechanisms. J Immunol 140: 899–904 23. Madi N, Steiger G, Estreicher J, Schifferli JA (1991) Defective immune adherence and elimination of hepatitis B surface Ag/Ab complexes in patients with mixed essential cryoglobulinaemia type II. J Immunol 147:495–502 24. Schifferli JA, Steiger G, Polla L et al (1985) Activation of the alternative pathway of complement by skin immune deposits. J Invest Dermatol 85:407–411 25. Watanabe N, Akikusa B, Park SY et  al (1999) Mast cells induce autoantibody-mediated vasculitis syndrome through tumor necrosis factor production upon triggering Fcgamma receptors. Blood 94:3855–3863 26. Guo S, Mühlfeld AS, Wietecha TA et al (2009) Deletion of activating Fcgamma receptors does not confer protection in murine cryoglobulinemia-associated membranoproliferative glomerulonephritis. Am J Pathol 175:107–118

89 27. Trendelenburg M, Fossati-Jimack L, Cortes-Hernandez J et  al (2005) The role of complement in cryoglobulininduced immune complex glomerulonephritis. J Immunol 175:6909–6914 28. Muhlfeld AS, Segerer S, Hudkins K et  al (2004) Overexpression of complement inhibitor Crry does not prevent cryoglobulin-associated membranoproliferative glomerulonephritis. Kidney Int 65:1214–1223 29. Wietecha TA, Hudkins KL, Iyoda M et al (2006) Deletion of murine factor B in thymic stromal lymphopoietin mice aggravates cryoglobulin-associated membranoproliferative glomerulonephritis. J Am Soc Nephrol 17:F-PO820 30. Wietecha TA, Hudkins KL, Iyoda M et al (2007) Inhibition of complement pathways of the murine protein crry and deletion of factor B in thymic stromal lymphopoietin mice aggravates cryoglobulin-associated membranoproliferative glomerulonephritis. J Am Soc Nephrol 18:SA-PO317

The Pivotal Role of C1qR in Mixed Cryoglobulinemia

11

Domenico Sansonno, Loredana Sansonno, and Franco Dammacco

11.1

Introduction

Cryoglobulinemic tissue damage is likely the consequence of a pathogenetic noxa that acts upon the host’s immune system, resulting in an altered regulation of the peripheral immune response [1]. Recent data have clearly established that the structural composition of cold-precipitating immune complexes (ICs) includes HCV core protein as the relevant ligand [2]. HCV nucleocapsid, devoid of enveloped proteins, has been detected in the bloodstream of HCV-infected patients and is a good indicator of circulating viral load, possibly reflecting overproduction during virogenesis. Non-enveloped HCV core protein was shown to be secreted by transfected hepatoma cell lines in culture and in HCV transgenic mice [3]. The core protein has been detected in the serum of most HCV chronic carriers with active liver disease and almost half of those with inactive disease. In addition, serum levels of HCV core protein change following antiviral therapy and become undetectable in responsive patients [4]. In HCV-related cryoglobulinemia, HCV core protein is cold-precipitated in the context of ICs and its colddependent insolubility seems to be the result of IgM rheumatoid factor (RF), which acts as an incomplete cryoglobulin [2]. The complement system is highly activated in patients with HCV-related cryoglobulinemia [5].

D. Sansonno (*) Department of Biomedical Sciences and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

Normal mean levels of C3 and C4 in the soluble phase correspond to very low amounts (if any) in the cryoprecipitate, suggesting the existence of two virtually distinct microenvironments in which complement is differentially activated. Complement is known to be a major interdependent regulator of IC size and composition and its binding to nascent ICs may decrease their size and maintain them in solution [6]. Compared with supernatant, significant differences in C1q and C1qbinding activity have been shown in insolubilized ICs [2]. Efficient engagement of C1q protein by cryoglobulins may be an important pathogenetic mechanism involved in the cryoglobulin-related pathway. HCV core protein has been shown to interact directly with the globular domain of the C1q receptor (gC1qR) [7]. This HCV core–gC1qR interaction is assumed to play a critical role in modulating the T-cell immune response [8]. HCV core-induced inhibition of T cell responsiveness may underlie the pathogenetic process that blocks the suppression of B-cell clones producing RF autoantibodies, generated by chronic antigenic challenge in HCV-related type II cryoglobulinemia [9]. Nonetheless, engagement of circulating HCV core protein with gC1qR displayed on the surface of B lymphocytes [10] can provide the virus with a direct means of alterin host immunity. The wide expression of gC1qR on the surface of both circulating blood cells and endothelial cells [11] favors their specific binding to HCV core-protein-containing ICs. HCV core deposition has indeed been reported in the skin [12] and kidney [13] of cryoglobulinemic patients, suggesting that HCV core-gC1qR interactions play a relevant role in cryoglobulin-related damage and in IC formation and cryoprecipitation.

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_11, © Springer-Verlag Italia 2012

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11.2

D. Sansonno et al.

gC1qR-HCV Core Interaction

The 33-kDa gC1qR is an acidic protein expressed on somatic cells. It binds to the globular heads of C1q and modulates complement activation [14]. Apart from its specific interaction with C1q, gC1qR binds to numerous cell-surface proteins, such as kininogen [15], vitronectin [16], nucleus-related like TFII B [17], lamin B receptor [18], splicing factor-2 (SF2) [19], mitochondrial-related cytochrome b2 [20], and BH3 only protein Hrk [21]. In addition to cellular proteins, gC1qR interacts with several bacterial and viral pathogenic proteins, such as adenovirus core protein [22] and HIV rev [23], suggesting that it is a part of the system that imports proteins to the cell. gC1qR is highly expressed on endothelial cells, implying a major role in cryoglobulinemic vascular damage. The binding of kininogen to gC1qR on the endothelial cell surface has been shown to serve as a platform for the assembly and activation of the intrinsic coagulation cascade that leads to the generation of bradykinin and thus to infiltration of vascular tissue by proinflammatory cells [24]. It was previously determined that HCV core protein binds the gC1qR region spanning amino acids 188– 259, while the interaction site on the core protein encompasses residues 26–124 [7]. Additionally, C1q examer, a highly positively charged molecule, binds, via its globular head complex, the NH2-terminal portion of gC1qR, spanning amino acids 74–95 [25]. Potential insights into the structural basis of these interactions may derive from the reported crystal structure of gC1qR, which revealed that three gC1qR molecules form a doughnut-shaped quaternary structure, with a sizable central channel and an asymmetric charge distribution on the surface, including exposed acidic residues in the COOH-terminal portion of the molecule and in the NH2-terminal a-helical domain. These negatively charged residues represent the binding site for the positively charged HCV core protein [26]. Data obtained from experiments performed in our laboratory indicate that the amount of gC1qR shed in the plasma as bioactive molecules is significantly higher in cryoglobulinemic patients than in HCVinfected patients without cryoglobulinemia or in healthy subjects. It has also been noted that there is a positive correlation between circulating gC1qR, plasma levels of IgM with RF activity, and C1q concentration in patients with mixed cryoglobulinemia

HCV CORE PROTEIN gC1qR C1q PROTEIN

IgM - RF

Fig. 11.1 Model of circulating immune complexes comprising gC1qR bound to HCV core protein and C1q protein, which in turn binds IgM with RF activity. These complexes possibly represent a portion of the spectrum of the cryoprecipitating immune complexes in MC patients

(MC). By contrast, no relation has been observed with circulating viral load, levels of non-enveloped HCV core protein, liver histology activity index, grade of liver fibrosis, and serum alanine aminotransferase levels. Soluble gC1qR circulates as a complexed form containing both C1q and HCV core proteins simultaneously bound to IgM molecules with RF activity. The nature and target of IgM binding have not been ascertained. However, according to preliminary data, IgM RF is not complexed with IgG molecules, suggesting that it is not likely directed to the Fc portion of IgGcontaining ICs. Instead, it can be inferred that IgM RF interacts directly with C1q, based on reports that the C1q domain interacts with IgM via the top of the molecule [27]. C1q has a rigid structure and is composed mainly of b-sheets. Interaction with IgM seems to be mediated by the globular head module B, due to its equatorial position [28]. A model of circulating ICs containing C1q, HCV core protein, and soluble gC1qR bound to IgM molecules is depicted in Fig. 11.1. In HCV-infected patients without MC, it was found that gC1qR simultaneously binds C1q and HCV core proteins, but not IgM molecules. These observations provide valuable information for understanding and dissecting the functions of gC1qR and related proteins, and point to a major impact of IgM RF in the activation of the complement system [29]. It has been proven that single IgM molecules activate the complement classical pathway by binding and activating the C1q fraction [30].

11

The Pivotal Role of C1qR in Mixed Cryoglobulinemia

93

Fig. 11.2 Localization of HCV core (a) and C4d protein (b) in a skin biopsy from an HCV-positive MC patient. Note that core protein is mainly located within the vessel lumen, whereas C4d is found along the vessel walls

11.3

gC1qR-HCV Core-Mediated Complement Activation

Binding to C1q protein is the primary requisite for the activation of complement via the classical pathway; this can be incontrovertibly demonstrated by measuring complement fragments generated during proteolytic cleavage of the C4 fraction. Indeed, C4d fragment is regarded as a reliable marker of complement activation [31] and C4d levels have been found in the sera of most patients with rheumatoid arthritis, an immune complex disease in which complement is activated via the classical pathway [32]. Median serum C4d levels are significantly higher in healthy controls than in MC patients but lower than in HCV-infected patients without MC. Lower levels of circulating C4d fragment in MC patients may indeed reflect its diversion near the sites of C4 activation. Thus, it seems possible that C4d fragment is detectable in different biological compartments, where C4 is involved in immune complex-mediated reactions. To test this hypothesis, skin biopsy samples from patients with cryoglobulinemic vasculitis were explored for the presence of HCV core and C4d deposits by indirect immunohistochemistry [33]. As shown in Fig. 11.2, both HCV core protein and C4d fragment were demonstrated in vascular structures, specifically, the vessel lumen, endothelium, and lamina propria. HCV core engagement of gC1qR on lymphocytes limits the induction of Th1 responses and may contribute to viral persistence [34]. In vitro effects of HCV core protein on lymphocytes from patients with and without

MC and from healthy subjects were shown to include the extensive inhibition of lymphocyte proliferation, ranging from 50% to 90%. Surprisingly, in the same samples, gC1qR concentrations are greatly increased in the supernatants of HCV-related MC patients, whereas a very limited increment was demonstrable in the nonMC group and no significant variations were found in healthy controls. These findings strongly support the contention that there is an intrinsic difference in the regulation and secretion of lymphocyte-derived gC1qR by HCV core proteins in MC patients. In line with higher gC1qR expression on lymphocytes of HCV-related MC patients, quantitative realtime RT-PCR confirmed the occurrence of greater amounts of gC1qR mRNA in the cells of these patients. Specific gC1qR mRNA expression was at least 6- to 10-fold higher in cells from MC patients than in those from the non-MC group and healthy controls. In vitro experiments showed that gC1qR mRNA expression progressed in a dose-dependent manner to the HCV core-induced suppressive effect of cell proliferation, raising the possibility that the magnitude of gC1qR expression is predisposed by an intrinsic difference in gC1qR gene regulation in patients with MC.

11.4

Conclusions

Soluble gC1qR may have a deep impact on the clinical features in patients with MC for several reasons, including the fact that it acts as a bridging molecule responsible for linking apoptotic cells is able to activate

94 Fig. 11.3 Pathogenetic model of cryoglobulinemic tissue damage. Complement regulates the size and composition of immune complexes, reducing their size and maintaining them in solution. Complement consumption resulting from C4 diversion contributes to vascular cryoglobulininduced damage by deposition of C1q-dependent immune complexes

D. Sansonno et al.

C4- HYPOCOMPLEMENTEMIA

C4 DIVERSION

CIRCULATING IMMUNE COMPLEXES

SKIN TISSUE DEPOSITS

HCV CORE PROTEIN

C4d gC1qR C1q PROTEIN

IgM

INSOLUBILITY OF CRYOPROTEINS

endothelial cells and platelets, and initiates the intrinsic blood coagulation and kinin-generating pathway [35]. These findings shed new light on our comprehension of the pathogenetic mechanisms underlying cryoglobulin-induced vascular damage. For example, soluble gC1qR may contribute to the vasculitic process by modulating complement activation and by enrichment of tissue deposits of HCV core protein. Measurement of circulating C4d has provided evidence of lower amounts in MC patients. Indeed, the exposure of a thioester group by split C4d leads to the formation of a covalent bond near the site of C4 activation [36]. Examination of skin biopsy tissues shows that C4d deposits are detectable in almost all MC patients, but in none of those without MC. Thus, low levels of circulating C4d in MC patients are likely the result of sequestered fragments. Furthermore, regression of cryoglobulinemic vasculitis, which has been observed following a successful therapeutic response, is characterized by a significant reduction of soluble gC1qR and a parallel increment of serum C4d fragment, strongly suggesting a disengagement of entangled C4 and inhibition of the C1 activation pathway. In this context, it seems reasonable to hypothesize a pathogenetic model whereby the dysregulated shedding of C1qR molecules modulates complement activation, which in turn leads to the consumption and deposition of C4d fragment-containing ICs within tissues, as summarized in Fig. 11.3.

Interestingly, HCV core protein generates C4d in serum depleted of C1q protein, indicating that it directly activates the complement cascade, likely via the mannan-binding lectin pathway. Thus, in the presence of high levels of circulating gC1qR, the HCV core protein can exacerbate the inflammatory condition by combined and simultaneous activation of both complement pathways. Under these conditions, endothelial cells are then activated with consequent initiation of a local inflammatory response. It has been shown that HCV core protein suppresses the host immune response by engaging gC1qR on the surface of immune cells. Core protein is readily detected in the plasma of HCV-infected patients with or without MC, and extensive inhibition of mitogenstimulated proliferation of peripheral blood lymphocytes can be demonstrated. In vitro experiments have indeed defined a unique property of MC patients, in that large amounts of soluble gC1qR are released in culture supernatants in step with HCV core inhibition of the lymphocyte proliferative response. Very limited increments were noted in non-MC patients and no changes from basal levels were demonstrated in healthy subjects. This strongly indicates that the mechanisms underlying gC1qR synthesis and release from lymphocytes are HCV core-mediated and negatively regulated by cell proliferation. No direct relation was found between the levels of non-enveloped HCV core protein and those of soluble gC1qR in patients

11

The Pivotal Role of C1qR in Mixed Cryoglobulinemia

with or without MC, indicating that the circulating amount of viral protein is not a critical factor. Why gC1qR expression levels differ in patients with as compared to those without MC is as yet unknown, but it might be the result of transcriptional control of the gC1qR gene and a genetic difference in the host gene regulation. Indeed, higher levels of soluble gC1qR are demonstrable in HCV-positive and HCV-negative MC patients. Quantitative real-time RT-PCR assay for specific gC1qR mRNA has shown that expression is 6- to 14-fold higher in cells from MC than in those from non-MC patients. These results parallel those obtained from analyses of gC1qR protein expression on lymphocytes. The percentages of cells expressing gC1qR in MC patients are 3- to 4-fold higher than those found in non-MC patients, suggesting that synthesis and release of gC1qR protein in cells is dependent on upregulation of the gC1qR gene. At variance from previous data showing that agents or proteins inducing cell proliferation are able to increase the amount of gC1qR in the surrounding milieu [37], these observations indicate that release of soluble gC1qR is regulated by core-mediated inhibition of cell proliferation. Moreover, they suggest that a novel mechanism capable of modulating gC1qR expression is operative. However, further investigations are needed to determine the precise activating conditions which result in its HCV core-mediated release. Once released, this receptor regulates complement activation and consumption of the C4 fraction in vascular bed and deposition of C4d fragment-containing ICs within tissues. In our proposed model, gC1qR initiates kinin generation as well as clotting on the endothelial surface via the contact system, resulting in damage to the vessel walls [38]. A genetic polymorphism of the gC1qR gene could explain dysregulation of its soluble levels in MC patients. However, thus far there are no such data and whether a gC1qR high-producer allele is associated with MC remains to be ascertained.

References 1. Saadoun D, Rosenzwajg M, Landau D et al (2008) Restoration of peripheral immune homeostasis after rituximab in mixed cryoglobulinemia vasculitis. Blood 111(11):5334–5341 2. Sansonno D, Lauletta G, Nisi L et al (2003) Non-enveloped HCV core protein as constitutive antigen of cold-precipitable immune complexes in type II mixed cryoglobulinaemia. Clin Exp Immunol 133(2):275–282

95 3. Sabile A, Perlemuter G, Bono F et al (1999) Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates. Hepatology 30:1064–1076 4. Kurtz JB, Boxall F, Qusir N et al (2001) The diagnostic significance of an assay for ‘total’ hepatitis C core antigen. J Virol Methods 96:127–132 5. Gorevic PD, Frangione B (1991) Mixed cryoglobulinemia cross-reactive idiotypes. Implications for the relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 28:79–94 6. Lindahl G, Sjobring U, Johnsson E (2000) Human complement regulators: a major target for pathogenic microorganisms. Curr Opin Immunol 12:44–51 7. Kittlesen DJ, Chianese-Bullock KA, Yao ZQ et al (2000) Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation. J Clin Invest 106:1239–1249 8. Yao ZQ, Nguyen DT, Hiotellis AI et al (2001) Hepatitis C core protein inhibits human T lymphocyte responses by a complement-dependent regulatory pathway. J Immunol 167: 5264–5272 9. Dammacco F, Sansonno D, Piccoli C et al (2000) The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinaemia, and overt B-cell malignancy. Semin Liver Dis 20:143–145 10. Yao Z-Q, Prayter D, Trabue C et al (2008) Differential regulation of SOCS-1 sognalling in B and T lymphocytes by hepatitis C virus core protein. Immunology 2:197–207 11. Lim BL, Reid KBM, Ghebrehiwet B et al (1996) The binding for globular heads of complement C1q, gC1qR. Functional expression and characterization as a novel vitronectin binding factor. J Biol Chem 271:26739–26744 12. Sansonno D, Cornacchiulo V, Iacobelli AR et al (1995) Localization of hepatitis C virus antigens in liver and skin tissues of chronic hepatitis C virus-infected patients with mixed cryoglobulinaemia. Hepatology 21:305–312 13. Sansonno D, Gesualdo L, Monno C et al (1997) Hepatitis C virus-related proteins in kidney tissue from hepatitic C virusinfected patients with cryoglobulinemic membranoproliferative glomerulonephritis. Hepatology 25:1237–1244 14. Ghebrehiwet B, Lim B-L, Kumar L et al (2001) gC1q-R/ p33: a member of a new class of multifunctional and multicompartimental cellular proteinsis involved in inflammation and infection. Immunol Rev 180:65–77 15. Joseph K, Ghebrehiwet B, Peerschke EI et al (1996) Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular “heads” of C1q (gC1q-R). Proc Natl Acad Sci U S A 93: 8552–8557 16. Lim BL, Reid KBM, Ghebrehiwet B et al (1996) The binding protein for globular heads of complement C1q, gC1qR. Functional expression and characterization as a novel vitronectin binding factor. J Biol Chem 271:26739–26744 17. Paul DB, Kuhns MC, McNamara AL et al (1995) Short-term stability of HIV provirus levels in the peripheral blood of HIV-infected individuals. J Med Virol 47:292–297 18. Simos G, Georgatos SD (1994) The lamin B receptor-associated protein p34 shares sequence homology and antigenic determinants with the splicing factor 2-associated protein p32 [letter]. FEBS 346:225–228

96 19. Krainer AR, Mayeda A, Kozak D et al (1991) Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70 K, and drosophila splicing regulators. Cell 66:383–394 20. Seytter T, Lottspeich F, Neupert W et al (1998) Mam33p, an oligomeric, acidic protein in the mitochondrial matrix of Saccharomyces cerevisiae is related to the human complement receptor gC1q-R. Yeast 14(4):303–310 21. Sunayama J, Ando Y, Itoh N et al (2004) Physical and functional interaction between BH3-only protein Hrk and mitochondrial pore forming protein p32. Cell Death Differ 11:771–781 22. Matthews DA, Russell WC (1998) Adenovirus core protein V interacts with p32 a protein which is associated with both the mitochondria and the nucleus. J Gen Virol 79: 1677–1685 23. Luo Y, Yu H, Peterlin BM (1994) Cellular protein modulates effects of human immunodeficiency virus type I Rev. J Virol 68:3850–3856 24. Joseph K, Ghebrehiwet B, Kaplan A-P (2001) Activation of the kininforming cascade on the surface of endothelial cells. Biol Chem 382:71 25. Peerschke EI, Ghebrehiwet B (1988) Identification and partial characterization of human platelet C1q binding sites. J Immunol 141(10):3505–3511 26. Jiang J, Zhang Y, Krainer AR et al (1999) Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Proc Natl Acad Sci USA 96(7):3572–3577 27. Zlatarova AS, Rouseva M, Roumenina LT et al (2006) Existence of different but overlapping IgG- and IgM-binding sites on the globular domain of human C1q. Biochemistry 45(33):9979–9988 28. Gaboriaud C, Jaunhuix J, Gruez A et al (2003) The crystal structure of globular head of complement protein C1q pro-

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Part IV Structural and Genetic Features, Cytokines and Chemokines in Cryoglobulinemia

Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance

12

Peter D. Gorevic

12.1

Introduction

The early history of mixed cryoglobulinemia (MC) included recognition of a bias toward oligoclonality or monoclonality apparent in either the IgM or IgG constituents of mixed cryoglobulins. In particular, IgM rheumatoid factor (RF) was found to be strikingly (>95%) skewed toward the utilization of k light chains, leading to the designation “type 2 cryoglobulinemia.” The percent contribution of type 2 cryoglobulinemia has increased in large series as the use of immunofixation has become routine in most clinical laboratories and with the recognition of the increased incidence of clonal IgMk RF associated with hepatitis C virus (HCV) infection and with primary SS. Intermediate forms of type 2 cryoglobulins have been described, spanning the spectrum between polyclonality and monoclonality [1], but are not necessarily part of a continuum that represents a temporal evolution of the immune reaction to HCV antigens [2]. The clonality of IgMk was confirmed by sequence analysis of Vk at the protein level, demonstrating conservation of hypervariable (CDR) and framework (FR) sequences among cryoglobulin IgMk proteins isolated from unrelated individuals. These studies focused on the light chain because of the relative ease of carrying out protein

P.D. Gorevic Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA e-mail: [email protected]

sequence analysis of k light chains whereas there is considerably less information regarding m VH protein sequences. Conservation of Vk and VH amino acid sequences provided, in part, a basis for the recognition of cross-reactive idiotypes (CRIs) among cryoglobulin IgMRF, as had been established serologically with polyclonal antibodies (Wa, Po, and later Bla). The latter were utilized to define broad subsets of cross-reactivity among type 2 cryoglobulins and correlated predominantly (but not exclusively) with light or heavy chain antigenic determinants. Further refinement of these CRIs came from the development of monoclonal antibodies (MAbs) to MC CRIs (17.109; 6B6.6; G6; Lc1; JG-B1; B6 and G8) and the use of synthetic peptides corresponding to conserved CDR2 (PLS2) and CDR3 (PSL3) sequences of mixed cryoglobulins to generate additional polyclonal reagents. The composite value of these antibodies for the definition of MC CRIs was to establish that: (a) cross-idiotypy may be a function of structural determinants of the variable region of IgMk, which may encompass the light and/or heavy chain, FR and CDR sequences, and even the antigen-binding site, and (b) MCI CRIs are significantly shared with primary SS and with specific lymphoproliferative diseases, some of which may be associated with MC or SS. The purpose of this chapter is to update an earlier review of MC CRI [3] to include the association of MC and certain lymphoproliferative diseases with HCV infection/exposure and to incorporate nomenclature for V-region genes that has been made possible by full sequencing of the VH and Vk gene loci [4].

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_12, © Springer-Verlag Italia 2012

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12.2

P.D. Gorevic

Further Studies of IgM RF Cross-Reactive Idiotypes

The polyclonal antibody Wa recognizes ~80% of monoclonal (m)IgMk mixed cryoglobulins. Its specificity is predominantly directed to the heavy chain but is also dependent on pairing with specific VkIII light-chain products. The VH specificity is directed to either VH1 (V1-69/DP-10/51p1) or VH3 (V3-7/ DP-53) heavy chains, the former coexpressed with V3-20 and the latter with V3-15k V-region genes; that is, there is a skewing of the combinatorial pairing of specific heavy- and light-chain V-region genes that is characteristic of type 2 cryo-mIgMk. Both Vk3 genes utilize a Jk1 joining segment for expression. These VH1 genes preferentially make use of the D3-22 diversity gene segments, with VH1-69 rearranging to JH4 and D region consensus 1, and V3-7 rearranging to JH3 or JH4 gene segments and D region consensus 2 [1]. B6 is an IgG1k MAb raised against a human IgM RF paraprotein; it is reactive with 24% of mRF (k) and 15% of non-RF mIgM, with predominantly H-chain specificity. Further analysis indicated that this MC CRI is encoded in a set of closely related (V3-11/DP-35/22-2B, V3-7, V3-30/DP-49,1.9III/ hv3005, V3-30.3/DP-46/56p1/GL-SJ2 and V3-33/ DP-50/3019b9) genes from the VH3 family, with crucial involvement of the lysine at position 57 of CDR2. Increased expression of the CRI was identified in primary SS but not in chronic lymphocytic leukemia (CLL) [3, 5]. G6 and G8 are MAbs produced in response to an IgMk RF. They are reactive with 35% of mRF (k) and 5% and 0% of non-RF mIgM, respectively, both with predominantly H-chain specificity. Further analysis indicated that these MC CRIs are encoded by the VH169 multiallelic gene locus and are directed to conformational epitopes requiring pairing with kIIIb (A27/ humkv325/DPK22) light chains. G6 and G8 bind distinct and overlapping allelic variants comprising the gene segment 51p1 (VH783) at the VH1-69 locus, with major idiotopes mapping to CDR2 but also involving FR1 and FR3, which are topographically related to the antigen-binding site. Increased expression of the CRIs for both these MAbs was identified in marginal B cells of lymphoid follicles and primary SS as well as in 20–22% patients with CLL and 13% of patients with lymphoma [3, 6].

12.3

Mixed Cryoglobulinemia Cross-Reactive Idiotypes and HCV Infection

In most series, 60–80% patients with MC are productively infected by HCV, and up to 60% of patients with HCV have cryoprecipitable and non-cryoprecipitable immune complexes, with both the laboratory phenomenon and overlapping syndromes of vasculitis, glomerulonephritis, arthropathy, and neuropathy correlating with disease that is more severe and of longer duration. In addition, a direct role for HCV, as well the immune response to virus and nucleocapsid, is indicated by the concentration of antibodies to HCV antigens, notably including core antigen and viral nucleic acid binding predominantly to the IgG fraction of mixed cryoglobulins [7]. Cc1 and Lc1 are MAbs produced to different mRFs that react with H chain determinants associated with VH1 and VH4 gene products, respectively. Cc1 is reactive with ~70% of mIgMk RF and Lc1 with ~25%. Both CRIs are significantly expressed in patients with primary SS [8] and among patients infected by HCV. These MAbs are reactive with MC CRIs expressed on the surface of B cells and (particularly Cc1) have been used to purify subsets in which HCV RNA could be demonstrated by RT-PCR [9].

12.4

Clonal Populations of B Cells in Liver, Peripheral Blood, and Bone Marrow in HCV Infection

In situ hybridization and sequencing of CDRH3 gene segments showed that oligoclonal or monoclonal expansions of B cells accounted for ~50% of the lymphoid follicles isolated by microdissection of the livers of HCV-infected patients. In addition, the presence of clonal RF-producing cells could be demonstrated by analysis of gene product. Similar expansions were also identified in bone marrow (6%) and peripheral blood (26%) [10, 11]. Three types of monoclonal B-cell lymphocytosis (MBL) have been identified by FACS analysis in the peripheral blood of ~30% patients infected by HCV: “atypical” CLL MBL (CD5+CD20bright), CLL-like (CD5bright, CD20dim), and CD5-MBL (<20% total). The incidence of these three types is increased relative to the general population, with a further increase when

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Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance

HCV progresses to cirrhosis and hepatocellular carcinoma. Evaluation of monoclonal V-region gene rearrangements from these cases confirmed both a bias of gene usage corresponding to that seen in non-Hodgkin lymphoma (NHL) complicating HCV and a somatic hypermutation suggestive of antigen selection [12]. The Wa CRI is expressed within the cryoprecipitates of patients with type 2 (but not type 3) cryoglobulinemia and HCV infection but may also be detected on the surface of peripheral B cells. Together with FACS analysis, it can be used to identify ~56% clonal B-cell expansions in HCV-infected individuals. In the peripheral blood, expansion of IgM+k+IgDlow/negCD21+ B cells, predominantly encoding VH1-69 and Vk3-20 or Vk3-15 gene segments has been shown; some of these cells prominently express the Wa CRI [13]. Other studies have implicated a preferential activation of naive B lymphocytes via CD81 as a mechanism for the induction of proliferation and hypermutation [14]. In patients with HCV infection and cryoglobulinemia, G6, G8, and 17.109 MAbs have been utilized to delineate the immunophenotype of B cells at various stages of differentiation that are expressing VH1-69 and kv325 gene products [15]. In that study, the B cells expanded in HCV infection were found to have a “memory” phenotype, in some cases replacing (but not increasing) the B-cell pool. Three patients with MC and splenic lymphoma were shown to have an absolute monoclonal VH1-69+ B lymphocytosis that persisted after regression of the lymphoma with antiviral therapy [15].

12.5

MC CRI and Lymphoproliferative Disease

12.5.1 Primary Sjögren’s Syndrome Lymphoma may complicate 7.5% cases of primary SS, and cryoglobulinemia is a significant predictive risk factor for its development. Salivary extranodal marginal B-cell lymphomas of the mucosa-associated lymphoid tissue (MALT)-type predominate, with diffuse large-cell lymphomas accounting for an additional 17.5% [16]. MAbs with predominantly light chain specificity (17.109, 6B6.6) were used to show that the majority of IgMk-producing B cells in primary SS express VH1 in conjunction with A27 (hum kv325/ Vk3-20) or, less commonly, VH4 in association with

101

L2 or L16 (hum kv328Vk3-15) [3]. V-region gene sequencing of monoclonal non-neoplastic proliferations of salivary B cells in patients with primary SS showed considerable overlap with the gene utilization apparent in HCV-associated NHL [17], very similar to the overlap of V-region gene usage noted for MBL associated with HCV [12]. These results contrast somewhat with an analysis of germline and rearranged V-region gene utilization and hypermutation analysis [18] by single-cell PCR of CD27+ and CD27- B cells from patients with primary SS, in some instances comparing B cells obtained from the inflamed parotid gland to those sorted from peripheral blood. Although these studies revealed increased expression of the light chain A27 gene in salivary gland relative to peripheral blood, this was not apparent for genes associated with B6, i.e., the heavy chain MC CRI shown in previous studies to be significantly expressed in glandular tissue of patients with primary SS [5, 18].

12.5.2 Hepatitis C Virus The structural restrictions noted above for the definition of the Wa CRI were used to analyze a series of published immunoglobulin V region genes for lymphomas occurring in HCV-infected patients. These studies predicted a 24% incidence of Ig gene products meeting the criteria for Wa reactivity in HCV-associated NHL [19]. Although it has been suggested that the E2 envelope antigen drives the expansion of Wa-positive B-cell clones [20] as well as somatic hypermutation by activation of cytidine deaminase [21], there did not appear to be sequence homology with clones exhibiting anti-E2 antibody activity or any indication of crossreactivity between Wa-positive mIgMk with the E2 antigen [1]. In one case, Wa CRI accounted for ~80% of the lymphocytosis noted in the peripheral blood of an HCV-infected patient with features of splenic lymphoma. The antigen was coexpressed with phenotypic markers for marginal zone cells, both of which regressed with antiviral therapy [22]. The expression of these antigens has been correlated with an association between marginal zone lymphomas and HCV and a 10-fold increase in the risk of developing NHL of the liver and salivary glands. In early studies, amplification of HCV-associated immunocytomas demonstrated somatic hypermutation,

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clonal diversity, and preferential expression of the VH51p1 (VH1-69)/VL kv325 gene segments [23]. Subsequently, HCV-positive NHL malignant cells were found to employ the same V-region gene sequences as the IgMRF-producing cells in MC; among five marginal zone lymphomas, three utilized a VH1-69 gene joined with D3-22 and JH4 segments with conservation of CDR sequences, suggestive of a common inciting antigen [24, 25]. A B-cell receptor repertoire shared between MC clonal B-cell proliferation and HCVassociated NHL includes V1-69, V3-7, and V4-59 VH genes and Vk3-20 and Vk3-15 k light chain genes with a limited hypermutation suggestive of a germinal center (GC) or post-GC origin [13, 17]. By contrast, the Vk315 and Vk3-20 genes, which have the highest CDR3 isoelectric point values among functional IgkV genes— a characteristic that has been correlated with the pathogenicity of autoantibodies (e.g., anti-DNA) causing disease—are underrepresented among follicular lymphomas/HCV [26]. Distinct biases of V-region gene usage distinguish splenic marginal zone lymphomas (MZL) from those cases of MZL and the diffuse large B-cell lymphomas complicating HCV infection [12].

12.5.3 MALT Lymphomas Primary SS or HCV infection may be associated with MALT lymphomas. A number of studies have shown a significant HCV seroprevalence among patients with diffuse large-cell lymphomas and MZLs. Among splenic cell, nodal, and extranodal MZLs, seroprevalences of 20%, 24%, and 35% have been reported [27]. In a large survey of B-receptor gene usage that included 24 cases of MALT lymphoma, it was noted that mantle cell lymphomas and CLL shared selected CDR3 homology that was not seen among a variety of other lymphomas. In addition, the MALT lymphomas were unique in sharing V-region gene usage employed by MC mIgMk clonal B cells. Whereas 18% of these MALT lymphomas overall exhibited this RF-associated gene usage pattern, including V1-69/JH4 gene rerrangements, the association was even more striking (41%) among the salivary gland (but not pulmonary) MALT lymphomas. In vitro expression of seven out of 14 MALT antibodies was found to correlate with RF-binding activity at the protein level [28].

P.D. Gorevic

12.5.4 Therapy Regression of clonality correlating with successful clearance of virus has been taken as indicative of an inciting role for HCV in the genesis of mRFk and may be reflected in the display of MC CRI on B-cell subsets in peripheral blood [15]. However, both the laboratory and clinical manifestations of cryoglobulinemia may persist even after a complete response to antiviral treatment, possibly due to residual lymphoproliferative disease [29]. The leukemic form of splenic lymphomas associated with villous lymphocytosis may regress following antiviral therapy, in some instances accompanied by the loss of clonal CRI on peripheral blood mononuclear cells and in others with persistence of CRI-bearing memory B cells, as demonstrated by FACS analysis [15, 22]. Cytoreduction of CD20 with MAbs has been examined as both monotherapy and combined with antiviral therapy with pegylated interferon-a and ribavirin. Depletion or disappearance of peripheral clones has been found in responders, as well as conversion from oligoclonality to polyclonality in liver and bone marrow, which may persist for up to 3 years following successful retreatment [30]. This is also reflected in the V-region repertoire of CRI-bearing B-cell subsets, again as determined by reactivity with the G6 Mab [31]. The persistence of clonal B cell expansions and of cryoglobulinemia following apparent clearance of virus has been linked to NHL in some instances, with systemic vasculitis in patients without detectable cryoglobulinemia linked to other (e.g., T cell) immune-mediated mechanisms triggered by the virus [32]. The therapeutic potential for antibodies reactive with idiotypic determinants has only been addressed in limited studies. Early reports documented the ability of anti-idiotypes to suppress B-cell RF production in vitro [3]. Since cryocomplex formation requires the binding of IgMk to IgG, definition of the antigen-binding site and specific antigenic determinants to which RF activity is directed might provide an additional therapeutic strategy to prevent these complexes from forming in vivo. The feasibility of this approach was demonstrated using the small molecule aspartame, which significantly inhibited the binding of three cryoIgMk to IgG in a calcium-dependent fashion [33]. Antibodies to MC CRIs, some of which (as noted

12

Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance

above) may significantly affect antigen binding or be directed to the antigen-binding site, might have a similar effect of interfering with cryocomplex formation. The G6 CRI was recently used to single-sort and clonally expand individual VH1-69/JH4/Vk3-20+ B-cells, the PCR product of which could be transfected into 293 T cells to obtain significant amounts of IgM with demonstrable RF activity. This system has the potential to evaluate shared epitopes for this paired gene usage in NHL and CLL and to assess structural determinants central to antiviral activity, binding to IgG, and requisite for cryocomplex formation [34]. Alternative strategies that have been considered include vaccination by pulsing dendritic cells with cryoglobulin [35], generation of CRI-specific MAbs for passive immunization, or the development of an idiotype vaccine [36].

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origin for the CRI-producing B cell and that some CRIs have an affinity for the antigen-binding site, a property that could be exploited to define the determinants to which these antiglobulins are directed and for therapeutic purposes. Other antibodies appear to be significantly influenced by conformational antigenic determinants, in which a CRI H- or L-chain specificity are only seen within the context of specific L- or H-chain pairing and may allow the possibility of crossreactivity with other non-IgG (e.g., FcR, HCV) antigens. A research agenda would thus include further analysis of: (a) the expression of these MC CRIs among specific B-cell subsets, (b) trafficking to tissues in which clonality is apparent and in which lymphoproliferation may occur, and (c) longitudinal studies in HCV-infected individuals.

References 12.6

Discussion

The complexity of the shared idiotypes that exist between mixed (IgM-IgG) cryoglobulins largely involves the variable region of the IgMk RF constituent that defines most type 2 mixed cryoglobulins. CRI encompass shared primary variable sequences of the m H and k L chains and are reflected in both the CDR and FR regions as well as the use of specific joining and diversity segments. MC occurring in association with HCV infection or primary SS shares selective usage of specific V-region H-chain (V1-69; V3-7; V4-59) and/or L-chain (V3-20; V3-15) genes, the expression of which has been correlated with polyclonal (Wa) or monoclonal (Ccl-1; Lcl-1; G6; G8; 17.109) antibodies to MC CRIs expressed on CD27+ memory B cells in the peripheral blood of patients productively infected by HCV. This has been corroborated by gene sequencing of V-region genes expressed by MZLs, MALT, or splenic lymphomas that may complicate MC, HCV, or SS and correlate with RF production by malignant B cells in some of these lymphomas. This observation relates in part to the expression of these CRIs, recognized by MAbs in the latter two disorders apart from MC, particularly with regard to salivary gland tissue in SS and possibly hepatic lymphoid follicles in HCV. Additional CRIs, particularly those conserved among CDRs and evaluated as R/S substitutions by mutational analysis, suggest a GC or post-GC

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104 9. Fornasieri A, Bernasconi P, Ribero ML, Sinico RA, Fasola M, Zhou J, Portera G, Tagger A, Gibelli A, D’Amico GD (2000) Hepatitis C virus (HCV) in lymphocyte subsets and in B lymphocytes expressing rheumatoid factor cross-reacting idiotype in type II mixed cryoglobulinemia. Clin Exp Immunol 122:400–403 10. Sansonno D, Lauletta G, De Re V, Tucci FA, Gatti P, Racanelli V, Boiocchi M, Dammacco F (2004) Intrahepatic B-cell clonal expansions and extrahepatic manifestations of chronic HCV infection. Eur J Immunol 34:126–136 11. Vallat L, Benhamou Y, Gutierrez M, Ghillani P, Hercher C, Thibault V, Charlotte F, Piette JC, Poynard T, Merle-Béral H, Davi F, Cacoub P (2004) Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection. Arthritis Rheum 50:3668–3678 12. Fazi C, Dagklis A, Cottini F, Scarfo L, Bertilaccio MTS, Finazzi R, Memoli M, Ghia P (2010) Monoclonal B cell lymphocytosis in hepatitis C virus infected individuals. Cytometry B Clin Cytom 78B(Suppl 1):S61–S68 13. Charles ED, Green RM, Marukian S, Talal AH, Lake-Bakaar GV, Jacobson IM, Rice CM, Dustin LB (2008) Clonal expansion of immunoglobulin M + CD27+ B cells in HCVassociated mixed cryoglobulinemia. Blood 111:1344–1356 14. Rosa D, Saletti G, De Gregorio E, Zorat F, Comar C, D’Oro U, Nuti S, Houghton M, Barnaba V, Pozzato G, Abrignani S (2005) Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis c virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 102: 18544–18549 15. Carbonari M, Caprini E, Tedesco T, Mazzettta F, Tocco V, Casato M, Russo G, Fiorilli M (2005) Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis. J Immunol 174:6532–6539 16. Baimpa E, Dahabreh IJ, Voulgarelis M, Moutsopoulos HM (2009) Hematologic manifestations and predictors of lymphoma development in primary sjögren syndrome: clinical and pathophysiologic aspects. Medicine 88:284–293 17. De Re V, De Vita S, Gasparotto D, Marzotto A, Carbone A, Ferraccioli G, Boiocchi M (2002) Salivary gland B cell lymphoproliferative disorders in Sjögren’s syndrome present a restricted use of antigen receptor gene segments similar to those used by hepatitis C virus-associated non-Hodgkins’s lymphomas. Eur J Immunol 32:903–910 18. Dörner T, Hansen A, Jacobi A, Lipsky PE (2004) Immunglobulin repertoire analysis provides new insights into the immunopathogenesis of Sjögren’s syndrome. Autoimmun Rev 1:119–124 19. Knight G, Gao L, Gragnani L, Elfahal MM, De Rosa FG, Gordon FD, Agnello V (2010) Detection of Wa B cells in hepatitis C virus infection. A potential prognostic marker for cryoglobulinemic vasculitis and B cell malignancies. Arthritis Rheum 62:2152–2159 20. Chan CH, Hadlock KG, Foung SK, Levy S (2001) VH1-69 Gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B-cells responding to the E2 viral antigen. Blood 97:1023–1026

P.D. Gorevic 21. Machida K, Cheng KT, Pavio N, Sung VM, Lai MM (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79: 8079–8089 22. Casato M, Mecucci C, Agnello V, Fiorilli M, Knight GB, Matteucci C (2002) Regression of lymphoproliferative disorder after treatment for hepatitis C virus infection in a patient with partial trisomy 3, Bcl-2 overexpression and type II cryoglobulinemia. Blood 99:2259–2261 23. Ivanoski M, Silvestri F, Pozzato G, Anand S, Mazzaro C, Burrone OR, Efremov DG (1998) Somatic hypermutation, clonal diversity, and preferential expression of the VH51p1/ VL kv325 immunoglobulin gene combination in hepatitis C virus-associated immunocytomas. Blood 91:2433–2442 24. De Re V, De Vita S, Marzotto A, Rupolo M, Gloghini A, Pivetta B, Gasparotto D, Carbone A, Boiocchi M (2000) Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 96:3578–3584 25. Marasca R, Vaccari P, Luppi M, Zucchini P, Castelli I, Barozzi P, Cuoghi A, Torelli G (2001) Immunoglobulin gene mutations and frequent use of VH1-69 and VH4-34 segments in hepatitis C virus-positive and hepatitis C virusnegative nodal marginal zone B-cell lymphoma. Am J Pathol 159:253–261 26. Smilevska T, Tsakou E, Hadzidimitriou A, Bikos V, Stavroyianni N, Laoutaris N, Fassas A, Agagnostopoulos A, Papadaki T, Belessi C, Stamatopoulos K (2008) Immunoglobulin kappa repertoire and somatic hypermutation patterns in follicular lymphoma blood cells. Blood Cells Mol Dis 41:215–218 27. Arcaini L, Bruno R (2010) Hepatitis C virus infection and antiviral treatment in marginal zone lymphomas. Curr Clin Pharmacol 5:74–81 28. Bende RJ, Aarts WM, Riedl RG, Daphne De Jong, Pals ST, van Noesel CJM (2005) Among B cell non-Hodgkins lymphomas, MALT lymphomas express a unique autoantibody repertoire with frequent rheumatoid factor reactivity. J Exp Med 201:1229–1241 29. Landau DA, Saadoun D, Halfon P, Martinot-Peignoux M, Marcellin P, Fois E, Cacoub P (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58:604–611 30. Dammacco F, Tucci FA, Lauletta G, Gatti P, De Re V, Conteduca V, Sansonno S, Russi S, Mariggiò MA, Chironna M, Sansonno D (2010) Pegylated interferon-{alpha}, ribavirin, and rituximab combined therapy of hepatitis C virusrelated mixed cryoglobulinemia: a long-term study. Blood 116:343–353 31. Saadoun D, Resche Rigon M, Sene D, Terrier B, Karras A, Perard L, Schoindre Y, Coppéré B, Blanc F, Musset L, Piette JC, Rosenzwajg M, Cacoub P (2010) Rituximab plus Peginterferon-alpha/ribavirin compared with Peg-interferonalpha/ribavirin in hepatitis C-related mixed cryoglobulinemia. Blood 116:326–334

12 Mixed Cryoglobulinemia (MC) Cross-Reactive Idiotypes (CRI): Structural and Clinical Significance 32. Terrier B, Sene D, Deechartres A, Saadoun D, Ortonne N, Rouvier P, Musset L, Rigon MR, Maisonobe T, Cacoub P (2011) Systemic vasculitis in patients with hepatitis C virus infection with and without detectable mixed cryoglobulinia. J Rheumatol 38:104–110 33. Ramsland PA, Movafagh BF, Reichlin M, Edmundson AB (1999) Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester. J Mol Recognit 12:249–257 34. Charles ED, Orloff MIM, Dustin LB (2011) A flow cytometry-based strategy to identify andf express IgM from VH1-69+ clonal peripheral B cells. J Immunol Methods 363:210–220

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35. Maeda M, Inaba S, Nomura A, Tokunaga Y, Sugio Y, Itoh Y, Iino T, Otsuka T, Okamura S, Niho Y (2000) Vaccination of a refractory essential monoclonal cryoglobulinemia patient with cryoglobulin-pulsed dendritic cells. Leuk Lymphoma 39:441–446 36. de Re V, Simula MP, Pavan A, Garziera M, Marin D, Dolcetti R, de Vita S, Sansonno D, Geremia S, Toffoli G (2009) Characterization of antibodies directed against the immunoglobulin light kappa chain variable chain region (VK) of hepatitis C virus-related type-II mixed cryoglobulinemia and B-cell proliferations. Ann N Y Acad Sci 1173: 152–160

Molecular Insights into the Disease Mechanisms of Type II Mixed Cryoglobulinemia

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Valli De Re and Marica Garziera

13.1

IgM Concentrations and Serum Factors May Be Involved in Cryoprecipitation

Serum type II cryoglobulins, characteristic and pathogenic of type II mixed cryoglobulinemic syndrome (MC-II), consist of a polyclonal component, usually IgG, and a mono/oligoclonal component, usually IgM, that shows rheumatoid factor (RF) activity. Neither the IgM nor the IgG fraction is cold-precipitable on its own. However, while the mechanisms responsible for cold-induced precipitation of mixed cryoglobulins are not well understood, at least two steps are known to be essential to the reaction. The first is efficient formation of the immune complexes and the second is a change in the physiochemical condition of the serum. In fact, as observed using dynamic light scattering (DLS), which determines the dynamic size-distribution profile of small particles in solution, there is a distinct relationship between IgM concentrations and mean hydrodynamic diameters or particle sizes. Different responses to changes in the aqueous environment were found, too, a feature that in turn suggests the importance of serum factor(s) for cryoprecipitation. Various agents have been proposed [1–4], but to date none have clearly revealed the cryoprecipitating component.

13.2

Other factors that may be critically linked to cryoprecipitation include differences in the carbohydrate residues expressed on the immunoglobulin regions. A major function of these sugars is to augment the stability of the proteins to which they are attached, but specific glycoforms are also involved in recognition events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated IgG may induce an association with mannose-binding lectins, thus contributing to the pathology by lectin-mediated activation of the complement cascade pathway [5]. In the context of cryoglobulins, a deficiency in sialytation has been shown to play a critical role in the cryoglobulin activity of lupus-like autoimmune syndrome, in which carbohydrates were shown to influence the flexibility between Ig domains [6]. Moreover, an unusual homogeneous glycosylation of the Fc region reportedly promotes the crystallization of IgG cryoglobulin [7]. However, although oligosaccharides are known to greatly influence solubility, to date the specific role of saccharides in type II cryoglobulins is still unknown.

13.3

V. De Re (*) Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine, (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy e-mail: [email protected]

Carbohydrate Residues of Ig May Be Involved in Cryoprecipitation

Distinct IgM and IgG Molecular Genetic Structures Associated with MC-II Cryoprecipitates

The molecular genetic structures that enable IgM and IgG to form immune complexes are also important parameters contributing to RF activity, immune complex formation, and thus to cryoprecipitation.

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_13, © Springer-Verlag Italia 2012

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Delineation of the peptides involved in complex formation by immunoblot analysis and peptide spectrometer identification, following IgM gel filtration and affinity chromatography of the cryoprecipitates, revealed the restricted use of IgM peptides [8]. MALDI-TOF spectrometry enabled the identification of the specific sequences common to several MC-II-IgM+k+ sequences included in heavy chain V1-69 and light chain V3-15 or V3-20 (e.g., ASQSVSSNLAWYQQ, LLIYGASTR, and EIVMTQSPATLSVSPGER). Therefore, we proposed that cryoglobulin was produced by a limited number of B-cell clones (mono/oligoclonality) able to pass immunological check-points in terms of B-cell selection in the bone marrow in the absence of allelic exclusion and class switching. The introduction of immunoglobulin VDJ PCR amplification, combined with mass spectrometry, led to the demonstration that the IgM component of the cryoprecipitates represents the circulating counterpart of the B-cell receptor (BCR) of the overexpanded B-cell population [8]. Ig-VH and Ig-VK genomic sequences of the overexpanded B-cell clones indirectly enable prediction of the amino acid sequence of IgM constituting the cryoprecipitate, by providing an easier approach than direct amino acid sequencing. Accordingly, the VH1, and especially the VH1-69 gene, as well as VH3 and VH4 were the most highly represented V(H) families, with a very high restriction in VK3 gene usage [9, 10]. The involvement of such restricted genes in the production of BCRs suggests an important role for the receptor in the pathogenesis of type II MC and IgM-IgG cryoprecipitation. Therefore, the study of V-Ig molecular variants could aid in determining the role of the charged residues and structural characteristics of the V-Ig region in the solubility of immunocomplexes [11–13]. Selective VDJ amplification was also found to be compatible with monoclonal or oligoclonal B-cell clonotypes in 80% (6/8) and 25% (2/8) of liver biopsies from hepatitis C virus (HCV)-infected patients with and without clinical evidence of cryoglobulinemia, indicating that HCV infection has an important role in determining B-cell expansion [14]. In fact, in HCV infection, the liver is the main site of inflammatory cell recruitment. Accordingly, it was shown that the intrahepatic B cells of HCV-infected patients undergo massive clonal expansion, which, although less frequently, is also detected in the circulation and bone marrow. Thus, over-representation of B-cell clone expansions in the liver strongly indicates that

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putative microenviromental factors are primarily responsible for their emergence and persistence. These notions are further corroborated by the observation that some HCV-related MCs and non-Hodgkin lymphomas (NHLs) are curable by virus eradication [15– 17], and a reduction in MC-II symptoms was shown after anti-B-cell treatment [18, 19]. In some cases, the expanded B-cell clones replace the entire pool of circulating B cells, although the absolute number of B cells remains within normal limits in the majority of MC-II patients. It has been proposed that homeostasis of B-cell number is maintained by the high rate of B-cell apoptosis induced by HCV, which balances B-cell expansion [20]. Immunophenotyping and cell-size analysis demonstrated that the expanded B-cell clones induced by HCV infection have a prevalent CD27+ “memory” phenotype [20, 21]. This finding, together with the evidence for immunoglobulin VH intraclonal variation and gene restriction [9], suggests that a limited set of antigens are associated with the expansion of activated B cells in MC-II.

13.4

Common Recognition of Restricted Epitopes in IgM Cryoglobulins

Besides celiac disease, MC-II is the only autoimmune disease in which a definite triggering antigen has been identified, HCV. Indeed, the concentration of HCV RNA in the cryoprecipitate was found to be 10- to 1000-fold greater than in the supernatant [22]. Nonetheless, the link between the production of IgM with RF activity, IgG, and HCV in a same cryoprecipitate remains unknown. In an attempt to answer this question, IgMs from the cryoprecipitate of patients with MC-II were isolated and used to define epitopes for IgM present in HCV proteins and in IgG. Several epitopes were recognized in non-structural region 3 of HCV (amino acids 1250–1334 NS3), in particular amino acids 1238–1279 and 1251–1270 and a third epitope in the crystallizable fragment constituting the constant domain of human IgG immunoglobulin (amino acids 345–355 of IgG Fc) [23]. The latter peptide is localized within the CH3 domain of IgG and is part of the dimeric interface of the immunoglobulin. Its sequence, EPQVYTLPPSR, is conserved among the different IgG classes except in IgG4, which has a single mutation. Its reactivity was confirmed in an ELISA using the synthetic EPQVYTLPPSR peptide.

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Molecular Insights into the Disease Mechanisms of Type II Mixed Cryoglobulinemia

MC-II IgM showed cross-recognition with the NS3 (amino acids 1238–1279) and the Fc (amino acids 345–355) domains; however, the high diversity of the NS3 and Fc amino acid sequences excluded linearsequence homology for mimicry. Instead, the T-cell mediated process of B-cell clonal selection and somatic mutation found in HCV+ MC-II may be induced by HCV-NS3 antigen, which could lead to the production of RF autoantibodies. A shift towards RF activity could reduce the affinity of IgM for HCV, resulting in advantageous conditions for infective agents. This mechanism is common to other microbial agents since increased RF levels have been found in other infections associated with B-cell expansion, such as Epstein-Barr virus infection [24, 25]. In this context, crystal-structure analysis of a complex between IgM with RF activity and its IgG autoantigen revealed relatively few antibody contact residues between the potential combining sites of IgM and IgG. Moreover, these residues are located on only one side of the combining site surface, indicating that IGM has another, entirely different, specificity than RF [26, 27]. In that case, IgM may have originated in response to another antigen and the reactivity with IgG Fc may be an unfortunate coincidence of cross-reactivity. In MC-II, NS3 HCV-antigen was found to be able to cross-link and cluster BCR molecules, both directly and indirectly through specific IgG anti-HCV-NS3 immunocomplexes [28]. Antigen-dependent signals from BCRs require clustering of the receptor [29] and the data support the activation of BCR by Fc-IGG and/or HCV-NS3 antigens, including signaling essential for B-cell proliferation and differentiation. Based on the above findings, we propose a model whereby BCR, by binding IgM/IgG/HCVNS3 immune complexes, deprives FcgIIR of its Fc-IgG natural ligand region [28]. This removes the brake inhibiting RF+ B-cell proliferation and promotes the selective accumulation of these cells. In the same way, endocytosis of NS3, mediated by BCR and by direct HCV infection, promotes caspase-8-mediated apoptosis and the activities of proteins that disrupt of NF-kB and IRF-3 activation [30, 31], which together could reduce the size of the B-cell population. In other studies, the high sequence homology between IGM-RF+ and HCV anti-E2 antibodies suggested that HCV-E2 is another antigen contributing to B-cell proliferation [32]. In vitro studies demonstrated binding between HCV E2 protein and CD81, a highly ubiquitous tetraspanning molecule well represented on

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the surface of B cells and able to decrease the threshold for BCR activation. This result suggested that the binding of BCR by viral antigens coupled with direct binding of CD-81 by HCV-E2 provides a strong proliferative signal associated with HCV+ B-cell polyclonal activation [33, 34]. Moreover, selected IgMk clones from a patient with MC-II were shown to react with the Fabs fragment of IgG1anti-HCV/E2 antibodies belonging to the VH1-69 subfamily and isolated from the same patient, suggesting that clones of some subfamilies are naturally prone to react against other VH gene subfamilies, such as VH 1–69 [35]. Another group proposed that, instead of E2, B cell expansion requires the dual engagement of BCR and toll-like receptor 7 by IgG and HCV RNA, respectively [36].

13.5

Evidence of Clustered HLA Molecules Associated with MC-II Syndrome

The pivotal role played by antigen stimulation in the expansion of IgMk RF+ B-cell clones relies on the ability of the immune system to distinguish between self and non-self. Remarkably, several lines of evidence indicate a significant association between DR5 supertype of HLA-class II DRB1 and DQ3 supertype HLA-class II DQB1 in HCV+ MC-II patients [37–39], implying that B-cell proliferation is affected by IgM stimulation and HLA presentation. In addition to specific B-cell antigenic stimulation, a contribution of sex hormones to the etiopathogenesis of HCV-related MC-II has been proposed, given the high prevalence of MC-II in female patients, A probable protective effect of Schistosoma mansoni coinfection, at least in the Egyptian population, also seems likely. However, the immune basis for either of these phenomena is unknown [40].

13.6

Differences and Similarities Between RF-IgM from Patients with MC-II and Rheumatoid Arthritis

The role of IGM RF+ activity in MC-II is strongly associated with the induction of cryoglobulin immune complexes. The structure of IgG recognized by RF+ IgM autoantibodies from MC-II patients differs significantly from that of RF obtained from patients with

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rheumatoid arthritis (RA). In most cases, RA-derived RF was able to bind IgG1, 2, and 4, but not 3 [41], while MC-II derived MC-II preferentially bound IgG1, 2, and 3 [23]. Moreover, most RA-derived RF bound IgG at a discontinuous epitope consisting of residues from both the CH2 and CH3 heavy-chain constant regions [42], while the Fc EPQVYTLPPSR peptide is localized in the CH3 region of IgG [23]. Finally, no case of serum sickness syndrome has been reported thus far in RA patients receiving high-dose IgG1 rituximab infusion, while rituximab may form a complex with RF-positive IgMk, leading to accelerated cryoprecipitation and to severe systemic reactions [43]. Thus, overall, the data suggest that the IgG structure recognized by RF+ activity in MC-II patients is different from that present in RA patients. This difference may explain the cryoglobulin precipitation seen in MC-II but not in RA patients.

13.7

Conclusions

In the last few years, there have been many advances in our understanding of MC-II pathogenesis, especially after the sequencing of the HCV genome in 1989. At the moment, the most probable hypothesis is that MC-II is sustained by the proliferation of a restricted set of memory (CD27+) IGMk+ B cells with a restricted usage of RF-encoding Ig gene segments. Oligoclonal/monoclonal B cells are found in the liver, peripheral blood, and bone marrow but the exact mechanism sustaining B-cell proliferation remains elusive. A distinct relationship between IgM concentrations, specific patterns of immunoglobulin glycosylation, and the limited use of certain genes encoding immunoglobulin regions may explain the cryoprecipitation of immunocomplexes. Characterization of the binding affinity of clonal IgM with RF activity evidenced a cross-recognition between HCV-NS3 peptide and a specific IgG Fc region present in the IgG CH3 region. There may be some differences between the IgG structure recognized by RF+ activity in MC-II patients and that found in RA patients, with cryoprecipation occurring only in the former. Continued patient-centered studies are necessary to elucidate the characteristics of cryoglobulins from MC-II patients and the mechanism that sustain their production.

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16. Vallisa D, Bernuzzi P, Arcaini L et al (2005) Role of antihepatitis C virus (HCV) treatment in HCV-related, lowgrade, B-cell, non-Hodgkin’s lymphoma: a multicenter Italian experience. J Clin Oncol 23:468–473 17. Hermine O, Lefrere F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med 347: 89–94 18. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 19. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101:3827–3834 20. Racanelli V, Frassanito MA, Leone P et al (2006) Antibody production and in vitro behavior of CD27-defined B-cell subsets: persistent hepatitis C virus infection changes the rules. J Virol 80:3923–3934 21. Charles ED, Green RM, Marukian S et al (2008) Clonal expansion of immunoglobulin M+CD27+ B cells in HCVassociated mixed cryoglobulinemia. Blood 111:1344–1356 22. Sansonno D, Carbone A, De Re V, Dammacco F (2007) Hepatitis C virus infection, cryoglobulinaemia, and beyond. Rheumatology (Oxford) 46:572–578 23. De Re V, Sansonno D, Simula MP et al (2006) HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 20:1145–1154 24. Carayannopoulos MO, Potter KN, Li Y et al (2000) Evidence that human immunoglobulin M rheumatoid factors can be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts. Scand J Immunol 51:327–336 25. Yang L, Hakoda M, Iwabuchi K et al (2004) Rheumatoid factors induce signaling from B cells, leading to EpsteinBarr virus and B-cell activation. J Virol 78(18):9918–9923 26. Duquerroy S, Stura EA, Bressanelli S et al (2007) Crystal structure of a human autoimmune complex between IgM rheumatoid factor RF61 and IgG1 Fc reveals a novel epitope and evidence for affinity maturation. J Mol Biol 368: 1321–1331 27. Corper AL, Sohi MK, Bonagura VR et al (1997) Structure of human IgM rheumatoid factor Fab bound to its autoantigen IgG Fc reveals a novel topology of antibody-antigen interaction. Nat Struct Biol 4:374–381 28. De Re V, Pavan A, Sansonno S et al (2009) Clonal CD27+ CD19+ B cell expansion through inhibition of FC gammaIIR in HCV(+) cryoglobulinemic patients. Ann N Y Acad Sci 1173:326–333

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29. Tamir I, Stolpa JC, Helgason CD et al (2000) The RasGAPbinding protein p62dok is a mediator of inhibitory FcgammaRIIB signals in B cells. Immunity 12:347–358 30. Prikhod’ko EA, Prikhod’ko GG, Siegel RM et al (2004) The NS3 protein of hepatitis C virus induces caspase-8-mediated apoptosis independent of its protease or helicase activities. Virology 329:53–67 31. Foy E, Li K, Wang C et al (2003) Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 300:1145–1148 32. Ferri S, Dal Pero F, Bortoletto G et al (2006) Detailed analysis of the E2-IgM complex in hepatitis C-related type II mixed cryoglobulinaemia. J Viral Hepat 13:166–176 33. Landau DA, Saadoun D, Calabrese LH, Cacoub P (2007) The pathophysiology of HCV induced B-cell clonal disorders. Autoimmun Rev 6:581–587 34. Rosa D, Saletti G, De Gregorio E et al (2005) Activation of naive B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 102:18544–18549 35. Perotti M, Ghidoli N, Altara R et al (2008) Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia. Autoimmun Rev 7:468–472 36. Charles ED, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76:818–824 37. Cacoub P, Renou C, Kerr G et al (2001) Influence of HLA-DR phenotype on the risk of hepatitis C virusassociated mixed cryoglobulinemia. Arthritis Rheum 44: 2118–2124 38. De Re V, Caggiari L, De Vita S et al (2007) Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus. Dig Liver Dis 39:S65–S71 39. De Re V, Caggiari L, Simula MP et al (2007) Role of the HLA class II: HCV-related disorders. Ann N Y Acad Sci 1107:308–318 40. Abbas OM, Omar NA, Zaghla HE, Faramawi MF (2009) Schistosoma mansoni coinfection could have a protective effect against mixed cryoglobulinaemia in hepatitis C patients. Liver Int 29:1065–1070 41. Artandi SE, Canfield SM, Tao MH et al (1991) Molecular analysis of IgM rheumatoid factor binding to chimeric IgG. J Immunol 146:603–610 42. Bonagura VR, Artandi SE, Davidson A et al (1993) Mapping studies reveal unique epitopes on IgG recognized by rheumatoid arthritis-derived monoclonal rheumatoid factors. J Immunol 151:3840–3852 43. Sene D, Ghillani-Dalbin P, Amoura Z et al (2009) Rituximab may form a complex with IGmkappa mixed cryoglobulin and induce severe systemic reactions in patients with hepatitis C virus-induced vasculitis. Arthritis Rheum 60:3848–3855

The Role of VCAM-1 in the Pathogenesis of Hepatitis-C-Associated Mixed Cryoglobulinemia Vasculitis

14

Gilles Kaplanski

The hallmark of inflammation is the recruitment of leukocytes in tissues. This is a complex multistep phenomenon involving several families of adhesion molecules, such as selectins, integrins, and the superfamily of immunoglobulins [1, 2]. The selectin family comprises three members: L-selectin, which is constitutively expressed on the surface of quiescent leukocytes; P-selectin, which is rapidly and transiently expressed on the surface of endothelial cells and platelets; and E-selectin, which is synthesized by endothelial cells 1–2 h following stimulation with interleukin (IL)-1 or tumor necrosis factor (TNF)-a and remains on the cell surface for 6–8 h [3]. These adhesion molecules interact with mucins through very transient and weak bonds and have been shown to mediate the first step of leukocyte adhesion to the endothelial monolayer, i.e., rolling [3]. Rolling is followed by the firm adhesion of leukocytes to the endothelium, which involves interactions between the b2 integrins (LFA-1, CD11a/CD18, and Mac-1, CD11b/CD18), expressed on all leukocytes, and two intercellular adhesion molecules, ICAM-1 and ICAM-2, on endothelium [2], as well as interactions of b1 and b7 integrins (a4b1, VLA-4 and a4b7, VLA-7), expressed by mononuclear cells with vascular cell adhesion molecule-1 (VCAM-1) on endothelium [2]. ICAM-1 is constitutively expressed on endothelial cells but is strongly up-regulated after 12–24 h of stimulation by IL-1, TNF-a, interferon (IFN)-g or thrombin [2, 4]. VCAM-1 is only expressed after a 24-h stimulation of endothelial cells with the G. Kaplanski Service de Médecine Interne, Hôpital de la Conception, Marseille, France e-mail: [email protected]

same inflammatory mediators [2]. Due to differences in b2 and b1 expression on leukocyte populations, ICAM-1 is considered to be involved in mediating adhesion to all types of leukocytes whereas VCAM-1 is thought to mainly support lymphocyte and monocyte adhesion [1, 2]. Mixed cryoglobulinemia (MC) is the most common extrahepatic manifestation of chronic hepatitis C virus (HCV) infection and a prototypic model of immunecomplex (IC)-mediated disease [5]. Most of the time, MC is totally asymptomatic but in some patients IC deposition induces mononuclear cell inflammation in small vessels, affecting the skin, joints, peripheral nerves, and kidneys clinically characterized by palpable purpura, arthralgia, subacute distal sensory polyneuropathy, and membranous glomerulopathy [6, 7]. A more severe form of the disease, similar to polyarteritis nodosa, rarely occurs; it is characterized by acute-onset fever, poor general condition, severe multifocal sensorimotor mononeuropathies, high blood pressure, and digestive tract vasculitis. This form is associated with necrotizing vasculitis of the mediumsized arteries, with a mixed polymorphonuclear and mononuclear cell infiltrate [8]. In 2005, to better understand the pathogenesis of MC and the possible involvement of the different adhesion molecules in the different clinical forms of the disease, we studied endothelial adhesion molecules in HCV-MC patients [9]. First, the soluble forms of E-selectin (ELAM), ICAM-1, and VCAM-1 were measured by ELISA in the serum of patients with HCV-MC. The results were compared with those obtained from the serum of HCV patients without MC and in healthy controls (Fig. 14.1). The concentrations of all three adhesion molecule were higher in HCV-MC patients

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Fig. 14.2 sVCAM-1 concentrations are elevated in severe forms of vasculitis. Box plot representation. LG low-grade mixed cryoglobulinemia, HG high-grade mixed cryoglobulinemia, PAN PAN-like cryoglobulinemia; **p < 0.001

800

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Fig. 14.1 Concentrations of (a) soluble (s)ELAM, (b) sICAM1, and (c) sVCAM-1 in healthy controls (control) and in HCV patients without mixed cryoglobulinemia (HCV+/Cryo-) or with mixed cryoglobulinemia (HCV/cryo+). Box plot representation, horizontal bars show the median (*p < 0.01, **p < 0.001)

than in controls, but only VCAM-1 concentrations were significantly higher in HCV-MC patients than in HCV patients without MC. The highest concentrations were measured in the serum of patients with the more severe forms of the disease and not in those with symptoms limited to purpura (Fig. 14.2). We then used histochemistry and specific monoclonal antibodies to examine the expression of ICAM-1 and VCAM-1 in peripheral nerve biopsies of patients with HCV-MC. ICAM-1 was very weakly expressed or negative in all the patients tested. VCAM-1 staining, by contrast, was frequently observed in epineural and perineural small-vessel endothelium in patients with HCV-MC, but not in all patients (Fig. 14.3). Interestingly, no antiVCAM-1 staining was seen in the nerve biopsies of HCV patients without MC but suffering a neuropathy unrelated to MC. Cryoprecipitate was then purified from the serum of several patients with HCV-MC and added in vitro to human umbilical vein endothelial cells (HUVEC) before adhesion molecule expression was determined using FACS analysis. All three molecules were induced by MC in this assay, with a 60 ± 30% increase for ELAM, a 30 ± 9% increase for VCAM-1, and a 16 ±5% increase for ICAM-1. The addition of an exogenous source of complement did not modify adhesion molecule expression and serum from HCV patients without MC demonstrated no effect at all. Since HUVEC do not express Fcg receptors but do express receptors for C1q, we asked whether

14

The Role of VCAM-1 in the Pathogenesis of Hepatitis-C-Associated Mixed Cryoglobulinemia Vasculitis VCAM-1

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b

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Patient 3

Patient 2

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Fig. 14.3 Immunolabeling of the superficial peroneal nerve (longitudinal section) using anti-ICAM-1 or anti-VCAM-1 monoclonal antibody (mAb). (a–d) HCV-MC patients 1 and 2 showing positive labeling of the internal wall of the perineural

vessel with anti-VCAM-1 mAb (a, c) but not with anti-ICAM-1 (b, d); magnification ×540. (e, f) HCV-MC patient 3: positive immunolabeling with anti-VCAM-1 (e) and no labeling using anti-ICAM-1 (f). Magnification ×50

cryoprecipitate from HCV-MC patients contains C1q. Indeed, C1q was detected in all of the cryoprecipitates, with concentrations ranging from 30 to 770 mg/ml, suggesting that C1q on MC induces adhesion molecule expression on HUVEC. The identification of VCAM-1 and not ICAM-1 as the main adhesion molecule involved in the pathogenesis of HCV-MC vasculitis was surprising, and the fact that VCAM-1 was especially elevated in severe cases

of the disease, related to a more necrotizing form of vasculitis involving neutrophils, even more so. VCAM-1 mainly supports mononuclear cell adhesion to endothelium whereas ICAM-1 is more generally involved in both polymorphonuclear and mononuclear cell adhesion. Moreover, in the Arthus reaction, which is considered the best model of IC-mediated diseases and thus of MC vasculitis, all members of the three adhesion molecule families were shown to participate

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in mediating leukocyte infiltration, with ICAM-1 being the most important [10, 11]. In addition, ICAM-1 is largely involved in the pathogenesis of HCV-induced liver inflammation [12] and was detected in the skin of a patient with a palpable purpura related to HCV-MC [13]. We thus expected ICAM-1 to be involved, at least as much as VCAM-1, in this disease. However, other models of the Arthus reaction have since been reported, involving the cremaster muscle, lungs, and the heart. In these models, VCAM-1 was the principal molecule mediating leukocyte infiltration [14–16]. It is likely that the roles of the various adhesion molecules in mediating leukocyte infiltration depend on the tissue concerned. Notably, in muscle and nerve, as studied in our patients with severe forms of HCV-MC, VCAM-1 is the most important mediator of leukocyte adhesion and infiltration [14]. The more severe forms of HCV-MC combine both mononuclear cell and neutrophil infiltration [8], thus the predominant role of VCAM-1 suggests that neutrophil infiltration is in some way mediated by VCAM-1. In support of this hypothesis, VLA-4, the ligand of VCAM-1, has been shown to be expressed not only on mononuclear cells but also on neutrophils [17]. VLA-4 is present on naïve neutrophils and, if stimulated by chemoattractants such as C5a, FMLP, or leukotriene B4, also on mature neutrophils [17, 18]. Neutrophil VLA-4 may be able to mediate firm adhesion as well as tissue migration, explaining why anti-a4 integrin treatment decreases the severity of tissue lesions in animal models of IC-mediated inflammation affecting the lungs, heart, and cremaster muscle. In addition, neutrophils were recently reported to express another b1 integrin, VLA-9 (a9b1), which is involved in adhesion to VCAM-1 on endothelium and in neutrophil trans-endothelial migration [19, 20]. During HCV-MC vasculitis, the expression of adhesion molecules on endothelium may be largely mediated by inflammatory cytokines, such as TNF-a, IL-1b, and IL-6, which are elevated in the serum or tissue of these patients and may be produced by mononuclear cells after IC fixation to immunoglobulin FcR [21–23]. Tissue infiltration by leukocytes may then be induced by various chemokines [24], with IL-6 participating in leukocyte recruitment by balancing chemokine production [25]. Our previous observations, however, identified C1q in MC as a primary inducer of selectin and integrin expression on HUVEC. C1q may also, in part, mediate the immunosuppression associated with

G. Kaplanski

HCV infection [26]. More recently, dysregulated shedding of the C1q receptor was shown in HCV-MC and suggested to participate in the vascular damage induced by MC [27]. These combined observations support an important role for C1q and its receptor in HCV-MC pathogenesis.

References 1. Butcher EC (1991) Leukocyte-endothelial cell recognition: three (or more) steps to specificity end diversity. Cell 67: 1033–1036 2. Springer TA (1994) Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76:301–314 3. Tedder TF, Li X (1999) The selectins and their ligands: adhesion molecules of the vasculature. Adv Mol Cell Biol 28:65–111 4. Kaplanski G, Marin V (1998) Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Blood 92:1259–1267 5. Cacoub P, Renou C (2000) Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. Medicine 79:47–56 6. Dammaco F, Sansonno D (2001) The cryoglobulins: an overview. Eur J Clin Invest 31:628–638 7. Charles E, Dustin LB (2009) Hepatitis C virus-induced cryoglobulinemia. Kidney Int 76:813–824 8. Cacoub P, Maisonobe T (2001) Systemic vasculitis in patients with hepatitis C. J Rheumatol 28:109–118 9. Kaplanski G, Maisonobe T (2005) Vascular cell adhesion molecule-1 (VCAM-1) plays a central role in the pathogenesis of severe forms of vasculitis due to hepatitis C-associated mixed cryoglobulinemia. J Hepatol 42:334–340 10. Mulligan MS, Watson SR (1993) Protective effects of selectin chimeras in neutrophil-mediated Lung injury. J Immunol 151:6410–6417 11. Mulligan MS, Wilson GP (1993) Role of b1, b2 integrins and ICAM-1 in lung injury after deposition of IgG and IgA immune complexes. J Immunol 150:2407–2417 12. Ballardini G, Groff P (1995) Hepatitis C virus (HCV) genotype, tissue HCV antigens, hepatocellular expression of HLA-a, B, C and intercellular adhesion-1 molecules clues to pathogenesis of hepatocellular damage and response to interferon treatment in patients with chronic hepatitis C. J Clin Invest 95:2067–2075 13. Bernacchi E, Civita LL (1999) Hepatitis C virus (HCV) in cryoglobulinaemic leukocytoclastic vasculitis (LCV): could the presence of HCV in skin lesions be related to CD8+ lymphocytes, HLA-DR and ICAM-1 expression? Exp Dermatol 95:2067–2075 14. Norman U, Van De Velde NC (2003) Overlapping roles of endothelial selectins and vascular cell adhesion molecule-1 in immune complex-induced leukocyte recruitment in the cremasteric microvasculature. Am J Pathol 163:1491–1503

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The Role of VCAM-1 in the Pathogenesis of Hepatitis-C-Associated Mixed Cryoglobulinemia Vasculitis

15. Burns JA, Issekutz TB (2001) The a4b1 (very late antigen VLA-4, CD49d/CD29) and a5b1 (VLA-5, CD49e/CD29) integrins mediate b2 (CD11/CD18) integrin-independent neutrophil recruitment to endotoxin-induced lung inflammation. J Immunol 166:4644–4649 16. Bowden RA, Ding ZM (2002) Role of a4 integrin and VCAM-1 in CD18-independent neutrophil migration across mouse cardiac endothelium. Circ Res 90:562–569 17. Johnston B, Kubes P (1999) The a4-integrin: an alternative pathway for neutrophil recruitment? Trends Immunol 20:545–550 18. Pereira S, Zhou M (2001) Resting murine neutrophils express functional a4 integrins that signal through Src family kinases. J Immunol 166:4115–4123 19. Shang T, Yednock T (1999) a9b1 Integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers. J Leukoc Biol 66:809–816 20. Ross EA, Douglas MR (2006) Interaction between integrin a9b1 and vascular cell adhesion molecule-1 (VCAM-1) inhibits neutrophil apoptosis. Blood 107:1178–1183 21. Kaplanski G, Marin V (2002) Increased soluble p55 and p75 tumor necrosis factor-a receptors in patients with hepatitis C-associated mixed cryoglobulinemia. Clin Exp Immunol 127:123–130

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22. Saadoun D, Bieche I (2007) Role of matrix matalloproteinases, proinflammatory cytokines, and strees-derived molecules in hepatitis C virus-associated mixed cryoglobulinemia vasculitis neuropathy. Arthritis Rheum 56:1315–1324 23. Antonelli A, Ferri C (2009) Serum levels of proinflammatory cytokines interleukin-1b, interleukin-6, and tumor necrosis factor a in mixed cryoglobulinemia. Arthritis Rheum 60: 3841–3847 24. Antonelli A, Ferri C (2008) High values of CXCL10 serum levels in mixed cryoglobulinemia associated with hepatitis C infection. Am J Gastroenterol 103:2488–2494 25. Kaplanski G, Marin V (2003) IL-6: a regulator of the transition from neutrophil to monocytic inflammation. Trends Immunol 24:25–29 26. Kittlesen DJ, Chianese-Bullock KA (2000) Interaction between complement receptor gC1qR and hepatitis C virus core protein inhibits T-lymphocyte proliferation. J Clin Invest 106:1239–1249 27. Sansonno D, Tucci FA (2009) Role of the receptor for the globular domain of C1q protein in the pathogenesis of hepatitis C virus-related cryoglobulin vascular damage. J Immunol 183:6013–6020

Up-Regulation of B-Lymphocyte Stimulator (BLyS) in Patients with Mixed Cryoglobulinemia

15

Martina Fabris and Salvatore De Vita

15.1

BLyS: A Key Regulator of B-Cell Survival

Our knowledge of the mechanisms underlying B-cell survival and proliferation has profoundly changed since the identification in 1999 of B lymphocyte stimulator (BLyS), also called B-cell activating factor (BAFF) or tumor necrosis factor (TNF)- and Apo-Lrelated leukocyte-expressed ligand-1 (TALL-1) [1–3]. BLyS is a 285-amino-acid protein encoded by a gene on chromosome 13q32–34 and secreted primarily by myeloid cells, including monocytes, macrophages, and dendritic cells (DC) [4, 5]. The number of cells known to secrete BLyS is increasing, however, and currently also includes non-myeloid cells, such as bone marrow stromal cells [6], synoviocytes [7], astrocytes [8], salivary gland epithelium [9], and gut epithelium [10]. BLyS expression by macrophages and DC is stimulated by interferon (IFN)-g and interleukin-10 (IL-10) during inflammatory states and/or chronic infections [4, 11]. BLyS promotes the transition of type 1 B cells into subsequent phases of development—reaching peripheral sites of the immune system—up to the acquisition of mature follicular and marginal zone B-cell phenotypes [12, 13]. It was also demonstrated that human soluble BLyS potently elevates the NK cell activity of murine splenic cells in vivo through the

S. De Vita (*) Clinic of Rheumatology, Deparment of Medical and Biological Sciences, Azienda Ospedaliero – Universitaria of Udine, Udine, Italy e-mail: [email protected]

up-regulation of CD4(+) T lymphocytes and IL-2 and IFN-g generation [14]. BLyS exerts its functions by its interaction with three receptors: B-cell maturation antigen (BCMA), transmembrane activator and calcium-modulating cyclophilin ligand (CAML) interactor (TACI), and, more importantly, B-cell-activating factor receptor (BAFF-R) [15–17]. BCMA and BAFF-R are predominantly expressed on B lymphocytes, whereas TACI is present on B cells and activated T cells [18]. BLyS shows high homology with another member of the TNF superfamily, APRIL (a proliferation-inducing ligand), which shares with BLyS two of its three receptors: TACI and BCMA [19]. BLyS signaling is essential for the survival of pre-immune B lymphocytes, whereas antigen-experienced B lymphocytes generally interact more avidly with APRIL [20]. Downstream mediators of the BAFF/BAFFR interaction include the classic and the alternative NF-kB pathways, providing key regulatory control of antiapoptotic cell survival and growth stimulation. In particular, BLyS favors the expression of several anti-apoptotic genes, including Bcl-xL, Mcl-1, A-1, Bcl-2, and Bim [21], through the induction of survival-promoting kinase systems, such as Pim 1/2 and Erk, as well as early cellcycle progression molecules, such as c-myc, p27 kip1, cyclin D1, and cyclin D2. In a recent study, Fu et al. [22] demonstrated that, both in normal and in neoplastic B cells, BAFF-R is expressed also in the nucleus, where it functions as a transcription factor. Through its association with c-Rel and IKKb, it binds to the NF-kB binding site in the promoters of several NF-kB target genes, including BLyS, CD40L, BCl-xL, and IL-8.

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BLyS in Autoimmune Diseases

The importance of BLyS in B-cell homeostasis has been definitively demonstrated in murine models. BLyS knock-out mice showed profound alterations in the pool of mature B lymphocytes [23], whereas transgenic mice overexpressing BLyS developed critical lymphoid proliferation in blood and in the marginal zones of lymph nodes, with the production of high titers of immunoglobulins (Igs) and autoantibodies, such as rheumatoid factor (RF), anti-DNA antibodies, and sometimes cryoglobulins. As these mice aged, they also developed a lupus-like (glomerulonephritis) or Sjögren’s-like (salivary gland inflammatory infiltration) syndrome and finally B-cell lymphomas [24–26]. Consistent with these observations, high levels of BLyS in the serum and/or in the affected tissue have been documented in several autoimmune diseases [27]. BLyS levels are particularly elevated in Sjögren’s syndrome (SS) and systemic lupus erythematosus (SLE), but have also been strongly implicated in rheumatoid arthritis, systemic sclerosis, multiple sclerosis, diabetes, celiac diseases, and autoimmune thyroiditis [28–35]. However, only a fraction of these patients (15–30% but as high as 70% in the case of celiac disease) have higher than normal BLyS serum levels and are considered to exhibit a “high BLyS phenotype.” In autoimmune diseases, increased BLyS levels are usually related to the levels of disease-specific autoantibodies and to the presence and degree of lymphocyte infiltration in the affected tissues (e.g., synovial membrane, salivary glands), especially when ectopic germinal centers are present [36–39]. Very recently, high-level expression of APRIL, BLyS, BCMA, and TACI was reported in the renal biopsies (glomeruli and tubulointerstitium) of lupus patients with proliferative nephritis [40]. The mechanism underlying BLyS-associated autoimmunity has been extensively investigated and many but certainly not all aspects have been clarified [41]. It seems that BLyS can rescue anergic autoreactive B cells from death, but only in the absence of competition from non-autoreactive B cells. Yet, high BLyS levels promote autoantibody formation in individuals possessing diverse B cells. In a study aimed at better understand how excess BLyS promotes autoimmunity, one group [42] recently showed that limiting BLyS signaling only slightly

selects against the maturation of autoreactive B cells with higher affinity, whereas BLyS overexpression leads to broad tolerance escape and to the positive selection of autoreactive cells.

15.3

BLyS in Patients with Mixed Cryoglobulinemia

The possible role of BLyS in mixed cryoglobulinemia syndrome (MCsn) was first suggested in a preliminary study of patients with HCV chronic infection carried out by Toubi et al. [43], then intensively investigated by our group in 2007 [44], and soon after by Cacoub et al. [45]. We evaluated patients with HCV-related MCsn, HCV-infected individuals without MCsn, and healthy controls. Serum BLyS levels were higher (mean value 3.70 ± 2.97 ng/mL) and detected more frequently (25/66, 37.9%) in MCsn patients than in healthy blood donors (2/48, 4.2%) (OR = 14.02; CI = 3.13–62.91, p < 0.0001; Fig. 15.1a). BLyS serum levels were assessed by an antigen-capture ELISA method based on a Fab capture reagent (Human Genome Sciences, Rockville, MD, USA). This approach avoids interference by high RF concentrations, which previously hindered efficient BLyS assays in MCsn. The lowest limit of quantitation of this assay is 0.85 ng/mL; thus, samples with BLyS levels below this limit were considered below the level of quantitation (Fig. 15.1a). Importantly, besides the demonstration of higher BLyS serum levels in MCsn HCV-related disease, increased BLyS serum levels were also determined in HCV chronic infection, in the absence of MCsn (Fig. 15.1b). This finding is consistent with the hypothesis that HCV acts as an important trigger for BLyS up-regulation. Indeed, up-regulation is noted in about one-third of HCV-infected patients, as recently confirmed by Tarantino et al. in a larger series of patients with acute (AHC) and chronic hepatitis C (CHC) [46]. In this recent work, BLyS median levels were significantly higher in AHC patients (1,485 pg/mL) than in either CHC patients (1,058 pg/mL) or healthy blood donors (980 pg/mL) (p < 0.001). More importantly, BLyS levels were higher in AHC patients with disease evolving to chronicity (1,980 pg/mL) than in those whose disease was self-limited (1,200 pg/mL; p = 0.02). Finally, higher BLyS levels were independently associated with the persistence of HCV infection,

Up-Regulation of B-Lymphocyte Stimulator (BLyS) in Patients with Mixed Cryoglobulinemia

a 100 Percentage of cases

Fig. 15.1 BLyS serum levels in patients with MCsn, in HCV-positive patients without MCsn (HCV + w/o MCsn), and in healthy blood donors (HBDs). (a) Compared with HBDs, BLyS positivity (>0.85 ng/mL, black bars) was significantly more frequent both in MCsn patients (*p < 0.0001) and in HCV-positive patients without MCsn (**p = 0.0026). BLOQ Below the level of quantitation (<0.85 ng/mL). (b) BLyS-positive cases. Horizontal bold lines Mean BLyS levels. BLyS levels were significantly higher in MCsn patients than in HCV-positive patients without MCsn (***p = 0.0044). Five out of 12 (41.7%) HCV-negative MCsn patients were positive (>0.85 ng/mL) for BLyS and two of them (arrows) had “high” BLyS levels (>2.82 ng/mL, i.e. mean + 2 SD of the MCsn patients’ distribution). (From [44], with the permission of Rheumatology, Oxford University Press)

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indicating that high BLyS serum levels at the onset of AHC are a predictor of disease evolution to chronic infection [46]. BLyS serum levels higher than in the general population were detected in a percentage of both MCsn patients and HCV-infected patients without MCsn (with or without serum cryoglobulins). Why such an increase is limited to a fraction of MCsn and HCV-positive cases is unknown. Similar observations were previously reported in RA, SLE, and SS, thus suggesting individual genetic predisposition [47]. Several polymorphisms in the BLyS gene have been described [48, 49], some of which influence BLyS expression. Accordingly, infection (by HCV or other infectious agents in HCV-negative MCsn, as well as in other autoimmune diseases) in a subset of genetically predisposed individuals would induce BLyS expression, possibly favoring the subsequent development of autoimmune and lymphoproliferative features in some of them.

HBDs (48)

While the etiologic role of chronic infection is well established in MCsn, such information is lacking in SLE, RA, and SS, in which the role of a putative infectious trigger may be transient, leading to a secondary, fully self-perpetuating autoimmune response. Seen in this light, BLyS up-regulation in HCV-positive MCsn, and in HCV infection overall, serves as a general model linking viral infection, B cell proliferation, and autoimmune disease [50]. Of note, BLyS expression is also elevated in SS, the leading autoimmune disease associated with HCVnegative mixed cryoglobulinemia (with or without MCsn). Furthermore, the B-cell clones proliferating in SS express immunoglobulin gene sequences with remarkable similarities to those expanded in HCVrelated MCsn [51]. T cells were shown to abnormally produce BLyS in SS salivary lesions, in which monoclonal RFs with the cryoglobulin idiotype are seen [52]. Epithelial salivary cells may also secrete

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BLyS in SS [9]. Thus, BLyS expression in SS-related MALT (mucosa-associated lymphoid tissue) lesions might be linked to local B-cell expansion and cryoglobulin production [53, 54]. BLyS is significantly elevated in the course of several human hematological diseases, such as Hodgkin’s and non-Hodgkin’s lymphoma, chronic lymphocytic leukemia, and multiple myeloma [55–58]. In our series [44], MCsn patients with an overt lymphoproliferative disease had high BLyS levels more frequently than patients with MCsn but without lymphoproliferative disease (p = 0.04), consistent with the reported association between BLyS and lymphoproliferation. In contrast, BLyS was not associated with any of the specific organ manifestations that characterize MCsn (neuropathy, nephritis, skin ulcers, etc.). These data were confirmed by French authors [45], who showed that BLyS serum levels were positively associated with markers of HCV-associated B-cell lymphoproliferation. In addition, serum levels of BLyS were higher in HCVinfected patients with type II MC, high MC levels, positive RF, and associated systemic vasculitis. Finally, serum BLyS concentrations were higher (two-fold increase) in HCV-related MC vasculitis than in either healthy controls or patients with chronic HCV infection without MC vasculitis, and HCV-related B-NHL was associated with an even greater increase (threefold) in serum BLyS [59]. The immunochemical types of MC may vary during the disease course [60]. Type III MC can evolve to an oligoclonal form and finally to type II MC, thus evidencing a monoclonal component [61, 62]. BLyS serum levels may follow this course, increasing as B-cell monoclonality (type II MC) appears [45, 59]. The relationship between serum BLyS and MC levels suggests a role for BLyS in sustaining a high level of immunoglobulin secretion by B cells, as demonstrated in mice overexpressing BLyS [24–26]. Regarding HCV-related MCsn, HCV infection could be the earlier, critical event that leads to BLyS elevation, rather than a triggering by downstream biological events, when serum cryoglobulins appear (detected in up to 40% of HCV-positive subjects), or coincident with the appearance of overt MC syndrome (occurring in 1–5% of HCV-positive subjects). In this scenario, BLyS would play a role as an early, chronic “background” stimulus for B-cell autoimmunity and lymphoproliferation in a subgroup of HCV-infected individuals [44, 50].

M. Fabris and S. De Vita

15.4

BLyS, HCV, and B-Cell Lymphomagenesis

Autocrine production of BLyS has been documented in cases of neoplastic B-cell proliferations [63]. A very recent study [64] demonstrated that both the canonical and alternative NF-kB pathways are constitutively activated in diffuse large B-cell lymphoma, in which NIK kinase aberrantly accumulates in the neoplastic cells due to constitutive activation of BAFF-R, through interaction with autochthonous BLyS ligand, finally leading to autonomous lymphoma cell growth and survival. These results are a very important contribution to elucidating the mechanisms involved in abnormal NF-kB activation in neoplastic B-cells and to better defining new therapeutic approaches for patients with lymphoma, and possibly also those with pre-lymphomatous diseases. A critical step could be the acquisition of autocrine BLyS expression capacity, with intervention at this point perhaps essential to reducing the risk of subsequent, fully irreversible neoplastic transformation. B-cell lymphoma is a relevant risk in MCsn patients [65]. This is consistent with the hypothesis that HCV initiates a multistep process of lymphomagenesis in which additional factors (i.e., genetic, environmental, immunological) are involved [44, 47, 48, 66]. The regression of established HCVassociated B-cell lymphoma in MCsn patients after antiviral treatment reflects the role of HCV in lymphomagenesis. However, in 14 patients with HCV-related non-Hodgkin’s lymphoma (splenic lymphomas) who received antiviral therapy, long-term clinico-pathologic and molecular follow-up showed clinical and pathological disease regression after successful antiviral treatment but also persistence of the lymphomatous clone. In fact, monoclonal B-cell expansion was still detectable in the peripheral blood lymphocytes of all the patients, demonstrating an absence of a molecular regression and the persistence of an antigen-driven B-cell proliferation, despite effective clearance of the HCV infection [67]. Therefore, HCV infection may represent the initiation step, which is followed by a perpetuation step in which autoantigens, infectious triggers (including HCV itself), cell deregulation at the molecular level, anti-apoptotic factors, and B-cell growth factors, such as BLyS, may sustain, to differing degrees, autoimmunity and B-cell lymphoproliferation [44, 68, 69].

15

Up-Regulation of B-Lymphocyte Stimulator (BLyS) in Patients with Mixed Cryoglobulinemia

INFECTION + AUTOIMMUNITY

HCV targeting

AUTOIMMUNE DISEASE

Direct B cell targeting

Indirect B cell targeting

Fig. 15.2 HCV targeting, direct and indirect B cell targeting. HCV infection and autoreactivity may play different roles in sustaining B-cell autoimmunity or autoimmune disease and lymphoproliferation in different cases of MCsn. There is a strong rationale for therapy directed against the infectious triggering antigen or for an approach based on direct or indirect B-cell targeting, depending on the specific case and the disease stage. Sequential or combination approaches also merit investigation

15.5

BLyS and HCV Infection: Implications for Treatment Strategies in MCsn

Consistent with the latter statement, direct targeting of the infectious trigger or the B-cell autoimmune and lymphoproliferative disorder (e.g., by means of anti-CD20 treatment) or indirect B-cell targeting (e.g., by drugs blocking B-cell growth/anti-apoptotic factors) may prove effective in HCV-related MCsn [50] (Fig. 15.2). Antiviral therapy against HCV has been shown to significantly increase BLyS levels in CHC patients. IFN-a, rather than ribavirin, may lead to increased BLyS, as previously demonstrated by in vitro studies [4]. This effect was independent from the concomitant HCV-RNA clearance, although two studies noted a trend toward BlyS normalization after suspension of antiviral treatment only in patients who achieved a virological response [44, 46]. Those studies reported that the up-regulation of BLyS levels during antiviral therapy appeared to be reversible [44, 46]. IFN therapy can be effective in HCV-related MCsn, mainly in patients who become HCV-RNA-negative [70]. However, MCsn patients may also show persistently active disease despite viral RNA negativization [71], with persistent positivity of RF and cryoglobulins, i.e. the autoimmune and lymphoproliferative disorder persists. Finally, some MCsn manifestations, such as neuropathy, nephritis, and skin ulcers, may worsen

123

[72]; indeed, the onset of MCsn was reported after IFN therapy effective against HCV infection [73, 74]. Therefore, it cannot be ruled out that IFN-a related BLyS up-regulation in part contributes to these effects in predisposed individuals. BLyS may favor the survival of RF-positive B-cells, which may undergo antigen stimulation by different immune complexes also in the absence of the original HCV trigger [50, 75]. Anti-CD20 therapy similarly up-regulates the BLyS system. An increase in BLyS soon after B-cell depletion therapy with rituximab (RTX) was reported in SLE [76], RA [77], and SS [54] patients, as well as in MCsn patients [59] (Fabris M, personal communication). Both antiviral therapy and RTX may then also favor biological mechanisms implicated in the persistence of B-cell expansion, through BLyS up-regulation. Accordingly, a therapeutic strategy specifically targeting BLyS may be appropriate in MCsn [50]. Anti-BLyS therapy with belimumab appears to be effective and safe in SLE [78, 79] and was partially effective in RA. A trial with belimumab in the treatment of SS patients is ongoing in our institution. Other factors or mechanisms sustaining B-cell expansion, e.g., IL-6, the CD40/40 L system, or the B7/CD28 system, may also be successful targets. Finally, the sequential or combined use of the three different treatment strategies (anti-viral, anti-B cell direct, anti-B cell indirect) also deserves attention [80].

15.6

Conclusion

The discovery of BLyS and thus its possible role in HCV infection, B-cell proliferation, and autoimmunity, including the development, persistence, and evolution of MCsn, is currently a topic of intensive research. Strikingly, these observations provide the basis for a model with which to better dissect the link between infection, autoimmunity, and lymphoproliferation in other autoimmune and lymphoproliferative diseases of unknown etiology. Furthermore, determinations of BLyS levels and genetics in patients with MCsn and other autoimmune diseases may allow better classification of these patients in addition to providing predictors of response. Finally, different anti-BLyS treatment options are available, with phase III trials successfully completed. The application of these treatments, as for other biologics, should result in a better understanding of disease biology.

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Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis

16

Sabino Russi, Silvia Sansonno, Gianfranco Lauletta, Domenico E. Sansonno, and Franco Dammacco

16.1 Introduction Chronic active liver disease is an inflammatory disorder in which several distinct etiologies and pathogenetic mechanisms have been recognized [1]. Within the inflamed liver, there is an accumulation of lymphoid and myeloid cells, including T and B cells [2]. Local activation of these cells is thought to play an essential role in perpetuating the chronic inflammatory process and enhancing liver damage [3]. T and B cells frequently accumulate in the portal tracts, where they become part of follicle-like structures with features of germinal centers [4]. At these sites, both local differentiation of follicular dendritic cells and plasma cells and antibody production may occur [5]. Moreover, the development of these germinal-center-like structures appears to contribute to the pathogenesis of the disease, as evidenced by functional and molecular analyses showing that they are characterized by B-cell oligoclonal/monoclonal expansions [6, 7]. These B-cell clonotypes are of B-cell origin, indicating that IgH VDJ mutational activity is up-regulated in the liver microenvironment [8]. It has also been shown that distinct B-cell expansions contribute to the formation of intraportal follicle-like structures that possibly represent peculiar features of hepatitis C virus (HCV) infection [9]. In this context, sequence analyses of IgH CDR-3 gene segments have revealed a wide range of variations likely to be the result of an antigen-driven response [10]. S. Russi (*) Department of Internal Medicine and Human Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

The occurrence of B-cell clonal expansions in the liver of HCV-infected patients has been found to deeply influence the clinical picture, in that they are strictly related to mixed cryoglobulinemia (MC) and, in general, to lymphoproliferative features, including the unexplained high serum levels of rheumatoid factor (RF) and a monoclonal gammopathy of undetermined significance [8]. The relationships between the emergence and persistence of intrahepatic or circulating B-cell clonotypes and HCV infection are not known. Accumulating evidence indicates that certain chemokines are critical to providing the appropriate environment for the activation and expansion of naïve lymphocytes in response to signals delivered by antigen-presenting cells [11]. B-cell-attracting chemokine-1 (BCA-1, CXCL13), also referred to as B-lymphocyte chemoattractant, is a member of the CXC subtype of the chemokine superfamily [12]. It is essential for secondary lymphoid tissue development and the attraction of lymphocytes within microenvironments [13]. The BCA-1 gene maps to chromosome segment 4q21 and encodes a putative protein of 109 amino acids [12]. Within the human BCA-1 sequence, an arginine residue separates two of the four conserved cysteine residues that are peculiar to CXC chemokines [12, 13]. The primary BCA-1 receptor is CXCR5, a seven-transmembrane G-protein expressed by B lymphocytes [14], follicular helper T cells [15], osteoblasts [16], podocytes [17], and skinderived dendritic cells [18]. Interestingly, BCA-1 and CXCR5 knockout mice exhibit similar abnormalities, including a deficient development of peripheral lymphoid organs, impaired B-cell translocation to the B/T cell boundary, and reduction of the antibody response [19, 20]. BCA-1 is constitutively expressed in the

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_16, © Springer-Verlag Italia 2012

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128

300 Serum BCA-1 (pg/mL)

B-cell follicles of secondary lymphoid organs [12], in pleural and peritoneal cavities [19], and in ectopic lymphoid follicles within the synovium of patients with rheumatoid arthritis [21]. BCA-1 protein and the transcription of its gene have been determined in MC patients, leading to the hypothesis that BCA-1 contributes to the pathogenesis of cryoglobulinemic vasculitis by B-cell deregulation.

S. Russi et al.

p < 0.02

p < 0.007

200 p < 0.01 100

16.2 BCA-1 Levels in the Serum of MC Patients With MC

16.3 BCA-1 Expression in Tissues BCA-1 protein, as studied by indirect immunofluorescence in skin biopsy samples of MC patients, is mostly expressed along the collagen bundles and involves the superficial dermis, with variable mild/ deep dermal extension in patients with active vasculitis (Fig.  16.3). By contrast, BCA-1-protein deposition was not detected in skin biopsy tissues from healthy subjects or from HCV-infected patients with-

Healthy Controls

Fig. 16.1  BCA-1 serum levels in HCV-infected patients with and without MC and in healthy controls Cryoglobulinemic Vasculitis 900

Active

Non-active

800 Serum BCA-1 (pg/mL)

Serum measurements of BCA-1 are reported in Fig. 16.1. Compared with healthy blood donors, mean serum BCA-1 levels were higher in patients with MC than in those without the disease. It was also shown that BCA-1 serum levels were significantly higher in MC patients than in patients without MC. No correlation has been established between BCA-1 concentration and circulating viral load, liver histology activity index, grade of liver fibrosis, ALT activities, cryocrit percentages, serum IgM concentration, RF activity, or C4 levels. We stratified MC patients according to the occurrence of active or non-active cutaneous vasculitis. Although not reaching significance, the average cryocrit value was lower in patients with active vasculitis, whereas type III MC occurred more frequently in those with non-active vasculitis. Serum IgM levels, RF activity, complement C4 concentration, and the frequency of nephropathy and peripheral neuropathy were not significantly different in the two subgroups. Conversely, a significantly higher (p < 0.001) BCA-1 serum concentration was demonstrated in patients with active cryoglobulinemic vasculitis (Fig. 16.2).

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Fig. 16.2  BCA-1 serum concentrations in MC patients with and without active cryoglobulinemic vasculitis

out MC. Consistent with the immunofluorescence results, RT-PCR evidenced a definite expression of BCA-1 mRNA in extracts of frozen skin samples prepared from patients with cryoglobulinemic vasculitis (data not shown) whereas amplifiable product was not demonstrated in patients with non-active vasculitis nor in controls. To establish the contributions of the different etiologies to the inflammatory process of cutaneous vasculitis, BCA-1 protein and mRNA expression were investigated in a total of 16 skin biopsy samples from HCV-negative patients: 3 with hypersensitivity ­vasculitis, 4 with HBV-associated necrotizing vas-

16  Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis

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thus established: (i) fine and coarse dot-like immunofluorescence, diffusely extending to the entire region of the portal tract or confined to defined areas (Fig.  16.4a); (ii) granular bead-like deposits mimicking follicle-like structures within portal tracts (Fig.  16.4b). Furthermore, sinusoidal inflammatory cells tested positive for BCA-1 (Fig. 16.4c). Indeed, a BCA-1 follicle-like labeling pattern and intrasinusoid positivity were more frequently observed in samples from patients with MC (83%) than in those from patients without the disease (31%). Sometimes, the two patterns of BCA-1 immunodetection coexisted within the same section. Fig. 16.3  Demonstration of BCA-1 protein in skin tissue samples by immunofluorescence staining. Deposits of immune reactants (green fluorescence) are found along collagen bundles of dermis

culitis, 5 with Henoch-Schönlein purpura, and 4 with HBV and HCV-negative cryoglobulinemic vasculitis. In these patients, BCA-1 protein was expressed in restricted areas of some sections, whereas in others it was localized within or around focal inflammatory infiltrates. In every case, protein expression paralleled that of specific mRNA. BCA-1 immunoreactive deposits were also identified in liver biopsy samples from patients with and without MC. Representative results of BCA-1 protein immunodetection are shown in Fig.  16.4. Hepatocytes and bile duct epithelium did not stain for BCA-1 whereas the protein was intensely expressed in inflammatory cells located within portal tracts and, less frequently, in the sinusoids. Two main immunofluorescence patterns of BCA-1 expression were

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Fig. 16.4  BCA-1 immune deposits in the liver of HCV-infected patients. (a) BCA-1 deposits are homogeneously distributed in an enlarged portal tract. (b) The follicle-like appearance of por-

16.4 Analysis of BCA-1 with Laser Capture Microdissection (LCM) in Liver Tissue Quantitative real-time RT-PCR for specific BCA-1 mRNA expression was performed using nucleic acids recovered from LCM-treated microsamples. After microscopic control of tissue preservation and demonstration of RNA integrity, liver biopsy sections were subjected to LCM. An example of the LCM-derived portal tract is shown in Fig. 16.5a–c. Quantitative differences in specific BCA-1 mRNA expression in the livers of MC patients were defined and the results compared with the amount of BCA-1 protein, normalized to b-actin levels, in portal tracts (Fig.  16.5d). In MC patients with active vasculitis, BCA-1 mRNA levels were four- to 15-fold higher than in MC patients with non-active vasculitis and five- to 30-fold higher than in patients without MC.

c tal tract structures containing BCA-1 protein. (c) BCA-1 is present on intrasinusoid inflammatory cells and along sinusoidal walls

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Fig. 16.5  BCA-1 mRNA levels in laser-capture microdissection-derived portal tracts. (a–c) Examples of portal tracts ­isolated by LCM technique. (a) Section of liver biopsy showing an enlarged portal tract with a heavy inflammatory infiltrate and lymphoid aggregate, resembling a lymphoid follicle. (b) Cleared area remaining in the context of the tissue section after complete

dissection of the portal structure. (c) Dissected portal tract in tube-cap containing lysis buffer for molecular analysis. (d) Quantitative real-time RT-PCR performed on nucleic acids extracted from microdissected portal tracts from HCV-infected patients with and without MC and from controls. Data are means plus or minus SD for each group

16.5 Effects of Therapies on BCA-1 Serum Levels

In spite of the dramatic improvement of cryoglobulin-related signs and symptoms, mean serum BCA-1 levels remained largely unchanged before (245 ± 86 pg/ mL) and after (256 ± 116 pg/mL) antiviral therapy. No significant differences between responders and nonresponders (266 ± 129 pg/mL) were found for baseline levels of BCA-1. A representative pattern is shown in Fig.  16.6a. Serial BCA-1 levels did not vary significantly during 12 months of pIFN-a/RBV combination therapy in an HCV-1b-responsive patient. Patients who were shown to be refractory or who relapsed after pIFN-a/RBV combination therapy were treated with rituximab, an anti-CD20 monoclonal antibody. An intravenous infusion of rituximab was administered once a week for four consecutive weeks, with the levels of BCA-1 serially determined at baseline and before the start of each rituximab ­infusion. A

Due to the potential importance of B cells in MC pathophysiology, changes in BCA-1 levels were investigated in patients who underwent different therapeutic protocols, including combined pegylated (p)IFN-a/ RBV, rituximab, and corticosteroids (CS) treatments. Among the 20 MC patients, 12 (60%) showed a complete response to pIFN-a/RBV. The therapeutic response was characterized by rapid improvement of clinical signs and symptoms, including the disappearance of purpura and a decline of the cryocrit. Ten (83%) of these patients achieved a sustained virological response, whereas in the remaining two patients relapse occurred within 3–5 months after discontinuation of therapy.

16  Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis

c­ omplete therapeutic response was achieved in 62% of the patients. In step with the reduction in cryocrit levels, there was a significant increase of BCA-1 in all patients but one. This, indeed, was a short-lived change, in that a rapid return to baseline occurred after the first week, after which the level remained unchanged. A representative result is shown in Fig. 16.6b. The three patients unresponsive to rituximab were treated with CS, and BCA-1 serum variations were serially measured. Remarkably, a significant decrease in the serum BCA-1 concentration was noted in all of them (Fig.  16.6c), accompanied by an objective improvement of purpuric lesions. A significant decrease in BCA-1 occurred during the second month of CS therapy, whereas no parallel changes in cryocrit levels could be demonstrated.

16.6 Discussion Similar to other viruses, HCV plays a critical role in a wide variety of inflammatory processes by regulating the expression of transcriptional factors and proinflammatory genes, including tumor necrosis factor and members of its superfamily, interleukins, and chemokines [22]. Chemokines have been shown to orchestrate migration to and preferential sequestration of B and T cells in HCV-infected compartments [23]. Indeed, an increased number of circulating B cells has been demonstrated in these patients, likely reflecting the deregulation of B-cell traffic [24, 25]. Homeostatic trafficking of B cells is mainly regulated by the chemokine BCA-1, through interaction with its only known receptor, CXCR5, expressed on all mature circulating B cells and on a subset of memory CD4 T cells in healthy people [13, 14]. BCA-1 levels were found to be elevated in those with chronic HCV infection compared with healthy controls, with the highest levels occurring in HCVinfected MC patients. Usually, chemokines associate with endothelial cells and the extracellular matrix near the site of their production, but elevated levels of specific chemokines have been reported in the serum of HCV-infected patients, including those that bind CXCR3 (CXCL9, CXCL10, CXCL11) [26]. It can be inferred that the high serum levels of these chemokines are a consequence of their high local production. In particular, BCA-1 expression may be induced by the

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ongoing hepatic inflammation, which maintains the pathological process in the tissue by attracting additional lymphocytes, leading to chronic damage. No direct relation has been demonstrated between serum concentrations of BCA-1 and either the fibrosis score or the inflammatory index of liver histology. This strongly emphasizes the major role of inflammatory cells in the production of large amounts of BCA-1 within the portal tracts. The mechanism(s) underlying the difference in BCA-1 production between HCV-infected patients with and without cryoglobulins remains to be elucidated. In this context, the formation of follicle-like structures in the portal tracts of the liver of patients with chronic HCV infection may be considered the morphological counterpart of ectopic lymphoid tissue, which includes naïve B-cells in the central zone surrounded by mature B and T cells [27]. The more frequent formation of intraportal lymphoid follicles in MC patients than in patients without MC has been reported [28]. It is assumed that these structures contribute to antigen presentation in situ, to clonal expansion of antigen-specific B and T cells, and to switching from an acute inflammation to a chronic one [29]. BCA-1 expression in MC patients has been observed in livers in which follicle-like structures have been morphologically recognized, suggesting a major role for the protein in the organization and maintenance of ectopic lymphoid tissue. BCA-1 chemokine may attract B cells and initiate the formation of germinal centers, thus contributing to the development of chronic inflammation [30, 31]. A genetic polymorphism of BCA-1 could explain the altered levels of BCA-1 in MC patients. Although there are no data in direct support of this possibility, several high-producer alleles of pro-inflammatory cytokines are associated with HCV infection [32, 33]. Whether a BCA-1 high-producer allele is present in MC patients remains to be explored. Another possibility is that T-regulatory CD4+ CD25+ cells, shown to be defective in MC patients [34], influence the production of chemokines by stromal cells. This hypothesis also deserves further investigation. Of note, no direct correlation between BCA-1 serum levels and circulating viral load has been reported. Although these findings may indicate the lack of a direct effect of the virus on the sources of BCA-1 production, they do not highlight the in situ

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Fig. 16.6  BCA-1 serum changes in MC patients after different therapeutic protocols. (a) Representative pattern of a responsive patient who underwent standard pIFN-a/ RBV combination therapy; (b) patient refractory to standard therapy who was treated with the anti-CD20 monoclonal antibody rituximab; (c) patient unresponsive to rituximab therapy who was treated with corticosteroids

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immunological disorders [35]. Indeed, evidence of productive HCV infection was unequivocally obtained in the lymphomonocytes of MC patients but not in those without MC, implying lymphoid compartmen-

16  Role of B-Cell-Attracting Chemokine-1 in HCV-Related Cryoglobulinemic Vasculitis

talization of active viral replication [36]. Thus, it can be speculated that productive lymphoid-cell infection by HCV particles in MC patients can result in BCA1-producing cells. The demonstration of BCA-1 in the skin of cryoglobulinemic patients with active cutaneous vasculitis indicates its involvement in the pathogenesis of tissue damage. No previous data on BCA-1 expression in the skin of these patients are available for comparison with our findings. At variance from cutaneous lymphoproliferative B-cell disorders, in which BCA-1 is detected as a cell-associated molecule consistently expressed by neoplastic B cells and follicular dendritic cells within lymphoid infiltrates [37], we observed a staining pattern consisting of diffuse expression of BCA-1 protein in the dermis along collagen bundles. This feature, together with variable expression in the different areas of the dermis, paralleled the specific mRNA signal, suggesting that BCA-1 is dependent on the upregulation of BCA-1-producing cells. The lack of BCA-1-positive cells in the skin may be the result of interference by cell-surface BCA-1-reacting molecules, e.g., through steric hindrance. Alternatively, skin-derived dendritic cells, under favorable pressure from chemokines and chemokine receptors, may rapidly migrate to lymph nodes [38]. A similar feature of BCA-1 immune reactant deposits has been described in normal and aberrant gut-associated lymphoid tissues [39]. BCA-1 was found to be mainly associated with extracellular fibrils and to a much lesser extent with cells displaying a follicular dendritic cell phenotype. Notably, monocytes/macrophages are potent inducible producers of BCA-1 [40] in inflammatory sites. It has been suggested that extravasated monocytes in inflammatory lesions give rise to progeny cells capable of producing the chemokine [40], as it was observed to occur in the skin of cryoglobulinemic patients with active vasculitis. Although monocytes/macrophages do not constitutively express BCA-1, following activation they are able to secrete it. A primary implication of our findings is the induction of a remarkable leukocyte adhesion by BCA-1 in the presence of continuous blood flow, thus contributing to the selective recruitment into the skin of circulating B and T cells. Direct evidence for the induction of BCA-1 in HCV infection draws attention to its role in affecting the outcome of HCV-associated cryoglobulinemic vasculitis.

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To establish whether BCA-1 changes contribute to the success of therapy, serum levels of this chemokine were monitored throughout the administration of different treatments, including pIFN-a/ RBV combination, rituximab, and CS. Chemokine levels did not change to any significant extent after either a successful response to antiviral therapy or B-cell depletion induced by rituximab whereas a significant decline was determined during and after CS treatment and resulted in resolution of the cutaneous vasculitis. These findings have deep implications for understanding the pathogenesis of HCV-related cryoglobulinemic damage. It must be emphasized that, irrespective of viral load or cryoglobulin levels, clinical signs and symptoms abated and vasculitis resolved in CS-treated patients. Indeed, the effective control of inflammation exerted by CS is largely mediated by inhibition of the transcriptional activity of several genes encoding pro-inflammatory cytokines and chemokines [41]. In this context, it has been reported that the BCA-1 gene is the main target of CS therapy [42].

16.7 Conclusions In summary, patients with HCV infection have increased plasma levels of BCA-1, possibly as the result of its overproduction in the liver and skin. In patients with MC, BCA-1 contributes to lymphoid homing in the liver by creating a local microenvironment supportive of focal B-cell aggregation and with structural features remarkably similar to ectopic lymphoid follicles. In these patients, BCA-1 production seems to be involved in the exacerbation of cryoglobulinemic vasculitis, likely through aberrant dissemination of “antigen-priming” information from the liver to extrahepatic sites. Thus, HCV-related vasculitic damage is the result of multifaceted pathogenetic mechanisms in which immune reactants are differentially regulated. Consequently, tailored therapeutic manipulations may be required to treat patients with cryoglobulinemic vasculitis. Acknowledgments This study was supported in part by grants from: the Italian Medical Agency (AIFA), Research and Develop­ment Working Group (contract FARM7SJX), the Italian  Association for Cancer Research (AIRC), and the University of Bari.

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Serum a-Chemokine CXCL10 and b-Chemokine CCL2 Levels in HCV-Positive Cryoglobulinemia

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Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, IIaria Ruffilli, Caterina Mancusi, Ele Ferrannini, and Poupak Fallahi

17.1

Introduction

Chemokines are a group of low-molecular-weight peptides that induce the chemotaxis of different leukocyte subtypes [1]. The major function of chemokines is the recruitment of leukocytes to sites of inflammation, but they also play a role in tumoral growth, angiogenesis, and organ sclerosis [2, 3]. At present, more than 50 chemokines, classified into four major families, have been described [4]. However, only two of these families have been extensively studied and characterized, namely, the CC and CXC chemokines. Chemokines from the CC family are generally chemoattractant to T lymphocytes, monocytes, and natural killer cells, while CXC chemokines attract neutrophils and promote their adherence to endothelial cells [4]. Monocyte chemo-attractant protein 1 (MCP-1/CCL2) is a prototype CC chemokine and has an important function in innate immunity [5]. CCL2 is also a crucial factor for the development of adaptive Th2 responses by directing the differentiation of Th0 cells to Th2 in vitro [6]. Among CXC chemokines, CXCL10 displays strong chemo-attractant activity for Th1 lymphocytes secreting interferon (IFN)-g, with experimental data suggesting that CXCL10 is a reliable marker of aggressive Th1-mediated autoimmune disease. It was previously reported that CXCL10 mRNA was expressed in the hepatocytes of chronic hepatitis C patients, as demonstrated by in situ hybridization, and that the serum CXCL10 level was significantly A. Antonelli (*) Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy e-mail: [email protected]

increased in these patients [7]. CXCL10 is specifically produced by hepatocytes in inflammatory areas of the liver and may help to recruit T cells to the hepatic lesions in chronic viral hepatitis [8]. Furthermore, the expression of CXCL10 by hepatocytes in chronic hepatitis C virus (HCV) infection was shown to correlate with histological disease severity and lobular inflammation [9–11]. More recently, high plasma CXCL10 levels were reported to correlate with a poor outcome of antiviral therapy in patients with hepatitis C [12–15].

17.2

Chemokines and Cryoglobulinemia

Recently, serum CXCL10, IFN-g and tumor necrosis factor (TNF)-a were assayed in 102 patients with hepatitis-C-associated mixed cryoglobulinemia (MC+HCV), in 102 sex- and age-matched patients with type C chronic hepatitis without cryoglobulinemia (HCV+), and in 102 sex- and age-matched healthy controls. Cryoglobulinemic patients showed significantly higher mean CXCL10 serum levels than either controls (p < 0.0001) or HCV+ patients (p < 0.0001) (397 ± 132, 92 ± 53 pg/mL, 280 ± 149 pg/mL, respectively). Moreover, CXCL10 was significantly higher in 30 cryoglobulinemic patients with than without active vasculitis (460 ± 104 vs. 369 ± 139, pg/mL, respectively; p < 0.001). In both groups of MC+HCV patients, i.e., with or without active vasculitis, serum CXCL10 was significantly higher than in HCV+ patients (p < 0.001, p = 0.02, respectively). IFN-g levels did not significantly differ between MC+HCV and either HCV+ patients or controls. Serum TNFa levels were significantly higher in MC+HCV than in HCV+

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individuals or in controls (median [IQR]: 12.0 [9.8], 5.7 [5.4], 1.3 [2.1] pg/mL, respectively; p < 0.0001; Mann-Whitney U-test). Thus, the study demonstrates high CXCL10 and TNF-a serum levels in patients with hepatitis-C-associated cryoglobulinemia. Moreover, in MC+HCV patients increased CXCL10 levels were significantly associated with the presence of active vasculitis [16]. In another study, serum CXCL10 and CCL2 were assayed in 70 consecutive cryoglobulinemic patients and in two control groups (1:1, gender- and agematched) consisting of healthy (controls), or of patients with chronic hepatitis C without cryoglobulinemia. Cryoglobulinemic patients had higher CXCL10 serum levels than either the healthy controls (p < 0.0001) or the hepatitis C patients (p = 0.001) (389 ± 141, 91 ± 51, 311 ± 142 pg/mL, respectively). CXCL10 levels were significantly (p < 0.01) increased in cryoglobulinemic patients with active vasculitis compared to those without (445 ± 108, 339 ± 161 pg/mL, respectively). Cryoglobulinemic patients had significantly higher CCL2 serum levels than controls (p < 0.01) whereas this was not the case compared to hepatitis C patients (541 ± 493, 387 ± 173, and 451 ± 281 pg/mL, respectively). Therefore, this study demonstrated high serum levels of CXCL10 and CCL2 chemokines in cryoglobulinemic patients. Circulating CXCL10 was higher overall in cryoglobulinemic patients with active vasculitis, suggesting a prevalence of the Th1 immune response in this phase of the disease [17]. More recently serum levels of IL-1b, IFN-g, and CXCL10 were evaluated in a series of patients with hepatitis-C-related MC and compared with the levels in 54 sex- and age-matched patients with type C chronic hepatitis without cryoglobulinemia and with those in 54 controls. MC+HCV patients had significantly higher mean serum IL-1b and CXCL10 levels than either the controls (p < 0.01) or the HCV+ patients (p < 0.01). CXCL10 was significantly higher in 14 cryoglobulinemic patients with active vasculitis (necrotizing vasculitis or vasculitic skin ulcers) than in those without (p < 0.001); IL-1b was also increased in cryoglobulinemic patients with active vasculitis (p = 0.06) whereas no differences were observed for serum IFN-g levels [18]. In conclusion, these studies demonstrate significantly higher serum levels of IL-1b in patients with MC+HCV than in either healthy controls or HCV+ patients, with overall higher levels in patients with signs of active vasculitis. Furthermore,

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in the presence of active vasculitis, serum CXCL10 levels were confirmed to be significantly higher in patients with MC+HCV than in healthy controls. Future studies in larger patient series are needed to evaluate the relevance of serum IL-1b and CXCL10 as clinico-prognostic markers of MC+HCV, as well as their usefulness in the therapeutic approach to these patients.

17.3

Chemokines, Cryoglobulinemia, and Autoimmune Thyroiditis

We previously demonstrated a high frequency of autoimmune thyroid disorders in cryoglobulinemic patients [19]. However, the immunological base of this association was not clear. The pattern of thyroid disorders observed in mixed cryoglobulinemia is characterized by the presence of increased circulating levels of antibodies against thyroid peroxidase (AbTPO) and an increased risk of hypothyroidism in AbTPO-positive individuals. This pattern is similar to that observed in IFN-a-treated patients [20, 21]. Furthermore, NS5A and core proteins, either alone or by the synergistic effect of cytokines (IFN-g and TNF-a), were shown to be capable of up-regulating CXCL10 and MIG (monokine induced by IFN-g) gene expression and secretion of the respective proteins in cultured human hepatocyte-derived cells [10]. This finding suggests that CXCL10 produced by HCV-infected hepatocytes plays a key role in regulating T-cell trafficking into a Th1-type inflammatory site, such as liver tissue during chronic HCV infection, by recruiting Th1 lymphocytes, which in turn secrete IFN-g and TNF-a, thus inducing CXCL10 secretion by hepatocytes and perpetuating the immune cascade [10]. The increased expression of intrathyroidal IFN-g is associated with hypothyroidism [22]. Furthermore, IFN-g induces CXCL10 secretion in thyrocytes [23], a process that might be involved in stimulating an autoimmune reaction [24]. Recently, high levels of CXCL10 were demonstrated in patients with autoimmune thyroiditis (AT) and in general in the presence of hypothyroidism [24]. In addition, the involvement of Th1 immune response in the induction of AT [25], Graves’ disease and Graves’ ophthalmopathy was reported [26]. Our preliminary data also support the involvement of IFN-g and CXCL10 in patients with

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HCV infection and MC+HCV [27, 28], in the presence of AT, and hypothyroidism. At the same time, HCV is known to be able to infect thyroid cells [29, 30] but the possible consequences of the infection on thyrocyte function, vitality, and immunogenicity remain to be clarified. Based on these findings, it has been speculated that HCV thyroid infection up-regulates CXCL10 gene expression and secretion of the chemokine in thyrocytes (as previously shown in human hepatocytes) [10], thereby recruiting Th1 lymphocytes secreting IFN-g and TNF-a, inducing CXCL10 secretion by thyrocytes, and thus perpetuating the immune cascade. This may ultimately lead to the appearance of thyroid autoimmune disorders in genetically predisposed individuals. Support for this hypothesis comes from a recent study that evaluated CXCL10 serum levels in MC+HCV patients, in the presence or absence of AT. CXCL10 was assayed in 50 MC+HCV patients without AT, in 40 MC+HCV patients with AT (MC+AT), in two gender- and age-matched control groups [50 healthy controls (without HCV or AT; controls); 40 controls with AT (without HCV and mixed cryoglobulinemia; controls +AT)]. CXCL10 was significantly higher: (1) in controls + AT than in controls; (2) in MC+HCV patients than in controls; and (3) in MC+AT patients than in controls, controls + AT, or in MC+HCV patients. CXCL10 was significantly higher in MC+AT patients with than without aggressive thyroiditis (thyroid hypoechogenicity, or hypothyroidism). In conclusion, our study was the first to demonstrate high serum levels of CXCL10 in MC + HCV patients and showed that serum CXCL10 is significantly higher in MC + AT patients than in MC+HCV patients [31, 32].

cytopathic effect of HCV at the islet cell level. However, more recently, a possible autoimmune induction of diabetes by HCV was proposed. In fact, the type of diabetes manifested by patients with HCV chronic infection is not the typical T2D. Three studies [34, 36, 37] reported that HCV+ patients with T2D were leaner than T2D controls and showed significantly lower LDL-cholesterol as well as systolic and diastolic blood pressure. Furthermore, non-organ-specific autoantibodies were detected more frequently in MC+HCV patients with T2D than in non-diabetic MC+HCV patients (34% vs. 18%) [34]. An immune-mediated mechanism for MC+HCV associated diabetes has been postulated [34]. HCV infection of islet cells [35] may act by up-regulating CXCL10 gene expression and secretion of the protein, thus recruiting Th1 lymphocytes, which secrete IFN-g and TNF-a, in turn inducing CXCL10 secretion by islet cells and thereby perpetuating the immune cascade. The end result may be the appearance of islet cell dysfunction in genetically predisposed individuals. This hypothesis recently gained support from the findings of a study carried out by our group, in which CXCL10 serum levels were evaluated in HCV+ patients, in the presence or absence of T2D. CXCL10 was assayed in 45 HCV+ patients without T2D, in 40 HCV+ patients with T2D, and in a gender- and agematched control group (45 healthy controls, without HCV or T2D). CXCL10 was significantly higher: (1) in HCV+ patients than in controls and (2) in HCV+ with T2D patients than in either controls or HCV+ patients without T2D. In conclusion, our study demonstrated higher serum CXCL10 levels in HCV+ patients with than without T2D [20].

17.4

17.5

Chemokines, Cryoglobulinemia, and Type 2 Diabetes

An association between HCV infection and diabetes has been reported by several clinical epidemiologic studies, beginning in 1994 [33]. More recently, an association between HCV infection, in patients with chronic hepatitis but without cirrhosis, and type 2 diabetes (T2D) was reported in MC+HCV patients [34]. The mechanisms involved in the association between HCV infection and diabetes are not well clarified. A direct destruction of islet cells by HCV has been hypothesized. Masini et al. [35] demonstrated a direct

Conclusion and Perspectives

The studies discussed above provide evidence for significantly high serum CXCL10 levels in patients with MC+HCV and, in general, in those with active vasculitis, thyroiditis or diabetes, suggesting a Th1 immune process as the immunological base of the association between HCV infection and the appearance of these disorders. Future studies in larger patient series are needed to evaluate the relevance of serum CXCL10 as a clinico-prognostic marker of MC+HCV, as well as its usefulness in the therapeutic approach to these patients.

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A. Antonelli et al. 17. Antonelli A, Ferri C, Fallahi P et al (2009) CXCL10 and CCL2 serum levels in patients with mixed cryoglobulinaemia and hepatitis C. Dig Liver Dis 41:42–48 18. Antonelli A, Ferri C, Ferrari SM et al (2010) Serum concentrations of interleukin 1beta, CXCL10, and interferongamma in mixed cryoglobulinemia associated with hepatitis C infection. J Rheumatol 37:91–97 19. Antonelli A, Ferri C, Fallahi P et al (2004) Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia. QJM 97:499–506 20. Antonelli A, Ferri C, Ferrari SM et al (2009) Endocrine manifestations of hepatitis C virus infection. Nat Clin Pract Endocrinol Metab 5:26–34 21. Antonelli A, Ferri C, Fallahi P (2009) Hepatitis C: thyroid dysfunction in patients with hepatitis C on IFN-alpha therapy. Nat Rev Gastroenterol Hepatol 6:633–6365 22. Caturegli P, Hejazi M, Suzuki K et al (2000) Hypothyroidism in transgenic mice expressing IFN-gamma in the thyroid. Proc Natl Acad Sci USA 97:1719–1724 23. Garcia-Lopez MA, Sancho D, Sánchez-Madrid F et al (2001) Thyrocytes from autoimmune thyroid disorders produce the chemokines IP-10 and Mig and attract CXCR3+ lymphocytes. J Clin Endocrinol Metab 86:5008–5016 24. Antonelli A, Rotondi M, Fallahi P et al (2004) High levels of circulating CXCL10 are associated with chronic autoimmune thyroiditis and hypothyroidism. J Clin Endocrinol Metab 89:5496–5499 25. Antonelli A, Rotondi M, Fallahi P et al (2005) Increase of interferon-g inducible a chemokine CXCL10 but not b chemokine CCL2 serum levels in chronic autoimmune thyroiditis. Eur J Endocrinol 152:171–177 26. Antonelli A, Rotondi M, Ferrari SM et al (2006) Interferongamma-inducible alpha-chemokine CXCL10 involvement in Graves’ ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. J Clin Endocrinol Metab 9:614–620 27. Antonelli A, Ferri C, Fallahi P et al (2006) Thyroid disorders in chronic hepatitis C virus infection. Thyroid 16: 563–572 28. Antonelli A, Ferri C, Fallahi P et al (2005) Extrahepatic manifestations of hepatitis C virus: the thyroid disorders. Recenti Prog Med 96:370–381 29. Gowans EJ (2000) Distribution of markers of hepatitis C virus infection throughout the body. Semin Liver Dis 20: 85–102 30. Bartolomé J (2008) Detection of hepatitis C virus in thyroid tissue from patients with chronic HCV infection. J Med Virol 80:1588–1594 31. Antonelli A, Ferri C, Fallahi P et al (2008) High values of CXCL10 serum levels in patients with hepatitis C associated mixed cryoglobulinemia in presence or absence of autoimmune thyroiditis. Cytokine 42:137–143 32. Antonelli A, Ferri C, Fallahi P et al (2008) Alpha-chemokine CXCL10 and beta-chemokine CCL2 serum levels in patients with hepatitis C-associated cryoglobulinemia in the presence or absence of autoimmune thyroiditis. Metabolism 57:1270–1277 33. Noto H, Raskin P (2006) Hepatitis C infection and diabetes. J Diabetes Complications 20:113–120

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36. Antonelli A, Ferri C, Fallahi P et al (2005) Hepatitis C virus infection: evidence for an association with type 2 diabetes. Diabetes Care 28:2548–2550 37. Skowronski M, Zozulinska D, Juszczyk J et al (2006) Hepatitis C virus infection: evidence for an association with type 2 diabetes. Diabetes Care 29:750; author reply 751

Part V Clinical Manifestations of Cryoglobulinemia

Experimental Models of Mixed Cryoglobulinemia

18

Charles E. Alpers, Tomasz A. Wietecha, and Kelly L. Hudkins

18.1

Introduction

The definition of cryoglobulins, and their classification into three broad categories based on the characterization of their immunoglobulin components as being of monoclonal or polyclonal origin, has been covered in other chapters of this monograph. In this chapter, we consider the availability of relevant animal models for studies of this injury process and recent insights that have been gleaned from these models, with special reference to cryoglobulinemic-associated renal injury. The urgency for good model systems is driven by the association of type II and type III cryoglobulinemia with preceding infection by hepatitis C virus (HCV). We and others have previously shown that, in humans, the principal renal manifestation of chronic HCV infection is development of a membranoproliferative glomerulonephritis (MPGN), most often associated with cryoglobulinemia [1–9]. Indeed, it is now understood that HCV is associated with the great majority of cases of what were previously thought to be idiopathic MPGN and essential mixed cryoglobulinemia [4, 8, 10]. The pathogenetic mechanisms for development of cryoglobulinemia or MPGN have not been defined, despite their clear linkage to chronic hepatitis C infection [11]. A major obstacle to better understanding the pathogenetic mechanisms of these diseases has been C.E. Alpers (*) Department of Pathology, University of Washington, Seattle, WA, USA Division of Nephrology, Department of Medicine, University of Washington, Seattle, WA, USA e-mail: [email protected]

the lack of good model systems for study. An animal model of HCV infection that both reproduces the spectrum of disease observed in chronically infected patients and is conducive to mechanistic studies, allowing testing of potential treatments, is currently lacking [12–16]. However, as will be discussed, there are well defined models of cryoglobulinemia in animal model systems, although their number remains few. Such models have generally been confined to murine systems; we are unaware of model systems of cryoglobulinemia involving mammals larger than rodents.

18.2

Modeling Human Cryoglobulinemia and Renal Disease

As reviewed previously, cryoglobulinemic renal injury in humans typically involves the glomeruli and is usually manifest as a MPGN pattern of injury, often with distinctive pathologic features that are highly suggestive of a cryoglobulinemic etiology [1, 3, 4, 10]. These features include accentuated lobulation of the tuft architecture and, depending on the acuteness/chronicity of the glomerular involvement, a marked infiltration of glomerular capillaries by leukocytes, typically monocytes (a characteristic feature of early and/or active glomerular involvement). Other features of early and/or active glomerular injury in this disorder include some combination of increased glomerular cell number (typically mesangial hypercellularity), mesangiolysis, capillary endothelial swelling, duplication of capillary basement membrane matrices, and, in some cases,

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intracapillary accumulations of globular eosinophilic material composed of precipitated immune complexes consisting, at least in part, of the cryoglobulins. As the disease evolves into a more chronic phase, the concomitant histologic changes become those of increased mesangial matrix, persistent increased mesangial cellularity, and more pronounced glomerular capillary basement membrane duplication (also referred to as “splitting” in many descriptions of this disorder). Electron microscopy of affected glomeruli may demonstrate that the deposited immune complexes, typically located in the mesangium and subendothelial portions of capillary walls, have a nondescript granular electron-dense appearance or a finely fibrillar and/or tactoid and/or microtubular pattern of substructural organization, each distinctive features of certain cryoglobulin deposits. Depending upon numerous host response factors, including engagement of complement and Fc receptors on leukocytes and activation states of the infiltrating leukocytes, the extent of immune complex deposition may appear relatively scanty, as a consequence of phagocytosis or other mechanisms for disposal of immune complexes. Uncommonly, cryoglobulins may deposit in small arteries, arterioles, or extraglomerular capillaries, where they may give rise to an acute and sometimes necrotizing vasculitis. Recapitulation of these features of human cryoglobulinemic MPGN, with mesangial and glomerular capillary deposits of cryoglobulin containing immune complexes and a monocytic inflammatory response, should be the goal for an ideal animal model of cryoglobulin induced renal injury. Such a model could then be used to investigate mechanisms of injury and test the efficacy of interventions that may be of therapeutic benefit in this disorder.

18.3

Murine Models of Cryoglobulinemia

As recently reviewed, several murine models of cryoglobulinemia have been produced [3]. One of these models is induced by infusion of murine hybridoma cells obtained from a mouse strain exhibiting features of systemic lupus and concurrent cryoglobulinemia. The hybridoma produces a monoclonal IgG3 that has properties of a type I monoclonal cryoglobulin. Further

refinement of this model included the creation of transgenic mice that overexpress this pathogenic IgG3. The model has been useful for studies of the neutrophil (not monocyte) inflammatory infiltrates in glomeruli that are characteristic of this system, studies of the physicochemical properties of the IgG3 cryoglobulin, and studies that identified the presence of serpins in glomeruli as a potential mediator of injury [17–19]. As a model of type I monoclonal cryoglobulinemia, with atypical inflammatory infiltrates compared with human cryoglobulinemic MPGN, this model has significant limitations for studies of HCV associated mixed cryoglobulinemia and renal disease. Most other murine models of cryoglobulinemia are models of systemic lupus (e.g., BXSB mice), in which the disorder develops as part of a broad program of autoimmune activity. The robustness of the cryoglobulinemia in those models is difficult to appreciate from published reports. In addition, the extensive autoimmune responses in these mice limits their utility for studies of mechanisms of injury consequent to HCV-induced mixed cryoglobulinemia. In recent years, the largest body of work on mechanisms of cryoglobulinemic MPGN in animal models has come from studies of the thymic stromal lymphopoietin transgenic (TSLP tg) model of cryoglobulinemia [3, 20–26]. This model involves constitutive overexpression of TSLP, a cytokine that overlaps in activity and receptor binding characteristics with interleukin 7 (IL-7) and which, similar to IL-7, promotes differentiation and expansion of some B lymphocyte populations. More recently discovered activities of TSLP include promotion of T helper type 2 cell responses, promotion of regulatory T cell responses in peripheral tissues and influencing effector functions of granulocytes [27, 28]. A serendipitous observation that the TSLP tg mice developed renal insufficiency led to close examination of their renal and systemic pathologic alterations [26]. These mice were found to have circulating mixed cryoglobulins (type III, containing polyclonal IgG and IgM), immune complex deposition in multiple organs (kidney, lung, liver, skin), and consequent MPGN, pneumonitis, hepatitis, and skin ulceration, all pathologies that may occur as systemic consequences of cryoglobulinemia in humans. Detailed examination of the renal disease in particular revealed all of the features characteristic of human cryoglobulinemic MPGN, including mesangial matrix

18 Experimental Models of Mixed Cryoglobulinemia

expansion, mesangial hypercellularity, thickening and duplication of glomerular capillary walls, glomerular influx of monocytes, intracapillary globular deposits, and mesangial and capillary wall deposits of polyclonal immunoglobulin, which included the intracapillary globules and which were demonstrable by immunofluorescence and by electron microscopy. Some of the deposited immune complexes exhibited a microtubular substructure detectable by electron microscopy. Other features that have established the TSLP tg mouse as an exceptionally good model for cryoglobulinemic injury in general and glomerulonephritis in particular are its robustness (all TSLP tg mice derived from the founder strain develop the disease), the predictable course of the disease (female mice reliably demonstrate early but overt manifestations of MPGN by 30 days of age; male mice by 50–70 days of age) and relatively rapid evolution (the disease is fully manifest in female mice by 50 days of age; in male mice this extends to 120 days of age). The predictability and speed of onset of disease allow interventional studies that can be accomplished within a relatively short time frame for in vivo studies and with relatively small cohorts within the experimental groups, while still retaining statistical power. These features are conducive to studies of interventions intended either to prevent disease progression or to reverse established structural injury. Although not exploited to date, the differences in timing and progression of injury between female and male mice (which do not, however, differ in type or severity of disease manifestations) offers an opportunity to identify processes that may underlie the increased susceptibility of human females to many autoimmune diseases. Studies of these mice to date have produced new mechanistic insights into the development of cryoglobulinemic MPGN in the following areas: the role of complement and Fc receptor engagement in disease progression; the role of monocytes/macrophages in promoting disease progression versus a beneficial role in disease resolution; identification of a potential role for innate immunity in glomerulonephritis, and the demonstration of therapeutic interventions that potentially can be translated into treatment of human cryoglobulinemic MPGN that can both prevent disease progression and, most dramatically, lead to reversal of established structural glomerular injury if the inciting cryoglobulinemic stimulus can be abrogated.

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18.4

Mediation of Glomerular Injury

A prevailing concept in glomerular pathophysiology is that engagement of the classical pathway of the complement cascades by deposited immune complexes leads to recruitment of inflammatory leukocytes. These leukocytes have the capacity to exacerbate pathology by release of injurious effector molecules and to ameliorate injury by contributing to the opsonization and enhanced disposal of immune complexes and/or by otherwise modulating the activities of intrinsic glomerular cells. Complement activation, with recruitment of leukocytes, is initiated through several nonoverlapping pathways that converge at a common step involving cleavage of the complement component C3. Interventions that target the C3 cleavage step are a means of testing the importance of complementmediated injury and has been accomplished by administration of neutralizing antibodies and infusion of inhibitors. An approach to inhibit cleavage of C3 in the TSLP tg model utilized the rodent protein CR1-related gene/ protein y (Crry), an analog of human CR1 that functions like human DAF and membrane co-factor protein in inhibiting C3 convertase, thereby inhibiting the conversion of C3 into its active metabolites [29]. Soluble Crry, which may be the most potent of the circulating complement inhibitors, was shown to be protective of glomerulonephritic injury in the murine anti-glomerular basement membrane (GBM) antibody [30] and MRL/lpr lupus models [31]. The basis for this finding is unclear, at least in the lupus nephritis model, since the clinical benefit in MRL/lpr mice was disassociated from morphological manifestations of injury [31]. Based on studies like these, which demonstrated the efficacy of Crry overexpression in inhibition of glomerulonephritis (GN) in model systems of immune injury, we crossed overexpressing Crry transgenic mice with TSLP tg mice. We found that Crry overexpression was ineffective in ameliorating MPGN or other organ involvement in the TSLP tg cryoglobulinemic model despite demonstrable elevated circulating and tissue levels of Crry [25]. We recognized the importance of concurrent activation of the alternative pathway of complement with its self-amplifying loop, a phenomenon termed “tickover,” which may overcome inhibitory interventions directed primarily against classical pathway activation [32, 33]. Therefore,

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we further tested a role for complement amplification of cryoglobulinemic MPGN by deleting a key element of this portion of the complement cascade, factor B, to eliminate engagement of this pathway in TSLP tg mice and in combined TSLP/Crry tg mice [34, 35]. These maneuvers were also ineffective in ameliorating disease. While these studies cannot be viewed as a definitive test of the value of complement inhibition in cryoglobulinemic MPGN, our results offer a cautionary note for this approach to the treatment of this disorder [36]. A second current concept in glomerular pathophysiology is that a key amplification step of glomerulonephritic inflammatory injury depends on the engagement of specific Fc receptors on leukocytes and potentially on intrinsic renal cells by deposited immunoglobulins. In the case of Fc receptor engagement, it is well established that individual leukocyte FcR can have either activating or inhibitory roles in regulating the inflammatory response. Two classes of these receptors, the inhibitory receptor FcgRIIb and the activating receptors (currently FcgRI, III, and IV, all with a common g-chain) have been well studied [37]. Differences in intracytoplasmic domains result in different intracellular signaling pathway activation; the consequence is that engagement of activating leukocyte FcgRs initiates pro-inflammatory activities, through an ITAM (immunomodulatory tyrosine activation motif), resulting in calcium mobilization, degranulation, macrophage phagocytosis, and cytokine release. Alternately, engagement of FcgRIIb has an opposite effect on inflammatory events, inhibiting calcium mobilization, cell proliferation, degranulation, macrophage phagocytosis, and cytokine release via an ITIM (immunomodulatory tyrosine inhibitory motif) [37, 38]. The importance of these systems for understanding the pathogenetic mechanisms underlying glomerulonephritis has been shown in studies of knockout mice deficient in FcgRIII that were bred with NZB/NZW mice, which develop a spontaneous lupus-like nephritis. These modified NZB/NZW mice still deposited glomerular immune complexes and activated complement but were protected from subsequent inflammatory responses [39]. Related studies in mice deficient in either FcgRIII or FcgRIIb (using an anti-GBM antibody injury model) revealed an accelerated inflammatory injury in FcgRIIb-deficient mice, while FcgRIII-deficient mice demonstrated markedly diminished inflammatory changes [40–42]. These studies suggest a paradigm

C.E. Alpers et al.

whereby coordinate and relative activation of these receptors helps regulate the degree of inflammatory response to a specific injury. Elegant studies using bone marrow chimeras, by Tarzi et al. [43], demonstrated that the principal determinants of renal inflammation in the nephrotoxic nephritis model were carried by FcRs present on leukocytes and not intrinsic renal cells. The relevance of these findings for potential therapies of human disease is accentuated by the development of several novel pharmacological agents targeting this class of receptors, including inhibitory peptides [44], neutralizing antibodies that have been humanized for use in patients [45], soluble receptor antagonists [46], and intravenous immunoglobulin [37, 47, 48]. We have extended the promising findings of FcR regulation of glomerular inflammatory injury in the case of the inhibitory receptor FcgRIIb to our model of murine MPGN and cryoglobulinemia [24] and to cryoglobulinemic hepatitis [49]. By crossing TSLP tg mice with FcgRIIb null mice, we found that renal disease is exacerbated (Fig. 18.1) [24]. We also demonstrated the limitations of an un-nuanced paradigm of a balance of activating and inhibitory Fc receptors as a key determinant of inflammatory disease in cryoglobulinemic MPGN by showing no improvement, and perhaps slight worsening, of renal disease parameters when the common g chain of the activating Fc receptors is constitutively deleted in these mice (Fig. 18.1) [21]. Since increased deposits of immune complexes were detected in the TSLP tg mice crossed with Fcg-chain-deficient mice, we conclude that the importance of the FcgR I, II, and IV in facilitating disposal/phagocytosis of immune complexes outweighs their role in promoting other pro-inflammatory activities. Studies of the TSLP tg mouse have uncovered a third mechanism by which MPGN may be mediated. We have shown that the Toll-like receptor 4 (TLR4) is present in podocytes and is up-regulated in these cells in the evolution of renal disease in these mice at both the mRNA and protein levels [50]. TLR4 recognizes lipopolysaccharide (gram-negative bacteria) and has been implicated in the recognition of endogenous molecules that are generated in the setting of tissue injury, such as fibronectin, fibrinogen, and hyaluronic acid and heparin sulfate fragments [51]. In the specific case of TLR4, engagement of this receptor, and its obligate co-receptor, CD14, activates a signaling pathway of MyD88/IRAK4/TRAF6 to activate NFkB and likely other transcription factors, which in turn regulate

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a

b

c

d

e

f

Fig. 18.1 Effect of modulating Fc receptor expression in the TSLP tg model of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN). (a and b) Two examples of glomeruli from TSLP tg mice showing mesangial expansion, hypercellularity, and segmental duplication of glomerular capillary basement membranes. (c and d) Marked exacerbation of MPGN occurs when the inhibitory Fc receptor, FcRIIb is constitutively

deleted. Intracapillary aggregates of cryoglobulins (arrows) are readily seen in (d). (e) Control TSLP tg mice show MPGN with monocyte influx and splitting of capillary basement membranes, while in (f) deletion of the common g chain of activating Fc receptors FcgRI, II, III, and IV results in either no improvement or slight exacerbation of the glomerular disease. All stained with silver methenamine/H&E counterstain

the release of chemokines and cytokines from both immune and non-immune cell types. Complementing our studies in TSLP tg mice showing prominent up-regulation of a subset of toll-like receptors, notably TLR4, we performed corresponding studies in cultured podocytes. The results showed that expression of TLR4 could be up-regulated following exposure of the cells to TLR4 ligands. The upregulated TLR4 led to the release of chemokines; this release could then be reduced by specific knockdown of TLR4 with siRNA. In aggregate, our studies showed that TLR4 is constitutively expressed by glomerular cells (podocytes), is up-regulated in cryoglobulinemic MPGN, and that its increased expression may mediate glomerular injury by expression of leukocyte-attracting chemokines. These studies thereby link elements of the innate immune system to cryoglobulin-induced injury in solid-organ systems. While the full pathogenetic

significance of this engagement of the innate immune system in cryoglobulinemic MPGN is uncertain at present, the TSLP tg model offers an opportunity to further dissect the role of innate immunity in mediating the monocyte influx that characterizes this disorder as well as other downstream manifestations of injury consequent to deposition of cryoglobulin-containing immune complexes.

18.5

Cryoglobulinemia Is Reversible: Studies Involving Therapeutic Interventions

An advantage of the TSLP tg model of cryoglobulinemia and the consequent multi-organ system involvement including MPGN is the robust and relatively early onset of the disease process. This has the effect of

150 Fig. 18.2 Prevention and reversal of cryoglobulinemic membranoproliferative glomerulonephritis (MPGN) in TSLP tg mice after 8 weeks of treatment with imatinib. (a) Advanced MPGN is present in mice at 19 weeks of age, treated with saline for 8 weeks. (b) All aspects of injury are prevented in mice treated for 8 weeks with imatinib beginning at age 11 weeks. (c and d) Reversal of the advanced MPGN that is present by 18 weeks. (c) A typical lesion of control mice at this age; (d) after 8 weeks of treatment with imatinib, there is full reversal of MPGN. All stained with silver methenamine/H&E counterstain

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a

b

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d

making the model well suited for studies of therapeutic interventions. Based on a prior body of work that has demonstrated the presence of growth factors such as platelet-derived growth factor receptor (PDGF) and transforming growth factor (TGF)-b in glomeruli as a consequence of the MPGN induced in this model [52], we have studied various interventions directed against these growth factors as well as other targets that might prove useful as treatment options for HCV-associated mixed cryoglobulinemia. Among the interventions tested by our laboratory are antibodies directed against PDGF and TGF-b [53, 54]; IFN-a, to test whether its partial efficacy in human disease might be the result of activities independent of its anti-viral effect [55]; and general therapies utilized for the treatment of chronic human kidney disease (renin-angiotensin-aldosterone inhibition using the angiotensin-converting enzyme inhibitor enalapril and the angiotensin receptor blocker losartan [20]). In our studies, direct neutralization of PDGF and TGF-b by systemic administration of

neutralizing antibodies had only limited benefit; benefits resulting from the use of IFN-a were also limited. A more noteworthy finding was that commonly used therapies for patients with chronic kidney disease (enalapril and losartan) could have a dramatic effect in ameliorating the structural and functional (proteinuria) abnormalities of the kidney disease in the TSLP tg model, while not affecting the levels of systemic cryoglobulinemia or significantly altering disease manifestations in some other organs, such as lung [20]. An extraordinary finding was that imatinib, a receptor tyrosine kinase inhibitor that blocks the activity of PDGF receptors as well as several other molecules, had a dramatic effect on the cryoglobulinemia in this model. Imatinib administered early in the lifespan of TSLP tg mice suppressed cryoglobulin production and markedly attenuated the cryoglobulin-induced injury in kidneys and other organ systems. (Fig. 18.2a, b). When imatinib was administered to TSLP tg mice later in life, when MPGN and other organ damage

18 Experimental Models of Mixed Cryoglobulinemia

was well advanced, cryoglobulin production was again markedly suppressed, which in turn remarkably led to reversal of the characteristic MPGN, pneumonitis, hepatitis, and skin ulcerations routinely encountered in these mice (Fig. 18.2c, d) [22]. The complete reversal of the MPGN is extraordinary, and we are unaware of any other instances where this has been accomplished in other animal models of this type of glomerular injury. One implication of these studies is that ongoing cryoglobulin deposition may be required for persistence of cryoglobulin-induced solid-organ injury, such that therapeutic efforts in treating complications of cryoglobulinemia might be most successful if primarily directed towards treating the cryoglobulinemic stimulus. The mechanism by which imatinib suppressed cryoglobulin production was not identified in these studies, but future studies of this process may lead to novel therapeutic approaches for the treatment of cryoglobulinemia as it occurs in humans.

18.6

Summary

While there is as yet no animal model of HCV infection that reproduces HCV-associated type II mixed cryoglobulinemia and its disease manifestations, we have developed a surrogate murine model of cryoglobulinemia that closely resembles HCV-associated diseases as they occur in humans. The model also includes systemic manifestations, the best studied of which is MPGN. The TSLP tg mouse is currently the best-studied animal model of disease manifestations consequent to mixed cryoglobulinemia, but it remains underexploited for both pathogenesis and interventional studies. Using the TSLP tg model, we demonstrated the importance of Fc Receptors (FcRs) in modulating disease [21, 24] and identified components of the innate immune system that may act as potentially important mediators of disease (especially TLR4 and its co-receptor CD14) [50, 56, 57]. Interventions directed against PDGF receptor b [54] and the renin-angiotensin system [20] also have been shown to ameliorate the renal injury of MPGN. Most importantly, we demonstrated that therapy directed against receptor tyrosine kinases in B lymphocytes can largely abrogate cryoglobulinemia and reverse its systemic disease manifestations, including MPGN, pneumonitis, and hepatitis [22]. Demonstration that reversibility of established cryoglobulinemic MPGN can be achieved is an important proof of principle that

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should define a similar goal as we seek and test new therapies for the human disease. Acknowledgements The work reported here was supported by grants from the US National Institutes of Health (DK68802) and an unrestricted grant from the Genzyme Renal Innovations Program.

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35. Wietecha TW, Hudkins KL, Iyoda M et al (2006) Deletion of the murine factor B in thymic stromal lymphopoietin transgenic mice aggravates cryoglobulin-associated membranoproliferative glomerulonephritis. American Society of Nephrology Annual Meeting. San Diego, CA. J Am Soc Nephrol 17:510A 36. Couser WG (2003) Complement inhibitors and glomerulonephritis: are we there yet? J Am Soc Nephrol 14:815–818 37. Nimmerjahn F, Ravetch JV (2008) Fcgamma receptors as regulators of immune responses. Nat Rev Immunol 8:34–47 38. Nimmerjahn F, Ravetch JV (2007) Fc-receptors as regulators of immunity. Adv Immunol 96:179–204 39. Clynes R, Dumitru C, Ravetch JV (1998) Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 279:1052–1054 40. Nakamura A, Yuasa T, Ujike A et al (2000) Fcgamma receptor iib-deficient mice develop goodpasture’s syndrome upon immunization with type iv collagen: a novel murine model for autoimmune glomerular basement membrane disease. J Exp Med 191:899–906 41. Suzuki Y, Shirato I, Okumura K et al (1998) Distinct contribution of Fc receptors and angiotensin ii-dependent pathways in anti-GBM glomerulonephritis. Kidney Int 54: 1166–1174 42. Park SY, Ueda S, Ohno H et al (1998) Resistance of Fc receptor- deficient mice to fatal glomerulonephritis. J Clin Invest 102:1229–1238 43. Tarzi RM, Davies KA, Robson MG et al (2002) Nephrotoxic nephritis is mediated by Fcgamma receptors on circulating leukocytes and not intrinsic renal cells. Kidney Int 62: 2087–2096 44. Ellsworth JL, Maurer M, Harder B et al (2008) Targeting immune complex-mediated hypersensitivity with recombinant soluble human FcgammaRIA (CD64A). J Immunol 180:580–589 45. Woodle ES, Xu D, Zivin RA et al (1999) Phase I trial of a humanized, Fc receptor nonbinding OKT3 antibody, huOKT3gamma1(Ala-Ala) in the treatment of acute renal allograft rejection. Transplantation 68:608–616 46. Marino M, Ruvo M, De Falco S et al (2000) Prevention of systemic lupus erythematosus in MRL/lpr mice by administration of an immunoglobulin-binding peptide. Nat Biotechnol 18:735–739 47. Anthony RM, Nimmerjahn F, Ashline DJ et al (2008) Recapitulation of IVIG anti-inflammatory activity with a recombinant IgG Fc. Science 320:373–376 48. Kaveri SV, Lacroix-Desmazes S, Bayry J (2008) The antiinflammatory IgG. N Engl J Med 359:307–309 49. Kowalewska J, Muhlfeld AS, Hudkins KL et al (2007) Thymic stromal lymphopoietin transgenic mice develop cryoglobulinemia and hepatitis with similarities to human hepatitis c liver disease. Am J Pathol 170:981–989 50. Banas MC, Banas B, Hudkins KL et al (2008) TLR4 links podocytes with the innate immune system to mediate glomerular injury. J Am Soc Nephrol 19:704–713 51. Johnson GB, Brunn GJ, Platt JL (2003) Activation of mammalian toll-like receptors by endogenous agonists. Crit Rev Immunol 23:15–44 52. Taneda S, Hudkins KL, Cui Y et al (2003) Growth factor expression in a murine model of cryoglobulinemia. Kidney Int 63:576–590

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Experimental Models of Mixed Cryoglobulinemia

53. Banas MC, Hudkins KL, Wietecha TA et al (2006) Treatment of experimental membranoproliferative glomerulonephritis with a neutralizing anti-TGF-beta1 antibody. American Society of Nephrology Annual Meeting, San Diego, CA. J Am Soc Nephrol 17:179A 54. Kowalewska J, Hudkins KL, Taneda S et al (2004) Treatment with PDGF r-beta antagonist does not ameliorate cryoglobulin-associated membraphoproliferative glomerulonephritis in thymic stromal lymphopoietin (TSLP) transgenic mice. American Society of Nephrology Annual Meeting, St Louis, MO. J Am Soc Nephrol 15:698A 55. Segerer S, Hudkins KL, Taneda S et al (2002) Oral interferon-alpha treatment of mice with cryoglobulinemic glomerulonephritis. Am J Kidney Dis 39:876–888

153 56. Guo S, Wietecha T, Hudkins K (2008) CD 14 is a mediator of kidney injury in murine cryoglobulinemia-associated membranoproliferative glomerulonephritis (MPGN), American Society of Nephrology Annual Meeting, Philadelphia, 2008 57. Kobayashi T, Wietecha T, Hudkins KL et al (2010) CD 14 mediates inflammation and kidney injury of MPGN in the TSLP model cryoglobulinemic glomerulonephritis independent of TLR4: American Society of Nephrology Annual Meeting, Denver, 2010

The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia

19

Clodoveo Ferri, Alessandro Antonelli, Marco Sebastiani, Michele Colaci, and Anna Linda Zignego

19.1

Introduction

The term “cryoglobulinemia” refers to the presence in serum of one or more immunoglobulins that precipitate at temperatures below 37°C and redissolve on rewarming [1–3]. Cryoglobulinemia is usually classified into three subgroups according to the Ig composition in the cryoprecipitate: type I cryoglobulinemia, composed of only one immunoglobulin isotype or subclass; type II, and type III mixed cryoglobulins. Type II and type III cryoglobulins are immune complexes composed of polyclonal IgGs, autoantigens, and mono- or polyclonal IgMs, respectively, with rheumatoid factor (RF) activity [3–6]. Type I cryoglobulinemia, frequently asymptomatic per se, is usually associated with well-known hematological disorders. Serum mixed cryoglobulins are detectable in a variety of infectious or systemic disorders, often as isolated in vitro phenomenonon without any clinical correlation [1–3]. “Essential” mixed cryoglobulinemia (MC) represents a distinct clinical syndrome [1–3] classified among the systemic vasculitides [1–3]. Cryoglobulinemic vasculitis is secondary to vascular deposition of circulating immune-complexes, mainly cryoglobulins, and complement, with the possible contribution of hemorheological and local factors [1–3]. According to its histopathological and clinical features, MC belongs to the subgroup of small-vessel vasculitides, which also includes cutaneous leukocytoclastic vasculitis and Henoch-Schönlein purpura [1–3].

C. Ferri (*) Rheumatology Unit, Department of Internal Medicine, University of Modena and Reggio Emilia, Medical School, Modena, Italy e-mail: [email protected]

19.2

Epidemiology

There are no adequate epidemiological studies regarding the overall prevalence of MC; generally, it is considered to be a rare disorder. Numerous cohort studies made up of patients from different countries suggested that the prevalence of MC is, geographically, extremely heterogeneous; for example, the disease is more common in Southern Europe than in Northern Europe or Northern America [1]. MC is also characterized by clinical polymorphisms, which may complicate epidemiological studies. Moreover, in MC, a single manifestation (skin vasculitis, hepatitis, nephritis, peripheral neuropathy, etc.) is often either the only apparent or the clinically predominant feature, so that these patients are often referred to different specialties.

19.3

Etiopathogenesis

During the past few decades, the observation of chronic hepatitis as one of the most frequent symptoms of MC suggested a role for hepatotropic viruses in the pathogenesis of the disease [1]. Soon after the discovery of hepatitis C virus (HCV) as the major etiologic agent of non-A-non-B chronic hepatitis, in 1989, a role of HCV infection in MC was proposed independently by two pioneering studies [1, 4]. This hypothesis was definitively proven in 1991, when the presence of HCV RNA was detected by means of polymerase chain reaction (PCR) in 86% of Italian MC patients [1, 4]. Subsequently, numerous studies, including clinico-epidemiological observations as well as histopathological and virological investigations

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_19, © Springer-Verlag Italia 2012

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(HCV RNA detection by PCR and/or in situ hybridization), confirmed the important role of HCV in the pathogenesis of MC [1, 4]. Therefore, the term “essential” is now used to refer to a minority of MC patients (in Italy < 5%) [1]. Given the frequent association between MC and HCV, the clinical characteristics and outcome of MC are closely linked to the natural history of chronic HCV infection [5]. Nevertheless, it is important to bear in mind the possible role of genetic and/or environmental cofactors, although as yet they remain largely unknown, in the pathogenesis of different MC phenotypes [1]. In 1992, the demonstration of HCV lymphotropism in patients with both type C hepatitis and MC was an important step in elucidating the pathogenetic cascade of HCV-associated extrahepatic manifestations [6, 7]. It is likely that HCV, an RNA virus without reverse transcriptase activity and which cannot integrate into the host genome, may exert a chronic stimulus of the immune system through different viral proteins, such as core protein [1, 6–8]. Chronic stimulation of the lymphatic system might be exerted through viral epitopes, autoantigen production, and/or molecular mimicry. The interaction between HCV E2 envelope protein and CD81, a highly ubiquitous tetraspannin present on the surface of B cells, may represent another important pathogenetic event, responsible for the strong and sustained polyclonal stimulation of the B-cell compartment [1]. Importantly, this may explain, for example, the t(14;18) translocation observed in B cells of HCVinfected individuals [1, 5–9] and thus the increased expression of Bcl-2, with consequent inhibition of apoptosis, and abnormally prolonged B-cell survival. Of interest, the t(14;18) translocation is also observed in over a third of patients with isolated hepatitis type C [1, 5–9]. It is possible to hypothesize that, during chronic HCV infection, the contribution of the abovementioned factors, through a multistep process that causes sustained B-cell activation (Fig. 19.1), favors both the t(14;18) translocation and Bcl-2 over-expression. The resulting B-lymphocyte expansion is then responsible for autoantibody production, including cryoglobulins [1, 5]. Moreover, prolonged B-cell survival can be regarded as a predisposing condition for further genetic aberrations, with the appearance of frank B-cell lymphoma as a late complication of MC syndrome as well as chronic HCV infection without cryoglobulinemia [1, 5].

C. Ferri et al.

19.4

The Expanding Spectrum of HCV-Associated MC Clinical Features

It is plausible that cryoglobulinemic syndrome shares a number of etiopathogenetic events and clinical features with other possible HCV-related autoimmunelymphoproliferative diseases, such as autoimmune hepatitis, Sjögren’s syndrome, polyarthritis, and B-cell lymphomas [1]. Given this possible overlap, it is necessary to precisely define the spectrum of clinical features associated with MC, as a correct differential diagnosis is critical for clinico-pathogenetic studies and therapeutical trials. According to the original description, MC syndrome, or cryoglobulinemic vasculitis, is classified on the basis of the clinical triad of purpura, weakness, and arthralgias, accompanied by other frequent symptoms, namely membranoproliferative glomerulonephritis (MPGN), peripheral neuropathy, skin ulcers, and diffuse vasculitis [1]. Recently, the MC clinical spectrum has expanded to include an increasing number of possible complications, with non-organ- and organ-specific manifestations [1], as recorded in the Italian patient population referred to our university-based Division of Rheumatology (Table 19.1). The variability of the symptom composition of the main MC series reported in the literature may be due to differences in patient recruitment at the different specialist centers and not secondarily to their geographic origin and/or the classification criteria used [1]. Indeed, validated classification criteria for MC syndrome are still lacking; however, our group has proposed preliminary criteria mainly based on our expert clinical experience [1]. The presenting symptoms of MC vary among cryoglobulinemic patients and different clinico-serological patterns are initially seen, varying from apparently isolated serum mixed cryoglobulins, in some cases associated with mild manifestations such as arthralgias and/or sporadic purpura, to severe cryoglobulinemic syndrome [1]. The disease is diagnosed based on a combination of serological findings (mixed cryoglobulins with RF activity and frequent low C4) and clinicopathological features (purpura, leukocytoclastic vasculitis with multiple organ involvement) [1]. Chronically HCV-infected individuals may show asymptomatic serum mixed cryoglobulins [1], a finding that may precede by years or even decades the clinical onset of the disease. Conversely, some patients

19 The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia

157

PATHOGENESIS OF HCV SYNDROME genetic-environmental co-factors

HCV infection HCV genotypes HCV-proteins: core, E2 NS3, NS4, NS5A

APC

CD8

Autoreactive T-cells Poly-oligoclonal B cell expansion

IMMUNOLOGICAL DISORDERS

CD4

T(14; 18) translocation

Bcl2 activation

B-cell

Autoantibodies, RF, IC cryoglobulins

arthritis, sicca s., porphyria c.t., thyroiditis, diabetes, hepatitis, glomerulonephritis, lung fibrosis, cardiomyopathy, etc.

Host autoantigens CD81, LDL, HLA, sex hormones

Inhibition of apoptosis Prolonged B-cell survival

Other genetic aberrations (c-myc, Bcl6, p53, etc.)

Mixed cyoglobulinemia

(Cryoglobulinemic Vasculitis)

B-cell lymphomas

Hepatocellular carcinoma Thyroid cancer

HCV SYNDROME Fig. 19.1 The pathogenesis of HCV syndrome is the result of HCV infection, which may act as a chronic stimulus on the immune system, against a background of various pathogenetic co-factors; their variable contributions are reflected in different clinical phenotypes. These co-factors include: molecular mimicry involving HCV antigens and host autoantigens; interactions between HCV envelope protein E2 and CD81 with hepatocytes and lymphocytes; the predisposing genetic, hormonal, and metabolic background of the host; and as yet unknown environmental factors. The t(14;18) translocation, with activation of the Bcl2 proto-oncogene, may lead to prolonged B-cell survival. “Benign” B-lymphocyte expansion may be responsible for the production of different autoantibodies, including rheumatoid

factor and cryo- and non-cryoprecipitable immune complexes (IC), resulting in several autoimmune (organ and non-organ specific) disorders as well as cryoglobulinemic vasculitis. Mixed cryoglobulinemia is characterized by indolent B-cell proliferation that may be complicated by frank malignant lymphoma in about 10% of patients. Other proto-oncogene activations may ultimately lead to primary B-cell lymphomas or other malignancies, even in the absence of cryoglobulinemic syndrome. The various HCV-related diseases overlap both clinico-serologically and pathologically. This symptom complex can be referred to as the “HCV syndrome.” In this setting, cryoglobulinemic vasculitis represents a cross-road between autoimmune and neoplastic disorders

show typical cryoglobulinemic syndrome but without serum cryoglobulins, normally the hallmark of the disease. This may be a transient phenomenon due to the variable percentage of serum cryoprecipitable immune complexes at the time of sampling; thus, repeated cryoglobulin determinations are necessary to correctly diagnose these patients [1]. Cutaneous lesions represent the most frequent manifestations of MC [1, 2]. Among these, orthostatic purpura is generally intermittent, with the dimension and the diffusion of the purpuric lesions ranging from sporadic isolated petechiae to severe vasculitic lesions, often complicated by torpid ulcers involving the legs and malleolar areas. Repeated purpuric episodes may lead to typical sock-like ochraceous coloration on the

legs. Cutaneous manifestations are the direct consequence of vasculitic alterations, with the possible contributions of cofactors such as chronic venous insufficiency, physical stress, prolonged standing, and/ or muggy weather, with some cases further complicated by hemorheological disturbances [1]. While arthralgias represent one of the typical symptoms of MC syndrome, clear signs of chronic arthritis (usually mild, non-erosive oligoarthritis) are less frequently observed [1]. Specifically, rheumatoid-like polyarthritis may be seen in rare cases of MC but is more frequent in HCV-positive patients without MC syndrome [1, 5]. The presence of RF- and HCVseropositive arthritis makes the differential diagnosis between MC- and HCV-related arthritis very difficult,

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Table 19.1 Demographic, clinical, and serological features of MC patientsa Demographic Age at disease onset, mean ± SD years (range) Female/Male ratio Disease duration, mean ± SD years (range) Clinical Purpura Weakness Arthralgias Arthritis (non-erosive) Raynaud’s phenomenon Sicca syndrome Peripheral neuropathy Renal involvement Liver involvement Thyroid involvement Diabetes type 2 B-cell non-Hodgkin’s lymphoma Hepatocellular carcinoma Serological Cryocrit, mean ± SD % Type II/type III mixed cryoglobulins C3, mean ± SD mg/dl (normal 60–130) C4, mean ± SD mg/dl (normal 20–55) Antinuclear antibodies Antimitochondrial antibodies Anti-smooth muscle antibodies Anti-extractable nuclear antigen antibodies Anti-HCV Ab ± HCV RNA, % Anti-HBV antibodies HBsAg

54 ± 13 (29–72) 3 12 ± 10 (1–40) 98% 98% 91% 8% 32% 51% 81% 31% 73% 35% 14% 11% 3% 4.4 ± 12 2:1 93 ± 30 10 ± 12 30% 9% 18% 8% 92% 32% 1%

a

Evaluated at the end of follow-up in a series of 250 patients

in addition to which the simple coexistence of HCV infection and classical rheumatoid arthritis must also be considered. Figure 19.2 shows both the possible overlap between some autoimmune-lymphoproliferative disorders and the factors that may help in the differential diagnosis. For example, Sjögren’s syndrome shares a number of clinico-serological features with MC syndrome, while simple sicca syndrome, i.e., xerostomia and xerophthalmia, is recorded in half the patients with MC as well as in some with type C hepatitis. However, only a few cases of cryoglobulinemic vasculitis meet the current criteria for the classification of primary Sjögren’s syndrome. Moreover, a possible causative role of HCV in primary Sjögren’s syndrome can be excluded on the basis of clinico-epidemiological studies; however, in single patients with “primary”

Sjögren’s syndrome, often with cryoglobulinemia and purpura, and concomitant HCV infection, a pathogenetic link cannot be definitely ruled out, considering the lymphotropism of this virus [1, 5, 10] (Table 19.1, Fig. 19.2). Another frequent manifestation of MC syndrome is peripheral neuropathy, presenting more often as mild sensory neuritis [1]. Typical symptoms are paresthesias with painful and/or burning sensations in the lower limbs, often with nocturnal exacerbation, that may severely compromise the patient’s quality of life because of their chronicity and poor response to therapeutic attempts. Severe sensory-motor manifestations, often asymmetric mononeuritis, may develop abruptly in a minority of cases. In predisposed individuals, peripheral neuropathy may complicate interferon (IFN)-a treatment [1]. Central nervous system involvement, including dysarthria and hemiplegia, is rarely observed [1, 2]. Chronic hepatitis, generally mild-to-moderate, occurs in over two-thirds of patients at any time during the natural history of MC [1, 2]. This manifestation, uncommon in other systemic vasculitides, often characterizes the clinical course of the disease as a direct consequence of HCV infection. Chronic hepatitis may evolve to cirrhosis in one-fourth of patients, while only seven patients in our large MC series developed hepatocellular carcinoma (Table 19.1). Hepato-renal syndrome may also develop in a few patients with concomitant renal failure due to chronic glomerulonephritis; this represents a major life-threatening complication. On the whole, the clinical course and prognostic value of chronic hepatitis seem to be less severe than in classical type C hepatitis without MC syndrome [1, 2]. In addition, hepatocellular carcinoma less frequently complicates MC syndrome than is the case in the whole population of HCV-positive individuals [1, 2]. It is very difficult to fully explain these observations. One hypothesis is that MC patients, more often women, develop a rather benign clinical course of liver involvement, possibly due to low alcohol consumption and to the relatively low prevalence of HCV genotype 1b [1, 2]. Glomerulonephritis is another important organ involvement and may severely affect patient prognosis and survival of the disease [1, 2]. Membranoproliferative glomerulonephritis type 1, the most frequent histopathological pattern, is a typical immune-complex-mediated glomerulonephritis, although other immunological mechanisms have also been hypothesized [1, 2].

19 The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia

159

HCV+ MC syndrome and other autoimmune-lymphoproliferative diseases

AIH

MC

HCV

B-NHL RA

Primary SS

MC syndrome

B-NHL autoimm. systemic symptoms +/– cryogl. +/–, RF+/–, ANA +/– HCV+(15–30%)

purpura/weakness/arthralgias hepatitis+, MPGN+ cryogl.+, RF+, low C4+, HCV+(>90%)

Primary SS salivary gland inv. anti-SSA/SSB+, RF+ HCV+ rare

Autoimm. hepatitis ALT+, ASMA+/ANA+, RF+/– autoimm. systemic symptoms HCV +/–

Rheumatoid Arthr. symmetrical erosive polyarthritis, anti-CCP+, RF+/–, rare HCV+

Fig. 19.2 The potential overlap between mixed cryoglobulinemia (MC) syndrome and some autoimmune-lymphoproliferative disorders, such as primary Sjögren’s syndrome (pSS), rheumatoid arthritis (RA), autoimmune hepatitis (AIH), and B-cell non-Hodgkin’s lymphoma (B-NHL). Each disease may exhibit, during its clinical course, one or more autoimmune features, such as vasculitic purpura, sicca syndrome, arthralgias/arthritis, and thyroiditis, with the expression of serum RF and other autoantibodies. In addition, HCV infection, detectable in >90% of MC patients, may be involved in a significant percentage of B-NHLs and in a small percentage of other diseases. Given the presence of these common clinico-serological and virological findings, it is difficult to correctly classify individual patients. For the differential diagnosis, certain important parameters may be usefully employed: in addition to the histopathological characteristics, severity of salivary gland involvement and specific autoantibodies (anti-RoSSA/LaSSB) are rarely found in MC patients; conversely, cutaneous leukocytoclastic vasculitis,

visceral organ involvement (MPGM: membranoproliferative glomerulonephritis, hepatitis), low C4, and HCV infection, typically seen in MC, are seldom in primary SS. In the setting of HCV infection, there may be B-NHL, but this malignancy can also be a late complication of MC syndrome and pSS. In these cases, the patient’s previous clinical history and serological markers may facilitate the diagnosis. Arthralgias and arthritis are frequent manifestations of different autoimmune diseases, but erosive symmetrical polyarthritis and serum anti-cyclic citrullinated peptide antibodies (anti-CCP) are specific findings of classical RA. Finally, AIH may share various extrahepatic symptoms with other disorders; however, the activity/severity of hepatitis along with the presence of specific serological markers may help to differentiate AIH from other conditions, mainly HCV+MC patients. The overlapping territory involving these disorders frequently represents a gray zone in which the diagnosis in single patient may remain doubtful

A small proportion of MC patients develop widespread vasculitis involving small to medium-sized arteries, capillaries, and venules, with multiple organ involvement, namely, the skin, kidneys, lungs, central nervous system, and gastrointestinal tract [1–3, 11, 12]. In rare cases, intestinal vasculitis, typically

presenting as pain simulating an acute abdomen, may suddenly complicate the disease, often in patients with renal and/or liver involvement. A timely diagnosis and aggressive steroid treatment are necessary to intervene in this life-threatening complication [1, 2].

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Clinically overt interstitial lung involvement has been anecdotally observed in MC syndrome as well as in patients with isolated HCV infection [1, 2]. More often, lung involvement in MC is characterized by subclinical alveolitis, as demonstrated by means of broncho-alveolar lavage in unselected patient series [1, 2]. Hyperviscosity syndrome, due to high levels of serum cryoglobulins, is another rare clinical manifestation of MC, while hemorheological alterations may contribute to clinical symptoms such as orthostatic purpura, skin ulcers, and renal involvement [1, 2]. The severity of clinical symptoms generally does not correlate with serum cryoglobulin levels and/or hemolytic complement consumption [1, 2]. The latter is characterized by a typical pattern that is independent of disease activity: low or undetectable C4 with normal or slightly reduced C3 serum levels (Table 19.1). In some cases, sudden variations of C4, increasing from very low to abnormally high levels, may be the presenting symptom of B-cell lymphoma complicating MC [1, 2]. Some endocrinological diseases are significantly more frequent in MC patients than in the general population, including thyroid disorders, diabetes, and gonadal dysfunction [1, 2, 13, 14]. The most common thyroid disorders are autoimmune thyroiditis, subclinical hypothyroidism, and thyroid cancer; overt hyperthyroidism is less frequent and may occur as a reversible complication of IFN treatment. In addition, the incidence of diabetes mellitus type 2 in HCV-positive patients with and without MC syndrome was shown to be a statistically higher than in the general population [14]. Finally, HCV-positive males with or without cryoglobulinemic vasculitis may develop erectile dysfunction, attributable to hormonal and/or neurovascular alterations [1, 2]. B-cell lymphoma is one of the most frequent malignancies complicating the clinical course of MC syndrome, often as a late disease manifestation [1, 2, 5, 15] (Figs. 19.1 and 19.2). This complication may be related to peripheral B-lymphocyte expansion as well as to the lymphoid infiltrates, which represent the pathological substrate of the disease and are observed in the liver and bone marrow [1, 2, 5, 8]. Indeed, these infiltrates are regarded by some authors as “early lymphomas,” since they are sustained by lymphoid components indistinguishable from those of B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma (B-CLL) and immunocytoma

C. Ferri et al.

(Ic) [1, 2, 5]. However, unlike frank malignant lymphomas, they tend to remain unmodified for years or even decades and are followed by overt lymphoid tumors only in about 10% of patients [1, 2, 5]. These characteristics justify the proposed term “monotypic lymphoproliferative disorder of undetermined significance” (MLDUS) [1, 2, 5, 8]. Interestingly enough, type II MC-related MLDUS has its highest incidence in the same geographic areas where about 30% of patients with “idiopathic” B-cell lymphomas also display HCV positivity and where an increased prevalence of HCV genotype 2a/c has been observed in both MC and lymphomas [1, 2, 5, 8]. Type II MC-associated MLDUS presents two main pathological patterns: B-CLL-like and Ic-like [1, 2, 5]. In clinical practice, MC patients with malignant B-cell lymphomas with mild clinical course are not atypical and are sometimes diagnosed unexpectedly during a routine evaluation. There may be a sudden decrease or disappearance of serum cryoglobulins and RF, sometimes associated with abnormally high levels of C4; indeed, these serological variations may be the presenting findings of complicating B-cell malignancy [1, 2]. Among the neoplastic manifestations of MC, hepatocellular carcinoma occurs less frequently than noncryoglobulinemic type C hepatitis whereas papillary thyroid cancer is a rare complication [1, 2, 5, 16]. On the whole, since MC syndrome can be regarded as a pre-neoplastic disorder, careful clinical monitoring of these patients is recommended, even in the presence of a mild clinical course [1, 2, 5, 8].

19.5

Cryoglobulinemic Vasculitis and HCV Syndrome

Mixed cryoglobulinemia can represent a crossroads between certain autoimmune diseases (autoimmune hepatitis, Sjögren’s syndrome, polyarthritis, glomerulonephritis, thyroiditis, type 2 diabetes, etc.) and malignancies (B-cell lymphomas, hepatocellular carcinoma) [1–3, 5, 10–17] (Figs. 19.1 and 19.2). Consistent with the striking association between MC and HCV infection, the clinical history of the disease mirrors the natural course of this viral infection. It is not rare for patients to experience a slow disease progression: from mild HCV-associated hepatitis to various extrahepatic manifestations (arthralgias, sicca syndrome, Raynaud’s

19 The Expanding Spectrum of Clinical Features in HCV-Related Mixed Cryoglobulinemia

phenomenon, RF positivity, etc.), and ultimately to overt MC syndrome with typical clinico-serological manifestations. In a minority of these patients, a malignancy may develop, generally after a prolonged follow-up [1, 2]. Since the early 1990s, after the discovery of the causative role of HCV in MC [4, 7], the involvement of HCV in other extrahepatic disorders has gained increasing acceptance [5, 8, 17]. A correlation between several immune-mediated, organ- and non-organ specific disorders and HCV infection has been established. One of these, porphyria cutanea tarda (PCT) [5, 8], has been investigated in studies performed worldwide, reporting a wide range of associations [5, 8]. The pathogenesis of HCV-related PCT may be related to metabolic factors, in particular altered genes involved in iron metabolism, and host vs. HCV antigen cross-reactivity [5]. HCV-related lichen planus, i.e., orally located disease, is another possible association with variable geographic prevalence [5, 17] as well as several muco-cutaneous manifestations, as noted in a few reports or anecdotal observations [5, 17]. Peripheral neuropathy is a common complication of HCV infection, mainly in cryoglobulinemic vasculitis [1, 2, 5, 17], whereas central nervous system involvement is less common and more often affects patients with overt cryoglobulinemic vasculitis [5]. Some cardiovascular manifestations, mainly cardiomyopathy, during HCV infection have been reported in patient populations from Asian countries [1, 5, 18]. While a possible etiopathogenetic role of HCV in autoimmune hepatitis has been proposed, it is as yet controversial [1, 5]. A significant number of patients with autoimmune hepatitis may present with mixed cryoglobulins, HCV infection, and extrahepatic manifestations, such as thyroiditis, sicca syndrome, arthritis; conversely, in patients with HCV infection, one or more non-organspecific auto-antibodies are typically detected. The antigenic target specificity of HCV-related autoantibodies generally differs only quantitatively from that of autoantibodies associated with “primary” autoimmune hepatitis [5, 17]. Of interest, HCV-associated autoimmune hepatitis shows a clear heterogeneous geographic distribution, suggesting the involvement of various pathogenetic cofactors. In this context, HCV might trigger a peculiar clinico-serological subset of autoimmune hepatitis, mainly in specific geographic areas. The strength of the association with HCV as well as the pathogenic role of the virus varies largely among the different diseases and for a given disease

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among patient series from different countries [1, 5, 8]. Each disease can be regarded as a syndrome that includes distinct clinico-serological subsets, which in turn are the resulting phenotypes of multiple (genetic, environmental, infectious) pathogenetic cofactors. In this scenario, HCV-related clinical syndromes may trigger distinct autoimmune or neoplastic disease subsets, with additional important pathogenetic contributions. HCV-related disorders, or the HCV syndrome, represent a continuum, as suggested by the clinical history of some patients, in whom the entire spectrum may be displayed [5, 17]. In this setting, the expanding spectrum of HCV-related MC clinical features frequently reproduces the entire symptom complex of HCV syndrome.

References 1. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 16(3):25, http://www.ojrd.com/content/3/1/25 2. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33:355–374 3. Dammacco F, Sansonno D (1997) Mixed cryoglobulinemia as a model of systemic vasculitis. Clin Rev Allergy Immunol 15:97–119 4. Ferri C, Monti M, La Civita L et al (1994) Hepatitis C virus infection in non-Hodgkin’s B-cell lymphoma complicating mixed cryoglobulinaemia. Eur J Clin Invest 24:781–784 5. Ferri C, Antonelli A, Mascia MT et al (2007) HCV-related autoimmune and neoplastic disorders: the HCV syndrome. Dig Liver Dis 39:S13–S21 6. Zignego AL, Macchia D, Monti M et al (1992) Infection of peripheral mononuclear blood cells by hepatitis C virus. J Hepatol 15:382–386 7. Ferri C, Monti M, La Civita L et al (1993) Infection of peripheral blood mononuclear cells by hepatitis C virus in mixed cryoglobulinemia. Blood 82:3701–3704 8. Zignego AL, Ferri C, Pileri SA, et al for the Italian Association of the Study of Liver (AISF) (2007) Commission on Extrahepatic Manifestations of HCV infection. Extrahepatic manifestations of hepatitis C virus infection: a general overview and guidelines for a clinical approach. Dig Liver Dis 39:2–17 9. Zignego AL, Ferri C, Giannelli F et al (2002) Prevalence of Bcl-2 rearrangement in hepatitis C virus-related mixed cryoglobulinemia with or without complicating B-cell lymphoma. Ann Intern Med 137:571–580 10. Ramos-Casals M, De Vita S, Tzioufas A (2005) Hepatitis C virus, Sjogren’s syndrome and B-cell lymphoma: linking infection, autoimmunity and cancer. Autoimmun Rev 4: 8–15 11. Agnello V, De Rosa FG (2004) Extrahepatic disease manifestations of HCV infection: some current issues. J Hepatol 40:341–352

162 12. Dammacco F, Lauletta G, Montrone M, Sansonno D (2007) Mixed cryoglobulinemia: a model of virus-related disease in internal medicine. Dig Liver Dis 39:S8–S12 13. Antonelli A, Ferri C, Fallahi R et al (2004) Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia. QJM 97:499–506 14. Antonelli A, Ferri C, Ferrari SM et al (2008) Immunopathogenesis of HCV-related endocrine manifestations in chronic hepatitis and mixed cryoglobuliemia. Autoimmun Rev 8:18–23 15. Monti G, Pioltelli R, Saccardo E et al (2005) Incidence and characteristics of non-Hodgkin lymphomas in a multicenter

C. Ferri et al. case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 165: 101–105 16. Antonelli A, Ferri C, Fallahi R (1999) Thyroid cancer in patients with hepatitis C infection. JAMA 281:1588 17. Ferri C, Mascia MT, Saadoun D, Cacoub P (2009) Cryoglobulinemia and systemic manifestations of hepatitis C virus. EULAR compendium on rheumatic diseases, Ed. BMJ Publishing Group Ltd, London. Chap. 42a accessed on May 1 2009 18. Matsumori A (2005) Hepatitis C virus infection and cardiomyopathies. Circ Res 96:144–147

Classification of Cryoglobulinemic Vasculitis

20

Salvatore De Vita and Luca Quartuccio

20.1

Introduction

Cryoglobulinemic syndrome or cryoglobulinemic vasculitis (CV) is a systemic vasculitis associated with serum positive cryoglobulins, usually linked to non-malignant B-cell lymphoproliferation [1–3] and often triggered by chronic hepatitis C virus (HCV) infection [4, 5]. Classification criteria developed according to an accepted methodology are presently lacking for CV. However, they are essential for epidemiologic studies and research, and in turn for clinical practice [6, 7]. Recent recommendations for the management of CV [8], for instance, cannot be well applied given the absence of a patient classification system.

20.2

Pre-existing Classification Criteria and the Need for New Criteria

Earlier classification criteria of CV were elaborated by individual experts or by a panel of experts [9–12]. The latter was the case for the 1995 criteria of the GISC (Italian Study Group on Cryoglobulinemia) [9], which required positive cryoglobulinemia (³1% cryocrit for at least 6 months), the presence of at least two of the three clinical manifestations of purpura, fatigue, and arthralgias, plus positive laboratory features including positive RF or low C4 (<8 mg/dL), for the classification of CV. These criteria certainly derive from the

S. De Vita (*) Clinic of Rheumatology, Department of Medical and Biological Sciences, Azienda Ospedaliero –Universitaria of Udine, Udine, Italy e-mail: [email protected]

experience of experts, and despite the lack of a statistical basis may perform well. However, they do not make use of the currently accepted methodology by which items to be included in the criteria optimize sensitivity and specificity. Thus, current criteria represent the old standard of excellence and, while they should be respected, a modern, statistically supported approach is needed.

20.3

Which Items Should Be Included in the Classification of Cryoglobulinemic Vasculitis?

As with other systemic diseases, CV is characterized by a great variety of symptoms and signs and by possible abnormalities in several laboratory and instrumental tests. Moreover, the same manifestations may be present in other diseases. Thus, there is no single test to correctly classify all CV cases and controls. Conversely, analogous to other diseases, the classification of CV should integrate a combination of different items. A validated questionnaire was successfully included as an item in the classification criteria for Sjögren’s syndrome [10–12]. Similarly, to investigate a particular clinical feature in CV, such as purpura, the question(s) should be properly structured. Purpura may have been present sporadically and only in the past. Thus, different questions with the same purpose (i.e., detecting previous episodes of purpura) may show very different sensitivities and specificities. Consequently, several questions should be tested and then validated. Furthermore, different symptoms and signs need to be explored by different questions. Overall, the questionnaire approach for classification is not easy and

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deserves a proper work plan with which to study its development and validation. However, when properly developed, a validated questionnaire is of major value in diseases characterized by fluctuating manifestations. Nonetheless, there are some manifestations of CV, such as nephritis, that are poorly investigated by means of a questionnaire. Thus, the spectrum of CV manifestations should also be explored by the clinician, and the clinical manifestations to be considered for the classification criteria can include both those determined anamnestically (from the patient, by means of a questionnaire) and those clinically verified (by the physician). The laboratory tests to be included in the classification criteria also represent a major aspect. For the classification of CV, serum cryoglobulins must be present, and confirmed by a repeated test. However, this is not so obvious, especially when the disease has not yet been investigated in the patient. Initial negativity of cryoglobulin testing in patients with a true CV can occur for various reasons, such as low cryoglobulin amounts, problems in blood sample handling or testing, or tissue deposition rather than blood circulation of the immune complexes. Patients in whom CV is suspected despite negative cryoglobulins on initial testing deserve a careful follow-up, with repeated cryoglobulin determination while also excluding other diseases. The relevance of sample handling and highquality laboratory referral for the assessment of cryoglobulinemia are underscored. As concerns other laboratory investigations, only tests that are widely available and well standardized can be included in classification criteria. Investigational tests should therefore be excluded since they cannot be widely employed. Finally, the instrumental tests to investigate the different patterns of organ involvement in CV are quite heterogeneous, and they may be rather difficult to perform in all cases and controls. The risk of missing data is high in classification studies, and the discriminating power of a definite instrumental test is likely to be low.

Depending on the physician’s individual background and medical specialty, the diagnosis of CV may be based on different considerations. Also the choice of the best treatment approach may depend on the preferences of the different specialists. In light of these problems in establishing a gold standard diagnostic approach that a priori identifies cases and controls and in which statistical analyses confirm the best combination of clinical items and tests for classification, one possible remedy is to include specialists of different backgrounds in the classification studies. This strategy will decrease the risk of a specialty-oriented classification bias.

20.5

A key question regarding a patient with positive serum cryoglobulins is, “When should CV be classified?” This is a particular challenge if the clinical history fails to reveal overt features of vasculitis. A second question, which is not infrequently asked in clinical practice is, “In a patient negative for serum cryoglobulins by initial testing, but with some disease features that support the inclusion of CV in the differential diagnosis, when should CV be suspected?” Serum cryoglobulins may prove negative in their first determination, but a CV may still be strongly suspected and clinical decisions must be made accordingly. While proper classification has mainly epidemiologic and research purposes and needs a higher specificity, a diagnosis of CV has a key clinical relevant can still be formulated in the absence of positive classification criteria, and a higher sensitivity (accompanied by an acceptable specificity) is needed. Classification criteria are often used for diagnostic purposes as well, but this is not an appropriate application, as exemplified by the patient with negative serum cryoglobulins by initial testing but in whom CV is still strongly suspected.

20.6 20.4

Specialist-Related Problems with the Gold Standard Diagnosis of Cryoglobulinemic Vasculitis

The patient with CV may be referred to, among others, rheumatologists, hepatologists, nephrologists, infectious disease specialists, hematologists, and internists.

Classification Versus Diagnosis

New Classification Criteria for Cryoglobulinemic Vasculitis

The need for modern CV classification criteria developed using a standard methodology was first discussed in June 2002 at the 9th Congress of the GISC, in Udine, Italy. The following year, at the Congress of the European League Against Rheumatism, held

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Classification of Cryoglobulinemic Vasculitis

in Lisbon, a general work plan was delineated, with final agreement in September 2003 at the 10th GISC Meeting, in Modena, Italy [13]. It was agreed to divide the study into two parts. The first was dedicated to the development of a questionnaire showing a satisfactory sensitivity and specificity for CV. This questionnaire would be included in the second part of the study (part II), in which a standard methodology for classification studies was used. This reproduced the procedure employed for the Classification Criteria for Sjögren’s Syndrome [10– 12], and was also based on the expertise developed in that study. According to the experts, classification criteria were necessary for all patients with CV, either HCV-related or unrelated, with the presence of serum cryoglobulins (type I, II, III, or not typeable) defined as “a condition sine qua non” for classification of the disease. Furthermore, it was agreed that the core set of items for the classification of CV should include a dedicated questionnaire along with certain clinical manifestations and easily accessible laboratory tests. Consequently, histopathology, flow cytometry studies, novel laboratory biomarkers, and other possible diagnostic tools were excluded from the core set. Agreement was also reached for the inclusion criteria of cases and controls, a dedicated paper chart for the study, a glossary for the study, statistical analysis, and data dissemination. There was no financial support for the study. The final study protocol (part I and part II) was then developed by the coordinating center in Udine, Italy. In part I, a questionnaire consisting of 17 selected questions was sent to 12 European centers, where it was translated into the local language. The second and formal part of the study was a cooperative study involving 16 centers, some of which were different from those participating in the first part of the study. Clinicians with different medical specialties, from different specialty departments, were again involved. A dedicated paper chart was developed and included the following core set of items for classification: (1) a questionnaire for CV (with the questions selected in study part I), (2) data (89 items) on the pattern of organ involvement (present and past), and (3) laboratory tests (28 items). The classification of cases and controls as CV-positive and CV-negative, respectively, was based on the gold standard of the expert clinician, who had never examined that case/control before. Cases and controls included additional consecutive and unse-

165

lected cases and controls not enrolled in part I of the study, subdivided in three groups, A, B, and C. Group A consisted of patients with CV, either “essential” or associated with other disorders (HCV-related or -unrelated), with type I, II, III or non-typeable circulating cryoglobulins, as confirmed by at least two tests conducted at an interval of ³12 weeks. Group B patients were positive for serum cryoglobulins (confirmed as above) but were lacking CV based on the gold standard judgment of the expert clinician. Thus, the comparison of data in group A with group B should be able to answer the question, “If a patient has positive serum cryoglobulins, when should CV be classified?” For the inclusion of controls in Group B, the minimum followup required was 1 year (i.e., the expert was unable to note any manifestations supporting CV with a minimum of a 12-month history needed). Group C was made up of patients without serum cryoglobulins (by at least two repeated tests during a follow-up of at least 1 year) but with clinical or laboratory features that can be observed in the course of CV, thus answering the question, “In a patient negative for serum cryoglobulins by initial testing but with some disease features that support the inclusion of CV in the differential diagnosis, when should CV be suspected?” Furthermore, a distinction in group C was made between patients with systemic vasculitis (group C1) and those with other diseases different from systemic vasculitis but which should be distinguished from CV in clinical practice (group C2). Patients in group C had a previous followup of at least 12 months to allow a definite diagnosis of a disease other than CV. The classification criteria for CV developed by comparing group A (CV) with group B (positive serum cryoglobulins without CV) were then also tested by comparing group A with group C (either as a whole or distinguishing C1 and C2). The results of this study were presented at the 2010 EULAR Congress and at the 2010 Annual Meeting of the American College of Rheumatology [14, 15], and the final paper was published [16]. Overall, 925 cases and controls were studied. Figure 20.1 reports the final criteria (16). The criteria showed a high specificity (93.6%) and good sensitivity (88.5%) for CV. Furthermore, in a comparison of group A with group C, the same classification criteria showed a sensitivity of 88.5% and a specificity of 97.0% for CV. Thus, positive criteria were rarely present for diseases that may mimic CV. Preliminary classification criteria for CV have thus been developed by a co-operative international study

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Preliminary Classification Criteria for the CS satisfied if at least 2 of the 3 items (questionnaire, clinical, laboratory) are positive the patient must be positive for serum cryos in at least 2 determinations at≥ 12 week interval i. Questionnaire item: at least 2 out of the following • Do you remember one or more episodes of small red spots on your skin, particularly involving the lower limbs? • Have you ever had red spots on your lower extremities which leave a brownish color after their disappearance? • Has a doctor ever told you that you have viral hepatitis? ii. Clinical item: at least 3 out of the following 4 (present or past) • Constitutional symptoms Fatigue Low grade fever (37-37.9°C, >10 days, no cause) Fever (>38°C, no cause) Fibromyalgia Arthralgias • Articular involvement Arthritis •

Vascular involvement

•

Neurologic involvement

Raynaud’s phenomenon Purpura Necrotizing vasculitis Skin ulcers Hyperviscosity syndrome Peripheral neuropathy Cranial nerve involvement Vasculitic CNS involvement

iii. Laboratory item: at least 2 out of the following 3 (present) • • •

Reduced serum C4 Positive serum rheumatoid factor Positive serum M component

Fig. 20.1 Preliminary classification criteria for cryoglobulinemic vasculitis

and using a standardized methodology in a large number of real cases. They now need formal validation, possibly involving experts from a larger number of countries. In addition, they should be further tested in HCV-related vs. HCV-unrelated CV. These studies as well as more descriptive analyses and sub-analyses are ongoing or have been planned.

References 1. Meltzer M, Franklin EC (1966) Cryoglobulinemia – a study of twenty-nine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecipitability. Am J Med 40(6): 828–836 2. Gorevic PD, Frangione B (1991) Mixed cryoglobulinemia cross-reactive idiotypes: implications for the relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 28(2):79–94 3. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do

4.

5.

6.

7.

8.

9.

10.

not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 43(1):94–102 Ferri C, Greco F, Longombardo G et al (1991) Antibodies to hepatitis C virus in patients with mixed cryoglobulinemia. Arthritis Rheum 34:1606–1610 Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327(21):1490–1495 Johnson SR, Goek ON, Singh-Grewal D et al (2007) Classification criteria in rheumatic diseases: a review of methodologic properties. Arthritis Rheum 57:1119–1133 Dougados M, Gossec L (2007) Classification criteria for rheumatic diseases: why and how? Arthritis Rheum 57: 1112–1115 Mukhtyar C, Guillevin L, Cid MC et al (2009) European Vasculitis Study Group. EULAR recommendations for the management of primary small and medium vessel vasculitis. Ann Rheum Dis 68:310–317 Invernizzi F, Pietrogrande M, Sagramoso B (1995) Classification of the cryoglobulinemic syndrome. Clin Exp Rheumatol 13(Suppl 13):S123–S128 Vitali C, Bombardieri S, Moutsopoulos HM et al (1993) Preliminary criteria for the classification of Sjögren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 36:340–347

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11. Vitali C, Bombardieri S, Moutsopoulos HM et al (1996) Assessment of the European classification criteria for Sjögren’s syndrome in a series of clinically defined cases: results of a prospective multicentre study. The European Study Group on Diagnostic Criteria for Sjögren’s Syndrome. Ann Rheum Dis 55:116–121 12. Vitali C, Bombardieri S, Jonsson R et al (2002) European Study Group on Classification Criteria for Sjögren’s Syndrome. Classification criteria for Sjögren’s syndrome: a revised version of the European criteria proposed by the AmericanEuropean Consensus Group. Ann Rheum Dis 61:554–558 13. Lamprecht P (2004) The cryoglobulinemic syndrome – report from the workshop on classification and on the 10th

167 conference of the Italian Society for the Treatment of Cryoglobulinemia, Modena, 29 Sept, 2003. Z Rheumatol 63:235–238 14. De Vita S, Soldano F, Isola M et al (2010) Preliminary classification criteria for the cryoglobulinemic syndrome. Ann Rheum Dis 69(Suppl 3):77 15. De Vita S, Soldano F, Isola M et al (2010) Preliminary classification criteria for the cryoglobulinemic syndrome. Arthritis Rheum 62(Suppl 10):S851–S852 16. De Vita S, Soldano F, Isola M et al (2011) Preliminary classification criteria for the cryoglobulinemic vasculitis. Ann Rheum Dis 70(7):1183–1190

Demographic and Survival Studies of Cryoglobulinemic Patients

21

Giuseppe Monti, Francesco Saccardo, and Laura Castelnovo

21.1

Introduction

Cryoglobulinemic syndrome (CS) is highly heterogeneous in terms of its clinical presentation, the extent and severity of organ involvement, immunological abnormalities, and clinical course. It refers to the presence in the serum of immunoglobulins (Ig) that precipitate at temperatures below 37°C. Type I cryoglobulins are usually associated with lymphoproliferative disorders, formerly referred to as Waldenstrom disease, and with multiple myeloma, while mixed cryoglobulinemias (MCs), involving either type II or type III cryoglobulins, may be associated with connective tissue diseases, lymphoproliferative disorders, and chronic infections. A related and typical clinical manifestation of CS is vasculitis of the small and medium-size vessels. As CS is considered to be a relatively rare disorder, there are as yet no epidemiological studies regarding its overall prevalence, but according to the EURORDIS definition it is <5/10,000 people [1]. The clinical polymorphism of the disease is such that a single manifestation is often the only apparent clinical feature, so that patients are referred to a variety of specialists.

21.2

Role of HCV

Although a viral origin of cryoglobulinemia was long suspected, it was only in the beginning of the 1990s that a close relationship between hepatitis C virus G. Monti (*) Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy e-mail: [email protected]

(HCV) infection and CS emerged. Indeed, a high prevalence (50–86%) [2, 3] of HCV viremia was demonstrated in a large series of cryoglobulinemic patients. Consequently, CS is considered the most common extrahepatic manifestation of chronic HCV infection. The spread of CS is therefore, not surprisingly, closely related to the worldwide distribution of HCV infection. According to WHO assessments published in 2004, worldwide, about 140 million people are infected with HCV, representing 2.2% of the global population, with a broad variability in geographic distribution [4, 5]. The majority of those infected with HCV live in Asian countries (Taiwan, Mongolia, Pakistan), sub-Saharan Africa (Cameroon, Burundi, Gabon), and the eastern Mediterranean (Egypt, with an infection rate of >20%, has the highest frequency) [6]. Moreover, the WHO has calculated that each year there are between three and four million new cases of hepatitis C [7]. Over the past 20 years, the incidence of HCV infection in Western countries has decreased due to greater safety in blood transfusions and improvements in health conditions; however, increased drug abuse and the immigration of people from areas with high distribution of the virus pose a challenge to preventing renewed increases in infection. In Northern Europe, the overall prevalence is between 0.1% and 1%. In central Europe, it is intermediate, ranging from 0.2% in the Netherlands to 1.2% in France, and in southern Europe, including Italy, the prevalence varies between 2.5% and 3.5% [6]. Given the worldwide presence of HCV infection, the overall prevalence of cryoglobulinemia is likely to be under-reported; moreover, a higher incidence of HCV-related mixed cryoglobulinemia can be expected,

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especially in underdeveloped countries. If measured with the appropriate methods, cryoglobulins are positive in about 47–73% of HCV patients. Viganò et al., in 2007 [8], detected cryoglobulins in 47% of their patients. In Ramos Casals’ study [9], 73% of patients had asymptomatic cryoglobulinemia while 27% presented with cryoglobulinemic symptoms. Our unpublished data show a prevalence of cryoglobulins in HCV patients of about 70%. Some studies report a small increased incidence of type II mixed cryoglobulinemia [10] whereas Viganò, in his 10-year prospective study, demonstrated a type III predominance (80%) [8]. Two studies [9, 11] reported a prevalence of HCV genotype 1, which was not confirmed in other reports [12–14]. Frank symptomatic MC is uncommon, probably occurring in <1–5% of patients with chronic HCV infection [3, 15]. Two different recent works demonstrated that it seems to be more frequent in cirrhotic patients [8, 10]. In addition, patients with cryoglobulinemia because of chronic HCV infection reportedly have a higher incidence of cirrhosis and increased fibrosis scores compared to patients without cryoglobulinemia. The presence of specific HLA phenotypes (DR11 and DR8) seems to be associated with a significantly increased risk for the development of type II MC in patients with chronic HCV infection [16]. By contrast, other phenotypes (HLA-DR7) appear to protect against this form of the disease [16]. These results suggest that host immune response genes play a role in the pathogenesis of HCV-associated MC [16].

21.3

although it could not be confirmed in a large series [17]. While HBV and HCV have very similar epidemiological characteristics, the few historical series examining cryoglobulinemia and HBV infection do not allow determination of the role of co-infection with HCV. A recent study evaluated whether HBV occult infection could cooperate with HCV infection in the pathogenesis of MC and/or whether it was implicated in its pathogenesis independent of HCV [18]. Both HCV and HIV have an affinity for B lymphocytes, inducing their proliferation and transformation in addition to increasing immunoglobulin synthesis. Viral replication within lymphocytes contributes to their neoplastic transformation or impaired function, manifested in the synthesis of pathological immunoglobulins such as cryoglobulins [19]. The clinical significance of cryoglobulinemia in patients with HIV infection remains uncertain even if cryoglobulins seem to have poor clinical significance in patients with HIV infection alone [20]. There is a similar incidence of cryoglobulinemia in HCV and HCV/HIV co-infected patients, again suggesting a marginal influence of HIV on cryoglobulin production [20]. An association between other viruses and mixed cryoglobulinemia has been described. These associations are invariably referred to in anecdotal observations, often without sufficient clinico-serological assessments. Cryoglobulins have been found in hepatitis A virus, herpes zoster virus, cytomegalovirus, varicella zoster virus and parvovirus B19 infections and usually disappeared after recovery from the viral infection [21–23].

HBV, HIV, and Other Viruses

While HCV represents the major etiological agent of the disease in the Mediterranean area, patients with HCV-negative MC are more commonly found in the same areas where the overall prevalence of the disease is significantly low and its association with HCV is less frequent. Thus, a large number of other infectious agents may be associated with cryoglobulinemia, often without particular clinical relevance, with the exception of hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The association between HBV and mixed cryoglobulinemia, however, remains controversial but it has been described in some historical series. Prior to the detection of HCV, HBV infection was considered to be involved in the pathogenesis of most cryoglobulinemias [17]

21.4

Bacterial Infections and Parasites

Some reports have claimed an association between cryoglobulins and bacterial infections, including infections with gram negative bacteria (Proteus mirabilis, Treponema pallidum, Rickettsiaceae, Borrelia burgdorferi) and in patients with Staphylococcus aureus endocarditis. In a study of lepromatous leprosy with or without erythema nodosum, cryoglobulinemia was recorded in up to 95% of the cases, but the cryoglobulins usually resolved after recovery from the infection [24]. In one case report, an association with parasites was claimed (Acanthamoeba spp., visceral leishmaniasis) [25].

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Demographic and Survival Studies of Cryoglobulinemic Patients

21.5

Autoimmune Diseases

In patients with cryoglobulinemia without infection, the disease is usually suspected based on rheumatoid factor activity. Sjögren’s syndrome (SS), systemic lupus erythematosus (SLE), rheumatoid arthritis, antiphospholipid syndrome, Behcet disease, and mixed connective tissue diseases are frequently found in association with MC. Monti et al. [26] described cryoglobulinemia in association with autoimmune diseases in 5% of patients. The most frequent conditions were SS (31%) and SLE (26%), but systemic sclerosis, rheumatoid arthritis, and polymyositis were recorded as well. In Ferri’s series [2, 27] “essential” MC accounted for a quarter of the cases, with other frequent conditions being SLE, SS, and systemic sclerosis. Leukocytoclastic or cryoglobulinemic vasculitis are the classic vasculitic manifestations of SS. A concomitant chronic HCV infection is seen in about 3% of patients with SS; when sicca features occur in association with another systemic autoimmune disease or HCV infection, the designation is associated SS. SS cryoglobulinemic patients differ from patients without HCV infection with respect to a higher prevalence of photosensitivity and cryoglobulins, without clinical manifestations of cryoglobulinemia. Monoclonal (type II) MC is seen more frequently in SS patients (70%) and in those with B-cell lymphomas (80%), and systemic vasculitides (100%), but in only one third of patients with “essential” MC. Among the hematological neoplasms associated with cryoglobulinemia, there is a clear predominance of lymphoproliferative disorders (mainly non-Hodgkin lymphoma), with substantial extranodal involvement and elevated immunological markers. HCV infection is the main etiologic factor associated with hematologic malignancies in patients with cryoglobulinemia, followed by specific systemic autoimmune diseases, such as SS and SLE, highlighting the close relationship between lymphoproliferation, autoimmunity, and viruses [27].

21.6

Lymphoproliferative Diseases and Other Associations

Bryce, in 2006 [28], reported higher rates of lymphoproliferative and autoimmune disorders, considered to account for MC in these patients. However, this conclusion likely reflects referral bias as well

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geographic variations in the prevalence of hepatitis C, because many studies of cryoglobulinemia are from areas with relatively high prevalence of HCV. Finally, cryoglobulins can be found in acute and chronic glomerulonephritis, renal transplantation, dysproteinemia, and secondary to vaccinations [27].

21.7

Essential Cryoglobulinemia

When MC is detected in the absence of a well-defined disease, the syndrome is referred to as “essential MC.” However, in such cases the role of viral infections (HCV and HBV) cannot be ruled out. For example, occult virus may be localized in peripheral B mononuclear cells (PBMCs), acting as a trigger for developing polyclonal B proliferation [29].

21.8

Gender

Despite the absence of a clear correlation between HCV infection and gender, MC seems to be more prevalent in females. One large series [26] reported a female/male ratio of about 2:1. These data were subsequently confirmed in two other studies [8, 10].

21.9

Natural History and Progression

In a report published in 1986, the mean age at the first diagnosis of HCV cryoglobulinemia was reported to be 53.28 years [30]. In 1995, two studies determined a mean age of 52.7 and 54.7 years, respectively [31, 32]. More recent studies have found that the mean age at the time of first diagnosis is 64.4 years [33, 34]. There are few data on the natural history of MC. The most common pattern of the disease is a mild, slow-progressive disorder with a relatively good prognosis and survival. In a smaller percentage of patients there is a moderate-severe clinical course. However, the clinical course can become quite dramatic, with multi-system involvement and life-threatening complications. In particular, life-threatening cryoglobulinemia consists of glomerulonephritis with renal failure, catastrophic gastrointestinal vasculitis, lung involvement with respiratory failure, or severe central nervous system, spinal cord, or cranial nerve involvement, all attributable to mixed cryoglobulinemia. The survival rate in these patients is 3.6 years [9].

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Cutaneous manifestations of MC are the most frequent manifestations of the disease, with purpura as the most common symptom, both at the time of diagnosis and during follow-up. In a few cases, severe multiple organ disease develops after a long follow-up period. According to Tarantino [31, 34], the median interval between first symptoms and renal involvement is 48 months. The typical pattern is a membranoproliferative glomerulonephritis. The prognosis of patients with MC can be severely affected by the presence of renal or liver failure, either isolated or concomitant. In a limited but significant percentage of patients (14%) [3], MC is complicated by malignancy. B-cell lymphoma is the most frequent neoplasm but other neoplastic complications, i.e., hepatocellular carcinoma and papillary thyroid cancer, also often occur as late manifestations. Due to the possible appearance of malignancy during its clinical course, MC is regarded as a pre-neoplastic disorder.

21.10 Survival Patients with MC have a higher mortality than the general population. A number of prognostic factors are invariably associated with higher mortality, in particular age, renal involvement, widespread vasculitis, and type of cryoglobulin. Nevertheless, the prognosis of MC has improved in recent years and nowadays the causes of death in MC patients differ from those reported in the past. Cumulative survival 10 years after diagnosis is 56–58% [3, 28], but according to our data are 82% [32]. The survival rate seems to be similar in HCVpositive and HCV-negative patients, although the real age at which HCV-related MC occurs is difficult to determine. At the same time, it is difficult to establish the disease duration because of diagnostic difficulties or incorrect patient classification. In 1986, we found that the average age at death was 54 years, based on the results of a multicentric study [30]. In 2007, the average age at death was 67.2 years [28]. This relevant difference may be due to a general improvement in survival, changes in associated risk factors (such as HIV and HBV co-infections or alcohol abuse), or to better clinical management of the disease. The clinical outset of MC can be retrospectively correlated with different developments. The lack of

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liver or kidney impairment at the time of the syndrome’s diagnosis correlates with a longer survival rate whereas patients with cirrhosis or nephropathy die earlier, as do patients with co-morbidities [33].

21.11 Causes of Death Several factors may influence the outcome of patients with MC. Many early anecdotal observations suggested that renal involvement, infections, and widespread vasculitis were the primary causes of death. Our 1986 study reported an association between type II/III cryoglobulins and increased mortality [28]. Over the last 20 years, we collected data on the causes of death of MC patients who had been followed by us and in hospitals and universities belonging to the GISC (Gruppo Italiano per lo studio delle Crioglobulnemie). Thus, the series consisted of 1,055 patients with cryoglobulinemia, with pooled reliable data on 184 patients who had died from the disease. We compared the major pathologies of these patients at the time of their first presentation with their cause of death. At MC onset, about 20% of patients had few symptoms, with purpura as the main sign. The remaining 80% had evidence of organ impairment. HCV positivity was determined in 158 patients and 14% had active HCV-related hepatopathy (cirrhosis in twothirds of these patients). In 24% of patients, there was important renal involvement, with hematological, neurological, and autoimmune diseases being less frequent. The average survival was 8 years. It was less in patients with hepatopathies (6.78 years), nephropathies (5.82 years), and involvement of both organs (4.06 years), but longer in those without hepatic or renal involvement (10.26 years). Among the identified causes of death, 38% of the patients died of hepatopathies, with 13% due to cirrhosis. Hepatocellular carcinoma developed in 7%. Death was due to sepsis in 23% and to cardiovascular events in 17%. The remaining patients died of other causes: neurological, hematological, neoplastic, or vasculitic. Lymphomas occurred in 7%. It was not possible to assess the impact of therapy on prognosis, nor were we able to compare these data with those of patients who survived because a large number of them were lost to follow-up.

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References 1. VV.AA (2009) The prevalence of rare diseases: a bibliographic study. Orphanet Report series – Rare diseases collection. http://www.orpha.net/orphacom/cahiers/docs/GB/ Prevalence_of_rare_diseases_by_alphabetical_list.pdf . Accessed Dec 2009 2. Ferri C, Mascia MT (2006) Cryoglobulinemic vasculitis. Curr Opin Rheumatol 18(1):54–63 3. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical and serologic features and survival in 231 patients. Semin Arthritis Rheum 33(6):355–374 4. VV.AA (2002) NIH Consensus Statement on Management of Hepatitis C: 2002. NIH Consens State Sci Statements 19(3):1–46 (Review). http://consensus.nih.gov/2002/2002H epatitisC2002116html.htm. Accessed Dec 2009 5. World Health Organization (2002) Hepatitis C. http://www. who.int/topics/hepatitis/en/. Accessed Dec 2009 6. Baldo V, Baldovin T, Trivello R et al (2008) Epidemiology of HCV infection. Curr Pharm Des 14:1646–1654 7. Weinbaum C, Lyerla R, Margolis HS (2003) Prevention and control of infections with hepatitis viruses in correctional settings. Centers for Disease Control and Prevention. MMWR Recomm Rep 52:1–36 8. Viganò M, Lampertico P, Rumi MG et al (2007) Natural history and clinical impact of cryoglobulins in chronic hepatitis C: 10-year prospective study of 343 patients. Gastroenterology 33(3):835–842 9. Ramos-Casals M, Forns X, Brito-Zerón P et al (2007) Cryoglobulinaemia associated with hepatitis C virus: influence of HCV genotypes, HCV-RNA viraemia and HIV coinfection. J Viral Hepat 14(10):736–742 10. Kayali Z, Buckwold VE, Zimmerman B, Schmidt WN (2002) Hepatitis C, cryoglobulinemia, and cirrhosis: a metaanalysis. Hepatology 36(4 Pt 1):978–985 11. Leone N, Pellicano R, Ariata Maiocco I et al (2002) Mixed cryoglobulinaemia and chronic hepatitis C virus infection: the rheumatic manifestations. J Med Virol 66(2):200–203 12. Willems M, Sheng L, Roskams T et al (1994) Hepatitis C virus and its genotypes in patients suffering from chronic hepatitis C with or without a cryoglobulinemia-related syndrome. J Med Virol 44(3):266–271 13. Yamada G, Tanaka E, Miura T et al (1995) Epidemiology of genotypes of hepatitis C virus in Japanese patients with type C chronic liver diseases: a multi-institution analysis. J Gastroenterol Hepatol 10(5):538–545 14. Cacoub P, Maisonobe T, Thibault V et al (2001) Systemic vasculitis in patients with hepatitis C. J Rheumatol 28(1): 109–118 15. Hoofnagle JH (2002) Course and outcome of hepatitis C. Hepatology 36(5 Suppl 1):S21–S29 (Review) 16. Cacoub P, Renou C, Kerr G et al (2001) Influence of HLA-DR phenotype on the risk of hepatitis C virusassociated mixed cryoglobulinemia. Arthritis Rheum 44(9): 2118–2124 17. Galli M, Monti G, Invernizzi F et al (1992) Hepatitis B virus-related markers in secondary and in essential mixed cryoglobulinemias: a multicentric study of 596 cases. The

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Italian Group for the Study of Cryoglobulinemias (GISC). Ann Ital Med Int 7(4):209–214 Cohen P (1999) Cryoglobulinemia related to the hepatitis B and C viruses. Pathol Biol 47(3):232–236 (Review) Lapinski TW, Parfieniuk A, Rogalska-Plonska M et al (2009) Prevalence of cryoglobulinaemia in hepatitis C virusand hepatitis C virus/human immunodeficiency virusinfected individuals: implications for renal function. Liver Int 29(8):1158–1161, Epub 2009 Jul 7 Saadoun D, Aaron L, Resche-Rigon M et al (2006) Cryoglobulinaemia vasculitis in patients coinfected with HIV and hepatitis C virus. GERMIVIC Study Group. AIDS 20(6):871–877 Pagnoux C, Cohen P, Guillevin L (2006) Vasculitides secondary to infections. Clin Exp Rheumatol 24(2 Suppl 41):S71–S81 (Review) Lortholary O, Généreau T, Guillevin I (1999) Viral vasculitis not related to HBV, HCV and HIV. Pathol Biol 47(3):248– 251 (Review) Galli M, Monti G, Cereda UG et al (1984) Transient symptomatic cryoglobulinemia in gram-negative bacteria infections. Boll Ist Sieroter Milan 63(1):57–60 Matthews LJ, Trautman JR (1965) Clinical and serological profiles in leprosy. Lancet 2(7419):915–917 Casato M, De Rosa FG, Pucillo L et al (1999) Mixed cryoglobulinemia secondary to visceral Leishmaniasis. Arthritis Rheum 42(9):2007–2011 Monti G, Galli M, Invernizzi F et al (1995) Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC – Italian Group for the Study of Cryoglobulinaemias. QJM 88(2): 115–126 Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 3:25 (Review) Saccardo F, Novati P, Sironi D et al (2007) Causes of death in symptomatic cryoglobulinemia: 30 years of observation in a Department of Internal Medicine. Dig Liver Dis 39(Suppl 1):S52–S54 Bryce AH, Kyle RA, Dispenzieri A et al (2006) Natural history and therapy of 66 patients with mixed cryoglobulinemia. Am J Hematol 81(7):511–518 Saccardo F, Massaro P, Monti G et al (1986) Causes of death in essential mixed cryoglobulinemia. Ric Clin Lab 16(2): 389–391 Tarantino A, Campise M, Banfi G et al (1995) Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis. Kidney Int 47(2):618–623 Monti G, Saccardo F, Pioltelli P et al (1995) The natural history of cryoglobulinemia: symptoms at onset and during follow-up. A report by the Italian Group for the Study of Cryoglobulinemias (GISC). Clin Exp Rheumatol 13(Suppl 13):S129–S133 Zehender G, De Maddalena C, Bernini F et al (2005) Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome. J Virol 79(14):9145–9156 Tarantino A, Moroni G, Banfi G et al (1994) Renal replacement therapy in cryoglobulinemic nephritis. Nephrol Dial Transplant 9(10):1426–1430

HCV-Associated Membranoproliferative Glomerulonephritis

22

Christos P. Argyropoulos, Sheldon Bastacky, and John Prentiss Johnson

22.1

Introduction

Hepatitis C virus (HCV) is a blood-borne and sexually transmitted single stranded RNA flavivirus that causes a wide spectrum of liver disease, ranging from acute to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The worldwide prevalence of HCV positivity has been estimated to be 2% [1] but is highly dependent on the geographic area, with a prevalence as low as 0.6% in Germany but as high as 22% in Egypt. HCV infection is associated with numerous renal diseases that are related either directly (e.g., membranoproliferative glomerulonephritis, MPGN) or indirectly (e.g., IgA nephropathy, diabetic renal disease) to seropositivity. Renal lesions are very common in autopsies of HCV(+) patients, underscoring the impact of virologic, immunologic, and hepatic dysfunction factors on the kidney [2]. The most common pattern of glomerular injury found in patients with chronic HCV infection is MPGN type I (50–85% in some series).

22.2

Epidemiology and Pathogenesis

Occult HCV infection is the most common secondary cause of MPGN; however, there is substantial geographic variation in the prevalence of HCV seropositivity among patients with MPGN, ranging from 20% in the USA [3] to 60% in Japan [4]. In Southern

C.P. Argyropoulos (*) Renal and Electrolyte Division, Department of Internal Medicine, University of Pittsburgh, Pittsburgh, PA, USA e-mail: [email protected]

Europe, up to 50% of patients with hepatitis C will test positive for cryoglobulins [5] but only 10% of HCVpositive patients in the USA developed mixed cryoglobulinemia over an 8-year period [6]. The prevalence appears to be higher in patients coinfected with HIV [2, 7, 8] although co-infected patients tend to have less severe disease. The relationship between HCV and cryoglobulinemia is very strong, with antibodies against HCV present in 42–86% of cryoglobulinemic patients in the early literature [9–12] and >90% in more recent studies [7] (70% for patients with type III cryglobulins). The majority (>50%) of HCV-positive patients with MPGN will also have cryoglobulins in their serum [13, 14] and 30% of patients with mixed cryoglobulinemia will have some form of renal involvement [3, 7, 12]. Host immunogenetics appear to play a role in the development of cryoglobulinemia. In two studies, from Spain [15] and Italy [14], the presence of HLA-DRB1*11 was associated with 2.5-fold elevated risk, while HLA-DRB1*07 decreases this risk by 67%. However the interpretation of such associations as causal is complicated by the higher prevalence of HLA-DRB1*11 in HCV (+) patients [16] with normal ALT levels and milder hepatic injury. It is thus possible that the aforementioned associations between HLA alleles and cryoglobulinemia reflect a length-time bias; patients with milder hepatic injury survive longer, allowing for polyclonal activation of B cells followed by the emergence of a dominant clone after many years of antigenic stimulation. These epidemiologic observations suggest that the association between HCV infection and MPGN is mediated by the development of cryoglobulinemia. The latter reflects the presence of one or more immunoglobulins that precipitate at temperatures below

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_22, © Springer-Verlag Italia 2012

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Fig. 22.1 Peripheral blood smear in a patient with chronic hepatitis C virus (HCV) infection and renal cryoglobulinemia, showing aggregates of cryoglobulin precipitate. Some of these precipitates were counted by an automated blood cell counter as white blood cells, erroneously elevating the automated white blood cell count (Wright-Giemsa stain; 400×). The renal biopsy shown in Fig. 21.5 evidenced numerous cryoglobulin thrombi in glomerular capillaries

37°C but resolve upon rewarming (see Fig. 1 in [17]), Cryoglobulins are occasionally observed in peripheral blood smears (Fig. 22.1); they are classified pathologically in three groups depending on the type and clonality of the cryoglobulins. Type I cryoglobulins are monoclonal IgG, IgM, or IgA that self-aggregate through the Fc portion of the immunoglobulin molecule. Type II and III cryoglobulins are of mixed type, composed of monoclonal (IgMk) or oligoclonal IgMs respectively, with intrinsic rheumatoid factor activity which are complexed with antigens and polyclonal IgGs. Non-HCV conditions associated with cryoglobulins include other infections (hepatitis B, bacterial endocarditis, infectious mononucleosis, post-streptococcal glomerulonephritis, chronic parasitic infections), neoplasms (chronic lymphocytic leukemia, lymphomas), and autoimmune conditions (systemic lupus erythematosus, rheumatoid arthritis). In chronic hepatitis C infection, it is thought that the development of cryoglobulinemia is the result of the activation and proliferation of multiple clones of rheumatoid-factorproducing B cells infected by the HCV virus through CD81 receptors. After many years of stimulation, a dominant pre-malignant clone emerges, transforming the cryoglobulinemia from type III to type II. This orderly progression is supported by a number of epidemiologic observations, including a 30–200%

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increase in the risk of non-Hodgkin lymphoma and a 3-fold increased risk for Waldenström’s macroglobulinemia in HCV(+) patients [6, 18]. Approximately 70% of cryoglobulins causing renal disease are type II and 24% are type III [14]. Cryoglobulins are thought to mediate renal injury by a typical immune complex mechanism. Polymeric IgGs (i.e., in type III cryoglobulinemia) [19] and the IgMk rheumatoid factor of type II [20] have an affinity for fibronectin, which is found in the mesangium matrix. After localization to this area of the kidney, local activation of the classical complement pathway leads to a typical immune-complex glomerulonephritis. Other mechanisms have also been implicated in the intense cellular proliferative response seen in patient biopsies. Viral proteins and RNA are intrinsic components of the immune complexes seen in HCV-associated MPGN [21–23] and renal parenchymal cells express receptors (e.g., CD81) for such proteins. Activation of the pattern-recognition Tolllike receptors (TLR) in monocytes could trigger the innate immune system response; activation of TLR3 seems to differentiate HCV-associated from primary MPGN [24].

22.3

Pathology

Type I MPGN is the most common hepatitis-C-associated glomerular pattern of injury, with both cryoglobulinemic (majority) and non-cryoglobulinemic forms occurring. Type I MPGN is characterized by diffuse global endocapillary proliferation, with lobular accentuation, mesangial matrix expansion, glomerular capillary endothelial cell swelling, and glomerular capillary wall thickening (Fig. 22.2). Thickening of the glomerular capillary wall is due to mesangial interposition, characterized by ingrowth of mesangial cells and matrix into the contiguous subendothelial space of the glomerular basement membrane (GBM) and the formation of a new layer of subendothelial GBM, producing a double contour (tram tracking), as highlighted by silver stain (Fig. 22.3). In the cryoglobulinemic form of type I MPGN, there is frequently prominent infiltration of glomerular capillaries by circulating monocytes (Fig. 22.4), and PAS-positive hyaline thrombi are often present in glomerular capillary lumina (Fig. 22.5). Occasionally, cryoglobulin thrombi will occlude arterioles and small arteries (Fig. 22.6), sometimes producing microinfarcts. Rare patients develop

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HCV-Associated Membranoproliferative Glomerulonephritis

Fig. 22.2 Glomerulus from a patient with chronic HCV infection and type II cryoglobulinemia, who had type I membranoproliferative glomerulonephritis. Note the prominent lobular endocapillary proliferation (H&E; 400×)

Fig. 22.3 Portion of a glomerulus in a patient with type I membranoproliferative glomerulonephritis. Note the prominent glomerular basement membrane double contours (tram tracking) corresponding to mesangial interposition (methenamine silver; 1,000× oil immersion)

necrotizing vasculitis and/or crescents (Fig. 22.7) [7–9]. In the chronic phase of MPGN, the cellularity diminishes and there is increased mesangial sclerosis. Immunofluorescence microscopy reveals granular GBM and, less commonly, granular mesangial staining for IgG, IgM, and C3 (Fig. 22.8); approximately 50% of biopsies are also positive for C1q. In patients with cryoglobulinemia, hyaline thrombi with an identical staining profile may be present. Ultrastructural examination demonstrates electron-dense deposits in the interposed mesangium within the lamina rara interna of the GBM,

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Fig. 22.4 Portion of a glomerulus from the patient described in Fig. 22.2. Note the abundant circulating monocytes in the glomerular capillaries (methenamine silver; 600×)

Fig. 22.5 Glomerulus from the patient described in Fig. 22.1. Note the abundant PAS-positive cryoglobulin thrombi within the glomerular capillaries (PAS; 400×)

surrounded by a new layer of subendothelial lamina densa (Fig. 22.9). Mesangial electron-dense deposits are frequently but inconsistently present. Scattered subepithelial electron-dense complex deposits occur in a small subset of patients, ranging from large deposits resembling post-infectious humps through more numerous, smaller deposits resembling superimposed membranous glomerulopathy (type III MPGN, Burkholder variant). In cryoglobulinemic MPGN, the deposits often have microtubular/annular, fibrillar, and/ or tactoid/paracrystalline substructures (Fig. 22.10), although a minority of cryoglobulinemic deposits have no substructure. Intravascular cryoglobulin thrombi with similar substructures are also observed [25].

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Fig. 22.6 Glomerulus from a HCV-seronegative patient with a history of type III cryoglobulin dating back to 1996. The initial renal biopsy in 1996 and a more recent renal biopsy from 2009 show cryoglobulin thrombi in glomerular arterioles and small arteries and veins, with associated microinfarcts in the initial biopsy (not depicted). Of note, the serum cryoglobulin screen concurrent with the 2009 biopsy was negative (PAS; 400×)

Fig. 22.7 Necrotizing granulomatous vasculitis in a renal pericapsular small blood vessel in a patient with chronic hepatitis C virus infection and type II cryoglobulinemia. Note the fibrinoid necrosis (orange) (methenamine silver; 400×)

22.4

Clinical and Laboratory Manifestations

Patients with HCV-associated MPGN may present with the following: • Proteinuria (>0.5 g/day) with microscopic hematuria is the most common presenting syndrome seen in MPNG patients (41–55%) [14, 26]. Such patients may have abnormal kidney function upon presenta-

Fig. 22.8 Immunofluorescence stain of a glomerulus from a patient with type III cryoglobulinemia and chronic HCV infection acquired following a liver transplant for end-stage chronic autoimmune hepatitis. Note the granular, predominantly glomerular basement membrane staining for IgM. C3 and IgG stains were also positive in a similar distribution (IgM immunofluorescence; 400×)

tion and in up to 20% of patients with glomerulonephritis the proteinuria will be in the nephrotic range. • Acute nephritic syndrome, consisting of microscopic hematuria, proteinuria, hypertension and worsening renal function with or without oliguria, is seen in approximately 14–25% of patients. It is also associated with extrarenal manifestations of cryoglobulinemic vasculitis (rash, arthralgias, sensorimotor neuropathy, mononeuritis multiplex). Signs of systemic vasculitis occur in 10% of patients. • Isolated urinary abnormalities will be seen in a small percentage of patients (20%). Such patients will have a milder histologic picture of segmental mesangial proliferation. • Other, less frequent renal manifestations include acute (9%) and chronic renal insufficiency without urine abnormalities (12%) [14]. Most patients present between the fifth and sixth decades of life. The median time between the first symptoms of mixed cryoglobulinemia and renal involvement is 48 months. Hypertension (>140/95 mmHg) is almost invariably present (48–86%) even in non-nephritic patients [27]. Creatinine elevations are documented in 47% of patients referred for renal biopsy [26]; however, this frequency is likely to be overstated due to confounding by indication. The most common extrarenal clinical manifestations upon presentation are: purpura

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Fig. 22.9 Electron photomicrograph of an isolated glomerular capillary with mesangial interposition and electron-dense cryoglobulin deposits in a patient with chronic HCV infection and type II cryoglobulinemia (5,600×)

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New GBM

Cryoglobulin

Mesangial Interposition

Fig. 22.10 Electron photomicrograph from a patient with chronic HCV infection and type II cryoglobulinemia. Note the electron-dense deposits with a fibrillar substructure in the interposed mesangium with the subendothelial glomerular basement membrane (13,000×)

(70%), hepatomegaly (49%), arthralgias (46%), splenomegaly (36%), peripheral neuropathy (22%) and fever of unknown origin (19%). The prevalence of such manifestations increases with the duration of followup, so that arthralgias and organomegaly will be seen in 80–90% during their lifetime. Purpura is an invariable feature of HCV-associated MPGN; in a large series

EM 1-1

from Italy, only 6% of patients never developed this symptom [26]. The classic Meltzer triad of generalized weakness, purpura, and arthralgia will be present in one-third of patients. Pulmonary involvement occurs in 2–5% of cases and can be dramatic, with nodular infiltrates, pulmonary hypertension, and congestive heart failure. Other cutaneous manifestations include ulcers and Raynaud’s phenomenon, with an unadjusted prevalence of 8 and 4% of cases, respectively [14]. Hepatic involvement (organomegaly, stigmata of liver disease) will be observed in 50–70% of cases and these patients will test positive for hepatitis C (antibody or PCR test). Patients will have detectable cryoglobulins (most likely type II), with rheumatoid factor activity in their serum but the percentage (cryocrit) will vary from patient to patient and from time to time. Serum complement levels will be depressed (C4 more than C3), and the early component of the complement cascade (C1q) will be depressed as well. In contrast, total complement hemolytic activity (CH50) will be normal. The prevalence of abnormal C3 and C4 levels varies according to the severity of the renal lesions, ranging from 20%/60% (mesangial proliferative pattern) to 30%/60% (focal MPGN). In patients with diffuse MPGN depressed C3, C4 levels are seen in 52 and 93% of cases, respectively.

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Table 22.1 Differential diagnosis of HCV-associated membranoproliferative glomerulonephritis (MPGN) pattern of injury on biopsy Infections (usually MPGN I) Hepatitis C (non-cryoglobulinemic associated with MPGN I and III), hepatitis B Infection (MPGN I in adult HBsAg (+) patients), schistosomiasis (class III associated with S. mansoni, class VI (cryoglobulinemic) seen in S. mansoni/HCV co-infection), chronic bacterial infections (atrioventricular shunt nephritis, osteomyelitis, intra-abdominal, pelvic, pleural infections, cyanotic heart disease), leprosy, parasitic infections (quartan malaria, onchocerciasis, trichinosis, elephantiasis) Autoimmune diseases Systemic lupus erythematosus (type I or IV MPGN), mixed connective tissue disease Complement disorders Hereditary complement deficiency (C1q, C2,C3,C4 : MPGN I), acquired complement deficiency (C4 nephritic factor: MPGN I, C3 nephritic factor : MPGN II), congenital or acquired factor H deficiency Monoclonal immune deposition disease Light chain deposition disease (MPGN II), heavy chain deposition disease, type I cryoglobulinemia Chronic thrombotic microangiopathies Hemolytic uremic syndrome, drug induced Chronic liver disease (non-viral) Alpha 1 antitrypsin deficiency, schistosomiasis Malignancies (without cryoglobulinemia) Chronic lymphocytic leukemia, lymphoma, thymoma, renal cell carcinoma

22.5

Differential Diagnosis

The differential diagnosis depends on the presenting clinical (nephritic or nephrotic) renal syndrome. HCVassociated cryoglobulinemic MPGN rarely gives rise to these two clinical syndromes and the diagnosis will usually be considered during the routine diagnostic investigation for secondary causes (e.g., positive hepatitis C and cryoglobulin tests). In adults, the two most common causes of nephrotic syndrome are membranous nephropathy and focal segmental glomerulonephritis, both of which are associated with hypertension and microhematuria (in 30% of cases) and have been described in patients with chronic HCV infection without cryoglobulinemia or rheumatoid factor positivity [28, 29].The differential diagnosis of the nephritic syndrome is very wide and includes both primary (e.g., IgA nephropathy) and secondary (e.g., systemic lupus erythematosus) conditions. A secondary form of IgA nephropathy has been reported in chronic liver disease, including in patients with chronic hepatitis C viral infection [30]. The pathogenesis is thought to be the overabundance of circulating IgA-containing immune complexes due to increased IgA production in the gastrointestinal mucosa coupled with impaired hepatic clearance. For patients presenting acutely with systemic symptoms and purpura, other small-vessel vasculitis syndromes will need to be considered in the differential. Henoch Schönlein purpura is character-

ized by dominant IgA deposits in the vascular walls of purpuric lesions. A positive anti-neutrophil cytoplasmic antibody (ANCA) test and minimal immunoglobulin deposits suggest one of the ANCA-positive vasculitides (Wegener’s, microscopic polyangiitis or Churg-Strauss syndrome). Depressed complement levels and renal dysfunction characterize a number of conditions, including athero-embolic renal disease (depressed C3 and eosinophilia), thrombotic microangiopathies, systemic lupus erythematosus (simultaneous depression of C3 and C4), post-streptococcal glomerulonephritis, shunt nephritis, and endocarditis (low C3, type III cryoglobulins). Complement levels may also be low in significant hepatic dysfunction due to depressed synthesis; in case of combined liver and renal dysfunction, the hepatorenal syndrome should be considered. Many other conditions will be associated with the MPGN pattern of renal injury and they should be included in the differential (Table 22.1). Fibrillary and immunotactoid glomerulonephritis have been reported [31] to occur in a small number of patients with chronic HCV infection, with light microscopy showing a spectrum ranging from mild mesangial proliferation with mesangial matrix expansion and glomerular capillary wall thickening through MPGN (types I and III), with some patients having cellular or fibrous crescents. Congo red staining is negative for amyloid, but amyloid P protein and fibronectin have been demonstrated in the protein deposits. Electron

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HCV-Associated Membranoproliferative Glomerulonephritis

microscopy reveals haphazardly oriented fibrils 16–24 nm in diameter in fibrillary glomerulonephritis, and microtubule-like structures with a mean diameter of 33–40 nm in immunotactoid glomerulonephritis. Serum complement levels are depressed in some patients, but cryoglobulin precipitates are not identified by routine laboratory serum testing.

22.6

Prognosis and Course

The clinical course is variable, characterized by exacerbations alternating with quiescence. Renal flares can be seen in up to 53% of patients and are often multiple (range 1–4 over 10 years). Remissions can be either spontaneous or following therapy in 15% of cases [27]. In a large Italian study, progression to end-stage renal disease (ESRD) was noted in 15% of patients over a 10-year period. In a more recent study, the five year cumulative incidence of chronic kidney disease stage 5 (estimated glomerular filtration rate <10 ml/min) was 11% [14]. ESRD censored patient survival at 10 years was 49% in an earlier cohort [26]; the most common causes of death were cardiovascular, infectious, and hepatic and malignant conditions. Survival appears to have improved in more recent years (90% at 5 years and 80% at 10 years). Negative predictors of renal and patient survival include older age (>50), nephrotic range proteinuria, nephritic presentation, elevated creatinine at the time of biopsy (>1.5 mg/l in the two large Italian studies). The number of flare ups is an independent risk factor for the development of renal dysfunction.

22.7

Therapy and Outcomes

Therapy of MPGN associated with HCV may be considered in at least three differing manners: therapy directed at proteinuria and hypertension common to progressive renal diseases; therapy directed at cryoglobulins and their effects; and therapy directed against HCV. These are not, of course, mutually exclusive and in some cases, as shall be seen, all three approaches have been combined. Several other factors complicate the discussion of treatment of HCVassociated MPGN. Most important among such them is the stage of renal and liver disease. Patients with chronic stable illness are often approached differently

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than those with more aggressive and active renal disease. Furthermore, when patients with very advanced renal disease near or even on dialysis are considered for treatment, the actual object of therapy changes. Therapy is, in such cases, not designed to treat the MPGN but rather to eliminate or minimize the HCV status of the patient prior to liver transplantation. This discussion will focus on treatment of the renal disease and only briefly consider treatment schemes in dialysis or transplant patients. MPGN is characterized by proteinuria, hypertension, and variable progression of renal insufficiency. There is general agreement that all patients should be managed with standard conservative approaches to progressive glomerular disease, including strict control of blood pressure centered around the use of angiotensin converting enzyme inhibitors or angiotensin receptor blockers (or both), diuretics, and lipid-lowering agents. Treating hypertension in this context is not an easy task; even with multiple (2–3) agents a large proportion of patients will have abnormal office blood pressure readings. Although many approaches have been tried, there is no clear evidence-based therapy that is generally efficacious for primary or idiopathic MPGN [32]. Therapy of HCV-associated MPGN is therefore directed against either HCV or the cryoglobulins associated with HCV and the associated MPGN and systemic vasculitis. Therapy directed against HCV per se initially centered around the use of interferon a (IFN-a) and showed modest effects on proteinuria and renal function (reviewed in [33, 34]). However, IFN-a in a variety of dosing schedules was, unfortunately, associated with a low level of sustained virologic remission (SVR) and a relatively high rate of relapse with viremia [35]. Rates of SVR became substantially higher with the advent of a long-acting IFN-a preparation, pegylated interferon (PEG-IFN), which is used in combination with the antiviral nucleoside antimetabolite ribavirin (RBV). PEG-IFN is given subcutaneously weekly and RBV daily by mouth for periods from 24 weeks to 1 year, depending, to some extent on the genotype of the HCV [35]. This has become the standard therapy for both HCV liver and renal disease, including MPGN [34, 36]. With this approach, the SVR may be as high as 90% [33], although rates are lower with genotype 1 virus [37]. RBV and its metabolites are principally excreted by kidney, so dose adjustments must be made for glomerular filtration rate (GFR) <50. Algorithms

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for dosing at reduced GFR are published [38]; additionally, therapeutic levels have been established so that blood levels may be followed, which allows for the use of RBV even in dialysis patients [39]. The impact of this therapy on the course of MPGN has been evaluated in a number of relatively small studies (reviewed in [34]). When SVR is achieved, there is generally a reduction in proteinuria and a stabilization or improvement in serum creatinine. Though not definitively established through large-scale controlled randomized trials, this therapeutic approach is the consensus recommendation for HCV-associated MPGN characterized by proteinuria and slowly progressive renal insufficiency [33, 34]. When HCV-associated cryoglobulinemia is associated with an aggressive systemic vasculitis and more severe proteinuria and progressive renal failure, more rapidly acting therapeutic strategies have been employed. These center around short courses of immunosuppressive agents (primarily corticosteroids and cyclophosphamide), which have been successfully used in idiopathic cryoglobulinemia [40]. Since this approach is not directed against HCV per se, it will not affect the ultimate course of the disease, but is used to control acute vasculitic flares so that slower-acting antiviral therapy may then be employed. A number of approaches are used, involving either daily oral or even pulse IV steroids, either alone or in combination with cyclophosphamide, and usually in doses of 1–2 mg/kg. When vasculitis is quite active or cryoglobulin levels very high, plasmapheresis has been added. In general this approach will result in declines in cryoglobulin levels and impressive clinical responses, particularly in terms of vasculitic injury, within weeks to a few months [27]. Prolonged immunosuppressive therapy may actually result in increases in HCV levels and is not efficacious in the long term. Immunosuppressive therapy is less useful in slowly progressive renal disease, where it was shown in direct comparisons to have less effect than antiviral therapy (see Table II in [33]). An interesting variation on the approach to more acute HCV-associated cryoglobulin-mediated renal disease and vasculitis is the use of the B-cell depleting monoclonal antibody rituximab in combination with ongoing antiviral therapy with PEG-IFN and RBV. Several modest-sized and uncontrolled trials suggest that this therapy is efficacious in controlling acute flairs, eliminating cryoglobulins, and attaining SVR [41, 42].

C.P. Argyropoulos et al.

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19. Rostagno AA, Gallo G, Gold LI (2002) Binding of polymeric IgG to fibronectin in extracellular matrices: an in vitro paradigm for immune-complex deposition. Mol Immunol 38(15):1101–1111 20. Fornasieri A, Armelloni S, Bernasconi P et al (1996) High binding of immunoglobulin M kappa rheumatoid factor from type II cryoglobulins to cellular fibronectin: a mechanism for induction of in situ immune complex glomerulonephritis? Am J Kidney Dis 27(4):476–483 21. Rodríguez-Iñigo E, Casqueiro M, Bartolomé J et al (2000) Hepatitis C virus RNA in kidney biopsies from infected patients with renal diseases. J Viral Hepat 7(1):23–29 22. Sansonno D, Gesualdo L, Manno C et al (1997) Hepatitis C virus-related proteins in kidney tissue from hepatitis C virusinfected patients with cryoglobulinemic membranoproliferative glomerulonephritis. Hepatology 25(5):1237–1244 23. Sabry AA, Sobh MA, Irving WL et al (2002) A comprehensive study of the association between hepatitis C virus and glomerulopathy. Nephrol Dial Transplant 17(2):239–245 24. Wornle M, Schmid H, Banas B et al (2006) Novel role of toll-like receptor 3 in hepatitis C-associated glomerulonephritis. Am J Pathol 168(2):370–385 25. Alpers CE, Kowalewska J (2007) Emerging paradigms in the renal pathology of viral diseases. Clin J Am Soc Nephrol 2(Suppl 1):S6–S12 26. Tarantino A, Campise M, Banfi G et al (1995) Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis. Kidney Int 47(2):618–623 27. D’Amico G (1998) Renal involvement in hepatitis C infection: cryoglobulinemic glomerulonephritis. Kidney Int 54(2): 650–671 28. Davda R, Peterson J, Weiner R, Croker B, Lau JY (1993) Membranous glomerulonephritis in association with hepatitis C virus infection. Am J Kidney Dis 22(3):452–455 29. Stehman-Breen C, Alpers CE, Fleet WP, Johnson RJ (1999) Focal segmental glomerular sclerosis among patients infected with hepatitis C virus. Nephron 81(1):37–40 30. Pouria S, Feehally J (1999) Glomerular IgA deposition in liver disease. Nephrol Dial Transplant 14(10):2279–2282 31. Markowitz GS, Cheng JT, Colvin RB et al (1998) Hepatitis C viral infection is associated with fibrillary glomerulonephritis

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Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia

23

Gianfranco Ferraccioli, Francesca Faustini, and Elisa Gremese

23.1 Introduction After the identification of hepatitis C virus (HCV), in 1989, as the causative agent of mixed cryoglobulinemia (MC) in the great majority of these patients, it became clear that, beyond hepatitis, several extrahepatic manifestations of MC are strictly related to the infection and therefore that cryoglobulins are not essential [1]. To date, however, direct demonstrations of the triggering molecular events leading to the extrahepatic comorbidities have not been fully explained, due to the lack of experimental animal models. The high rate of HCV chronicity (50–85%) clearly evidences the capacity of the virus to overcome initial host immune control, thus favoring the occurrence of a chronic infectious-inflammatory state that can involve several organs over time [2]. The multifaceted manifestations related to the infection have been defined as “HCV-related syndrome.” It occurs at a mean age of 54 years (range 29–72) with a female/male ratio of 3:1 [3]. The ability of an infectious agent to trigger a chronic inflammatory state likely depends upon two major features: the chance of infecting immune cells (B cell or monocytes/macrophages) and the capacity to escape immune control. The obvious consequence, once both conditions have been satisfied, is a persistent, chronic, relapsing-remitting inflammatory state. Eventually, this can lead to persistent immunological activation, ending in either autoimmune (local or systemic) disease or a lymphoproliferative disorder.

G. Ferraccioli (*) Division of Rheumatology, Institute of Rheumatology and Affine Sciences (IRSA), School of Medicine, CIC – Catholic University of the Sacred Heart, Rome, Italy e-mail: [email protected]

23.2 Rheumatologic Manifestations in Cryoglobulinemia By definition, cryoglobulins are cryoprecipitable proteins; indeed, they not only precipitate in vitro at 4°C inside test tubes, but also as plugs inside small blood vessels, thus inducing signs and symptoms of small-vessel vasculitis. Since all organs and tissues have small vessels, they are vulnerable to damage by underlying vasculitis. The typical form associated with Meltzer-Franklin purpura is leukocytoclastic vasculitis. IgM immune complexes (ICs) with rheumatoid factor (RF) activity or those consisting of IgG/HCV proteins account for the persistence of the vascular inflammation [4]. These ICs strongly activate monocytes/macrophages, leading to the synthesis and release of several cytokines. Among them, CXCL10 (interferon-g-induced protein 10-IP10) [5] and MCP-1 (monocyte chemoattractant protein-1) [6] continuously recruit monocytes/macrophages and T cells while others, such as interleukin (IL)-6 and B-cell activating factor (BAFF) [7], keep B cells persistently activated and ensure their long half-life (Fig. 23.1). The underlying vasculitis, the persistent and chronic inflammatory state, and the lymphoproliferative milieu present as a broad spectrum of symptoms and signs. The pathogenesis and biomolecular events that cause these clinical manifestations are still mostly unknown. However, the most common one of rheumatologic interest with respect to MC can be explained by a persistent IC vasculitis and by persistent chronic inflammation, mainly involving monocytes/dendritic cells, as antigen-presenting cells, and B cells producing RF and IgG against HCV proteins (Table 23.1).

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_23, © Springer-Verlag Italia 2012

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Monocytes DC

IL6

BAFF T IL2 Soluble BAFF BAFF receptors B

RF synthesis IgG production

Fig. 23.1  Immunologic cooperation during chronic HCV infection Table 23.1  Rheumatologic manifestations during chronic cryoglobulinemic HCV infection Rheumatic manifestations Arthritis-Arthralgias Sjögrens’-Sicca syndrome Acrocyanosis-Raynaud’s phenomenon Leukocytoclastic vasculitis-Ulcers Myalgias-Fibromyalgias APS (antiphospholipid syndrome) Mono/Multineuropathies Interstitial lung disease Osteosclerosis with bone pain

Associated autoantibodies RF ANA; Anti-Ro, Anti-La ANA pANCA-cANCA RF; Thyroid disease ANA; Anti-cardiolipin pANCA; c-ANCA; Anti-cardiolipin; ANA RF; ANA –

23.2.1 Arthralgias and Arthritis The prevalence of arthralgias was reported to be 23%, based on a prospective study of 1,614 patients with HCV infection [8]. Generally, symptoms such as morning stiffness and pain are bilateral, affecting the small joints; rheumatoid-like characteristics are seen in twothirds of patients. Arthritis is non-erosive and nondeforming, but mono-oligoarthritis, usually involving the large joints, is also observed [9–11]. Usually, RF is the autoimmune feature, while ACPA (anti-citrullinated peptide auto-antibodies) are negative. In a recent study, ACPA were found in 5.7% of HCV carriers with arthralgias, but in 78% of a control RA (rheumatoid arthritis) population [10], and in another study in >84% of RA and 8.8% of HCV carriers [11, 12]. In some

patients, even though the ACR criteria for RA are often satisfied, nodules and erosions may be lacking. The best possible explanation for this form of arthritis is an IC-related arthropathy. Since RA can include ACPA and erosions, it becomes very important to define whether in HCV chronic infection the occurrence of arthritis is truly MC arthritis and not RA in a patient with chronic HCV infection. While RA that is seropositive either for RF and/or ACPA needs to be aggressively treated with methotrexate (MTX) and/or a tumor necrosis factor (TNF)-a blocker, irrespective of HCV infection, cryoglobulinemic arthropathy requires a much less aggressive approach, with antimalarials and/ or MTX, sometimes with small doses of glucocorticoids, in short courses. The biologic evidence is that TNF-a and the imbalance between Th1 and Th2 responses contributes to make HCV patients refractory to interferon (IFN). All these issues are very important from a clinical perspective since MTX, as well as nonsteroidal anti-inflammatory drugs (NSAIDs), can be hepatotoxic, while glucocorticoids may favor viral replication [13]. Therefore, one has to consider the cost-benefit ratio of any therapeutic choice in these patients. Furthermore, IFN therapy can lead to the occurrence of autoimmunity and to a relapse of the arthritis [14]. According to these statements, the key messages are: 1. All patients with recent onset of arthritis should be routinely tested for HCV (and other pre-existing infections if previously transfused), before starting disease modifying anti-rheumatic drugs (DMARDs) therapy. 2. In case of HCV antibodies positivity, the viral RNA should be investigated and genotyped. 3. RF and ACPA should be determined simultaneously in order to differentiate between RA and HCV-related arthropathy. 4. Erosions should be looked-for radiographically (X-rays) and ultrasonographically in order to confirm or exclude their presence. These data will allow HCV-related arthropathy to be differentiated from RF-positive RA and thus for the disease to be treated accordingly. Data obtained from several studies indicate that HCV-infected RA patients tolerate TNF-a blockers along with MTX, avoiding viral replication and liver damage [15]. This, of course, is very reassuring for the many patients in Mediterranean countries who carry an occult HCV infection. Patients poorly responsive to TNF-a blockers plus MTX can be

23  Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia

treated with B-cell-depleting biologics [16] or the antiIL-6 biologic tocilizumab (a monoclonal antibody directed against the IL-6 receptor), thus allowing control of both the RA autoimmune inflammation and the B-cell activation occurring in HCV chronic infection. If patients, on the other hand, are infected with continuously replicating viral species, a possible option is treatment with cyclosporine A (CsA). Experimental evidence has shown that cyclophylin-B, which is a target of CsA, is capable of regulating the viral protein NS5B, an HCV RNA-polymerase that plays a major role in HCV replication. Successful clinical use of CsA has been reported in such cases [17].

23.2.2 Sicca Syndrome and Sjögren’s Syndrome In 1994, we demonstrated the possible involvement of HCV in the development of chronic sialoadenitis [18]. In 1997, we showed that a lymphocytic infiltration mimicking clear-cut Sjögren’s syndrome (SS) was frequent in patients with HCV chronic infection [19]. In the same year, salivary and lacrimal gland inflammation strongly similar to Sjögren’s sialoadenitis was replicated in HCV transgenic mice, suggesting that HCV is not only lymphotropic but also sialotropic [20]. The presence of HCV RNA in the salivary glands of patients with SS was later confirmed in two clinical studies [21, 22]. This led us to hypothesize that HCV plays a direct role in driving autoimmune inflammation in the salivary glands, truly mimicking SS. In a monocentric analysis of our SS patients, we were able to show that HCV-infected patients with SS diagnosed according to the European-ACR criteria had several immunological features in common with primary SS patients [23, 24]. This was confirmed in a multicenter study in which 137 cases were collected and analyzed. It was shown that 58% of patients had diverse extraglandular manifestations, with the most frequent phenotypic presentations being arthritis-arthralgias (44%), vasculitis (20%), and neuropathy (16%). Of interest, parotidomegaly was observed in 16% of the case series, with labial histology in agreement with the diagnosis in 73% of the analyzed specimens. ANA were observed in 65%, low complement levels in 51%, and cryoglobulins were strongly associated with vasculitis and the presence of RF. Positive anti-Ro or anti-La auto-antibodies were detected in 23%. Splenomegaly and hepatomegaly

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were often seen (78%), whereas mild increases of ALT were noted in 68%; 42% of patients had sicca syndrome only. When the authors compared patients who were Ro- and/or La-positive with those who were negative for these antibodies, positive patients had a higher frequency of parotidomegaly (28% vs. 12%) and more arthritis (62% vs. 38%), but less frequent liver function test abnormalities (50% vs. 86%). Moreover, monoclonal bands were demonstrated in 43% of the patients, which is clearly higher than in primary SS, in which 20% of patients have only a monoclonal gammopathy [25]. Of interest, an association of HLA-DQ1*02 with sicca syndrome and with viral persistence was seen. All these findings together with the clinical and histopathological features, almost indistinguishable from primary SS, indicate that HCV-related SS is the first known infectious-related autoimmune disease.

23.2.3 Myalgias/Fibromyalgia Fibromyalgia syndrome (FS) is a clinical entity characterized by widespread muscular pain, several (>11 sites) tender points, and diffuse tenderness that is very sensitive to meteorological changes. In particular, windy weather and low barometric pressure are generally associated with increased tenderness all over the body, along with poor, unsatisfactory, non-relaxing sleep. FS was examined in patients with HCV chronic infection and found to be strongly increased in nonHCV related cirrhosis and in the main controls [26]. In particular, fatigue, which is a cardinal feature of FS, is a characterizing clinical hallmark of HCV-infected patients [27]. Studies have shown a general prevalence of FS of 17% (range 16–18.9) vs. 3% in controls (range 0–5). Since thyroid disease with anti-thyroid peroxidase and anti-thyroid microsomes auto-antibodies is present in 2–48% of chronic HCV carriers and subclinical hypothyroidism has been demonstrated in 2–9% [28], a multifactorial cause as the basis of the myalgiafibromyalgia syndrome has to be postulated. The obvious consequence is that therapy must address all the underlying possible mechanisms of these symptoms.

23.2.4 Mono/Multineuropathy Leukocytoclastic vasculitis is a prototypic vasculitis of the small vessels that can affect even the vasa ­nervorum.

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This vasculitis is the classical peripheral neuropathy, occurring in 70–80% of MC patients [29]. In rare cases, anti-neutrophil cytoplasmic auto-antibodies (ANCA) are detected. Subjective symptoms have been reported in up to 91% of patients. Sensory fibers are more commonly affected than motor fibers, with pure motor neuropathy in approximately 5% of patients. Since a significant increase of plasma anti-neuronal (anti-GM1, anti-sulfatide) auto-antibodies has been detected in MC, the vasa nervorum vasculitis, the thrombophilia sometimes associated with anti-cardiolipin auto-antibodies, and the anti-neuronal autoimmunity could together explain why neuropathy is frequent in MC and why plasma-exchange may be a therapeutic alternative to immunosuppressive therapies, especially in some acute cases [30].

G. Ferraccioli et al.

should raise the suspicion of a lymphoplasmacellular disorder or of a Sjögren’s-like evolution of the chronic infection [36–44].

23.2.7 Interstitial Lung Disease Italian patients with interstitial lung disease (ILD) have shown an increased prevalence of HCV infection [45]. On the other hand, MC patients often present with ILD [46, 47], which remains stable over time and is not accompanied by a decline of lung function [48]. Considering that MC can present as SS or as ANCApositive vasculitis and the great majority of such cases as an ongoing immune-complex vasculitis, all patients presenting with systemic complaints in the course of MC should be screened clinically and functionally (or though imaging techniques) for ILD.

23.2.5 Anti-phospholipid Syndrome Anti-cardiolipin auto-antibodies are detected in 20–27% of HCV-infected patients, without the occurrence of either anti-b2-glicoprotein I auto-antibodies or arterial or venous thrombosis. Moreover, ischemic lesions in the CNS were not higher in HCV patients with autoantibodies than in those without anti-cardiolipin, leading to the conclusion that the latter are the likely cause of the B-cell polyclonal activation [31, 32].

23.2.6 Acrocyanosis-Raynaud’s Increased plasma viscosity leading to impaired vascular supply and impaired vasal motility is observed mainly in type I cryoglobulinemia, associated with lymphoplasmacellular dyscrasias (myeloma, macroglobulinemia, lymphoma), but can also be observed in renal complications of MC, manifesting as an acute renal failure due to an acute nephritis. This scenario is seen in up to 31% of patients and can severely impact both the quality of life and survival [33, 34]. In these cases, manifestations include acrocyanosis, livedo reticularis, retinal hemorrhage, and severe Raynaud’s phenomenon with digital ulceration, purpura, and arterial thrombosis. Eventually ulcerations may occur, as commonly evidenced by nail-fold capillary abnormalities such as dilation, capillary shortening, and neo-angiogenesis [35]. The occurrence of these features in patients with type II and type III disease

23.2.8 Osteosclerosis Some patients with HCV chronic infection present with diffuse, persistent, sometimes throbbing bone pain. Twelve cases of bone osteosclerosis have been described. In these patients, cortical bone sclerosis and thickening of periosteal surfaces in the long bones are seen on X-ray. Bone turnover markers show an increased turnover; in some cases, anti-resorptive agents have been of clinical benefit. An imbalance between RANK-L/osteoprotegerin, with the latter prevailing, is the current interpretation of these still obscure manifestations [49, 50].

23.3 Conclusion and Discussion Rheumatic manifestations are common in MC and can present as a local manifestation of an ongoing IC disease (arthritis, ILD) or as a systemic autoimmuneinflammatory disease (SS, systemic vasculitis, mono/ multineuropathy, acrocyanosis, myalgia/myositis). The therapeutic approach depends upon the severity of the manifestations. According to current opinion, eradication of the virus should be the main goal in any case [51]. The minor clinical presentations (arthralgias, myalgias, sicca syndrome) can benefit from antimalarials and/or small daily doses of glucocorticoids. Once there is an association between erosive and

23  Rheumatologic Symptoms in Patients with Mixed Cryoglobulinemia

s­ eropositive RA and HCV infection, MTX can be used in combination with TNF-a blockers. The most severe manifestations (acute renal failure in nephritis, resistant skin ulcers) may require a prompt lavage of the IC-plugged vessel, using plasma exchange associated with immunosuppressants. Persistent inflammatory conditions (extracapillary nephritis, severe neuropathies, severe ulcers, systemic vasculitis) may require higher doses of glucocorticoids along with B-cell depletion through monoclonal antibodies [18] or more targeted biologics therapy, such as anti-IL6.

References 1. Trendelenburg M, Schifferli JA (1998) Cryoglobulins are not essential. Ann Rheum Dis 57:3–5 2. Vassilopoulos D, Calabrese LH (2005) Extrahepatic immunological complications of HCV infection. AIDS 19(Suppl 3):S123–S127 3. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serological features, and survival in 231 patients. Semin Arthritis Rheum 33:355–374 4. Sansonno D, Dammacco F (2005) Hepatitis C virus, cryoglobulinaemia, and vasculitis: immune complex relations. Lancet Infect Dis 5:227–236 5. Antonelli A, Ferri C, Fallahi P et  al (2008) High values of CXCL10 serum levels in mixed cryoglobulinemia associated with hepatitis C infection. Am J Gastroenterol 103:2488–2494 6. Antonelli A, Ferri C, Fallahi P et  al (2009) CXCL10 and CCL2 chemokine serum levels in patients with hepatitis C associated with autoimmune thyroiditis. J Interferon Cytokine Res 29:345–351 7. Fabris M, Quartuccio L, Sacco S et al (2007) B-Lymphocyte stimulator (BLyS) up-regulation in mixed cryoglobulinaemia syndrome and hepatitis-C virus infection. Rheumatology (Oxford) 46:37–43 8. Cacoub P, Poynard T, Ghillani P et al (1999) Extrahepatic manifestations of HCV. Arthritis Rheum 42:2204–2212 9. Buskila D (2000) Hepatitis C-associated arthritis. Curr Opin Rheumatol 12:295–299 10. Sène D, Ghillani-Dalbin P, Limal N et al (2006) Anti-cyclic citrullinated peptide antibodies in hepatitis C virus associated rheumatological manifestations and Sjogren’s syndrome. Ann Rheum Dis 65:394–397 11. Liu FC, Chao YC, Hou TY et al (2008) Usefulness of antiCCP antibodies in patients with hepatitis C virus infection with or without arthritis, rheumatoid factor, or cryoglobulinemia. Clin Rheumatol 27:463–467 12. Wener MH, Hutchinson K, Morishima C et  al (2004) Absence of antibodies to cyclic citrullinated peptide in sera of patients with hepatitis C virus infection and cryoglobulinemia. Arthritis Rheum 50:2305–2308 13. Magy N, Cribier B, Schmitt C et al (1999) Effects of corticosteroids on HCV infection. Int J Immunopharmacol 21(4):253–261

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14. Nissen MJ, Fontanges E, Allam Y et al (2005) Rheumatological manifestations of hepatitis C: incidence in a rheumatology and non-rheumatology setting and the effect of methotrexate and interferon. Rheumatology 44:1016–1020 15. Ferri C, Ferraccioli G, Ferrari D, GISEA Group et al (2008) Safety of anti-tumor necrosis factor-alpha therapy in patients with rheumatoid arthritis and chronic hepatitis C virus infection. J Rheumatol 35:1944–1949 16. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101: 3827–3834 17. Galeazzi M, Bellisai F, Manganelli S et  al (2006) Cyclosporine A for the treatment of autoimmune disorders in HCV infected patients. Autoimmun Rev 5:493–498 18. Pirisi M, Scott C, Fabris C et al (1994) Mild sialoadenitis: a common finding in patients with hepatitis C virus infection. Scand J Gastroenterol 29:940–942 19. Scott CA, Avellini C, Desinan L et al (1997) Chronic lymphocytic sialoadenitis in HCV-related chronic liver disease: comparison with Sjogren’s syndrome. Histopathology 30: 41–48 20. Koike K, Moriya K, Ishibashi K et  al (1997) Sialadenitis histologically resembling Sjogren syndrome in mice transgenic for hepatitis C virus envelope genes. Proc Natl Acad Sci USA 94:233–236 21. Toussirot E, Le Huédé G, Mougin C et al (2002) Presence of hepatitis C virus RNA in the salivary glands of patients with Sjögren’s syndrome and hepatitis C virus infection. J Rheumatol 29:2382–2385 22. Arrieta JJ, Rodríguez-Iñigo E, Ortiz-Movilla N et al (2001) In situ detection of hepatitis C virus RNA in salivary glands. Am J Pathol 158:259–264 23. De Vita S, Damato R, De Marchi G et al (2002) True primary Sjögren’s syndrome in a subset of patients with hepatitis C infection: a model linking chronic infection to chronic sialadenitis. Isr Med Assoc J 4:1101–1105 24. Vitali C, Bombardieri S, Jonsson R, European Study Group on Classification Criteria for Sjögren’s Syndrome et al (2002) Classification criteria for Sjogren’s syndrome: a revised version of the European criteria proposed by the AmericanEuropean Consensus Group. Ann Rheum Dis 61:554–558 25. Ramos-Casals M, Loustaud-Ratti V, De Vita S, SS-HCV Study Group et al (2005) Sjögren syndrome associated with hepatitis C virus: a multicenter analysis of 137 cases. Medicine 84:81–89 26. Buskila D, Shnaider A, Neumann L et al (1997) Fibromyalgia in hepatitis C virus infection. Another infectious disease relationship. Arch Intern Med 157:2497–2500 27. Kozanoglu E, Canataroglu A, Abayli B et  al (2003) Fibromyalgia syndrome in patients with hepatitis C infection. Rheumatol Int 23:248–251 28. Rocco A, Gargano S, Provenzano A et al (2001) Incidence of autoimmune thyroiditis in interferon alpha treated and untreated patients with chronic hepatitis C virus infection. Neuro Endocrinol Lett 22:39–44 29. Gemignani F, Pavesi G, Fiocchi A et  al (1992) Peripheral neuropathy in essential mixed cryoglobulinaemia. J Neurol Neurosurg Psychiatry 55:116–120 30. Alpa M, Ferrero B, Cavallo R et al (2008) Antineuronal antibodies in patients with HCV related mixed cryoglobulinemia. Autoimmun Rev 8:56–58

190 31. Ramos-Casals M, Cervera R, Lagrutta M, Hispanoamerican Study Group of Autoimmune Manifestations of Chronic Viral Disease (HISPAMEC) et  al (2004) Clinical features related to antiphospholipid syndrome in patients with chronic viral infections (hepatitis C virus/HIV infection): description of 82 cases. Clin Infect Dis 38:1009–1016 32. Sène D, Limal N, Cacoub P (2004) Hepatitis C virus-associated extrahepatic manifestations: a review. Metab Brain Dis 19:357–381 33. Ferri C, Moriconi L, Gremignai G et al (1986) Treatment of the renal involvement in mixed cryoglobulinemia with prolonged plasma exchange. Nephron 43:246–253 34. Madore F, Lazarus JM, Brady HR (1996) Therapeutic plasma exchange in renal diseases. J Am Soc Nephrol 7:367–386 35. Rossi D, Mansouri M, Baldovino S et  al (2004) Nail fold videocapillaroscopy in mixed cryoglobulinemia. Nephrol Dial Transplant 19:2245–2249 36. Inokuchi M, Ito T, Uchikoshi M et  al (2009) Infection of B  cells with hepatitis C virus for the development of ­lymphoproliferative disorders in patients with chronic ­hepatitis C. J Med Virol 81:619–627 37. Silvestri F, Pipan C, Barillari G et al (1996) Prevalence of hepatitis C virus infection in patients with lymphoproliferative disorders. Blood 87:4296–4301 38. de Sanjose S, Benavente Y, Vajdic CM et al (2008) Hepatitis C and non-Hodgkin lymphoma among 4784 cases and 6269 controls from the International Lymphoma Epidemiology Consortium. Clin Gastroenterol Hepatol 6:451–458 39. Charles ED, Green RM, Marukian S et  al (2008) Clonal expansion of immunoglobulin M+CD27+ B cells in HCVassociated mixed cryoglobulinemia. Blood 111:1344–1356 40. Zignego AL, Giannini C, Ferri C (2007) Hepatitis C virusrelated lymphoproliferative disorders: an overview. World J Gastroenterol 13:2467–2478

G. Ferraccioli et al. 41. De Vita S, Sacco C, Sansonno D et al (1997) Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection. Blood 90:776–782 42. Ferri C, Caracciolo F, La Civita L et al (1994) Hepatitis C virus infection and B-cell lymphomas. Eur J Cancer 30A:1591–1592 43. Ferri C, Monti M, La Civita L et al (1994) Hepatitis C virus infection in non-Hodgkin’s B-cell lymphoma complicating mixed cryoglobulinaemia. Eur J Clin Invest 24:781–784 44. Zignego AL, Giannelli F, Marrocchi ME et  al (2000) T (14;18) translocation in chronic hepatitis C virus infection. Hepatology 31:474–479 45. Meliconi R, Andreone P, Fasano L et al (1996) Incidence of hepatitis C virus infection in Italian patients with idiopathic pulmonary fibrosis. Thorax 51(3):315–317 46. Bombardieri S, Paoletti P, Ferri C et al (1979) Lung involvement in essential mixed cryoglobulinemia. Am J Med 66:748–756 47. Bertorelli G, Pesci A, Manganelli P et al (1991) Subclinical pulmonary involvement in essential mixed cryoglobulinemia assessed by bronchoalveolar lavage. Chest 100:1478–1479 48. Viegi G, Fornai E, Ferri C et  al (2005) Lung function in essential mixed cryoglobulinemia: a short-term follow-up. Clin Rheumatol 8:331–338 49. Manganelli P, Giuliani N, Fietta P et al (2005) OPG/RANKL system imbalance in a case of hepatitis C-associated osteosclerosis: the pathogenetic key? Clin Rheumatol 24:296–300 50. Fiore CE, Riccobene S, Mangiafico R et al (2005) Hepatitis C associated osteosclerosis (HCAO): report of a new case with involvement of the OPG/RANKL system. Osteoporos Int 16:2180–2184 51. Saadoun D, Resche-Rigon M, Thibault V et  al (2006) Antiviral therapy for hepatitis C virus – associated mixed cryoglobulinemia vasculitis: a long-term follow-up study. Arthritis Rheum 54:3696–3706

Endocrine Manifestations of HCV-Positive Cryoglobulinemia

24

Alessandro Antonelli, Clodoveo Ferri, Silvia Martina Ferrari, Michele Colaci, Alda Corrado, Andrea Di Domenicantonio, and Poupak Fallahi

24.1

Introduction

Hepatitis C virus (HCV) is known to be responsible for both hepatic and extrahepatic diseases. Among the systemic HCV-related extrahepatic diseases (HCVEHDs), mixed cryoglobulinemia (MC) has been extensively studied by clinico-epidemiological, immunological, and virological approaches [1–4]. However, recently, an increased prevalence of endocrine disorders has been observed in large series of HCV-infected patients and in those with MC. The most frequent and clinically important endocrine disorders of hepatitis C chronic infection are thyroid disorders and type 2 diabetes mellitus.

24.2

Autoimmune Thyroid Disorders

Many studies have examined the prevalence of autoimmune thyroid disorders (AITD) in HCV-positive (HCV+) patients. From a meta-analysis of the literature, a significant association between HCV infection and AITD has been reported [5]. The frequency of high levels of anti-thyroid antibodies in HCV+ patients ranges from 8% to 48% in different studies [6], while that of hypothyroidism ranges from 2% to 13%.

A. Antonelli (*) Metabolism Unit, Department of Internal Medicine, University of Pisa School of Medicine, Pisa, Italy e-mail: [email protected]

Recently [6], the prevalence of thyroid disorders was investigated in 630 consecutive HCV+ patients with chronic hepatitis. Patients with chronic hepatitis C (CH) were more likely to have hypothyroidism (13%), anti-thyroglobulin antibodies (AbTg) (17%), and antithyroperoxidase antibodies (AbTPO) (21%) compared to the control groups. Thyroid autoimmunity was also investigated in 93 MC patients [7], matched by sex and age to 93 patients with CH without MC and 93 healthy (HCV-negative) controls. The following thyroid abnormalities were significantly more frequent in MC patients than in HCV-negative controls: serum AbTPO (28% vs. 9%, p = 0.001); serum AbTPO and/ or AbTg (31% vs. 12%, p = 0.004); subclinical hypothyroidism (11% vs. 2%, p = 0.038); thyroid autoimmunity (35% vs. 16%, p = 0.006). Serum AbTPO were also significantly more frequent in MC patients than in CH controls (28% vs. 14%, p = 0.035). This study therefore demonstrated an increased prevalence of thyroid disorders in patients with HCV-related MC. These results are in agreement with those of a recent retrospective cohort study of users of the US Veterans Affairs health care facilities from 1997 to 2004, which included 146,394 patients infected with HCV. These patients had a significantly increased risk of thyroiditis [8]. Both differences in genetic variability and environmental co-factors, such as iodine intake or infectious agents other than HCV, could play an important role in the development of AITD [9]. Female gender is a risk factor for AITD development, while major risk factors for the development of hypothyroidism are again female gender in addition to the presence of AbTPO [9, 10]. HCV RNA has been detected in the thyroid of chronically infected patients [11, 12] but the possible

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consequences of HCV thyroid infection on thyrocyte function, vitality, and immunogenicity remain to be clarified. In the thyroid disorders observed in HCV infection and in MC, there are increased circulating levels of AbTPO, with the risk of hypothyroidism being higher in AbTPO-positive individuals [5]. A similar pattern was observed in patients treated with interferon (IFN)a [13]. Furthermore, it has been shown that viral NS5A and core proteins, alone or by the synergistic effect of cytokines [IFNg and tumor necrosis factor(TNF)a], are capable of up-regulating chemokine (C-X-C motif) ligand 10 (CXCL10) expression and secretion in cultured human hepatocyte-derived cells [14]. This suggests a key role for the CXCL10 produced by HCV-infected hepatocytes in regulating T-cell trafficking in the liver during chronic HCV infection, by recruiting Th1 lymphocytes, which secrete IFNg and TNFa, inducing CXCL10 secretion by hepatocytes, thus perpetuating the immune cascade [14]. Recently, high levels of CXCL10 were demonstrated in patients with autoimmune thyroiditis (AT) and overall in the presence of hypothyroidism [15], with a Th1 immune response shown to be involved in the induction of AT [16, 17]. Accordingly, a scenario can be proposed in which HCV thyroid infection up-regulates CXCL10 gene expression and secretion in thyrocytes (as previously shown in human hepatocytes), thus recruiting Th1 lymphocytes, which in turn secrete IFNg and TNFa, thus inducing CXCL10 secretion by thyrocytes and perpetuating the immune cascade, finally leading to the appearance of AITD in genetically predisposed subjects. A recent study [18] evaluated CXCL10 serum levels in HCV+ patients associated with MC, in the presence or absence of AT: in 50 MC patients without AT; in 40 MC patients with AT (MC+AT); in two genderand age-matched control groups [50 healthy controls (without HCV or AT; control); 40 controls with AT (without HCV and MC; control+AT)]. CXCL10 was significantly higher: (1) in control+AT than in control; (2) in MC patients than in control; (3) in MC+AT patients than in control or control+AT, or in MC. This study therefore evidenced high serum levels of CXCL10 in MC and showed that CXCL10 levels in MC+AT patients are significantly higher than in MC patients [18, 19].

A. Antonelli et al.

24.3

Thyroid Cancer, HCV Infection, and MC

A preliminary study reported a high prevalence of thyroid cancer complicating HCV-related hepatitis [20]. Subsequently, the prevalence of thyroid cancer in a series of unselected patients with HCV-related MC was investigated and compared with that of a control group [21]. Among 94 consecutive patients with MC, two had papillary thyroid cancer, while no such case was determined among controls (n = 2,401; p = 0.0019). In contrast, the prevalence of thyroid nodules was higher, although not significantly, in controls than in MC patients (65.3% vs. 54.8%). These data were subsequently confirmed in case–control studies, which concluded that HCV infection was associated with a high risk for thyroid cancer [5, 22]. However, a recent retrospective cohort study of users of US Veterans Affairs health care facilities, which included patients infected with HCV, failed to confirm an increased risk of thyroid cancer in these patients [8]. The exact mechanisms that transduce the HCV carcinogenic potential in thyroid cancer remain to be investigated; however, chronic AT is regarded as a preneoplastic condition [23]. In our studies in HCVinfected [22] and MC (MC-HCV+) [21] patients, features of AITD were seen significantly more often in patients with thyroid papillary cancer than in the other HCV+ patients. These findings suggest that AITD is a predisposing condition for thyroid papillary cancer.

24.4

HCV and Type 2 Diabetes

Several clinical epidemiologic studies [24] have reported an association between HCV infection and diabetes. However, almost all these studies consisted of HCV patients with and without cirrhosis [24], and it is well known that cirrhosis, of whatever etiology, is a risk factor for type 2 diabetes mellitus (T2D). Nonetheless, we have recently shown an association between HCV infection (in patients without cirrhosis), and T2D, both in HCV-related chronic liver [25] disease and in MC [26]. In our study, 229 consecutively recruited MC-HCV+ patients were compared with 217 sex- and age-matched controls without HCV infection [26]. The prevalence of T2D was significantly higher in MC-HCV+ patients than in controls (14.4% vs. 6.9%, p < 0.01). Diabetic MC-HCV+ patients were leaner than diabetic patients without MC-HCV

24

Endocrine Manifestations of HCV-Positive Cryoglobulinemia

(p < 0.0001) and had significantly lower total and low-density lipoprotein cholesterol levels (p < 0.001) and lower systolic (p = 0.01) and diastolic (p = 0.005) blood pressure. Non-organ-specific autoantibodies were present more frequently in MC-HCV+ diabetic patients than in non-diabetic MC-HCV+ patients (34% vs. 18%, p = 0.032). A population study (National Health and Nutrition Examination Survey-NHANES III 1988–1994) showed an adjusted odds ratio of 3.8 for T2D for individuals who were age >40 years and HCV+ [27] and an increased incidence of T2D [28]. There have also been a few reports in which IFN treatment of HCV infection was shown to improve glucose tolerance [26, 29] following the eradication of HCV infection; however, another study did not confirm these results [30]. In conclusion, these data indicate that HCV chronic infection is a risk factor for developing T2D in MC, although the mechanisms involved in the association between HCV infection and diabetes are not well clarified. It is speculated that insulin resistance (IR) (as a consequence of hepatic steatosis, which is present in about 50% HCV–infected individuals) [24], and/or elevated expression of TNFa (strongly correlated with the degree of liver diseases and the level of IR) [24] lead to the development of T2D [24]. Moreover, direct islet cell destruction by HCV was hypothesized by Masini et al. [31], in a study showing a direct cytopathic effect of HCV on islet beta cells. Indeed, virus-like particles were observed in islet cells from HCV+ pancreases, mainly close to the membranes of the Golgi apparatus, which was hyperplastic and dilated. These morphological changes were accompanied by reduced in vitro glucose-stimulated insulin release; however, apoptosis was not increased. More recently, the autoimmune induction of diabetes by HCV has been hypothesized; but, in fact, the type of diabetes manifested by patients with HCV chronic infection is not the typical form of T2D. Three studies reported [25, 26, 32] that HCV+ patients with T2D were leaner than T2D controls and showed significantly lower LDL-cholesterol, systolic and diastolic blood pressure. Furthermore, non-organspecific-autoantibodies were more frequently detected in MC-HCV+ patients with T2D than in non-diabetic MC-HCV+ patients (34% vs. 18%) [26]. We have postulated that MC-HCV+ diabetes has an immune-mediated pathogenesis [26]. In fact, HCV is able to infect islet cells [31] and may up-regulate expression of the CXCL10 gene. As discussed above,

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CXCL10 secretion by islet cells results in the recruitment of Th1 lymphocytes, which produce IFNg and TNFa. In the pancreas, this in turn induces CXCL10 secretion by pancreatic islet cells, thus perpetuating the immune cascade and leading to the appearance of islet cells dysfunction. We recently confirmed this hypothesis: in that CXCL10 serum levels were found to be higher in HCV+ patients with T2D than in a gender- and age-matched control group of T2D without HCV infection [4, 9].

24.5

Research Perspectives

Many aspects remain to be clarified regarding the immunopathogenesis of these disorders. An increasing number of studies have linked a Th1 immune response with HCV infection, MC, AITD, and diabetes. These data suggest a common immunological Th1 pattern as the pathophysiological basis of the association; however, this remains to be confirmed in further studies.

References 1. Ferri C, Antonelli A, Mascia MT et al (2007) B-cells and mixed cryoglobulinemia. Autoimmun Rev 7:114–120 2. Sansonno D, Carbone A, De Re V et al (2007) Hepatitis C virus infection, cryoglobulinaemia, and beyond. Rheumatology (Oxford) 46:572–578 3. Antonelli A, Ferri C, Galeazzi M et al (2008) HCV infection: pathogenesis, clinical manifestations and therapy. Clin Exp Rheumatol 26:S39–S47 4. Antonelli A, Ferri C, Ferrari SM et al (2008) Immunopathogenesis of HCV-related endocrine manifestations in chronic hepatitis and mixed cryoglobulinemia. Autoimmun Rev 8:18–23 5. Antonelli A, Ferri C, Fallahi P et al (2006) Thyroid disorders in chronic hepatitis C virus infection. Thyroid 16:563–572 6. Antonelli A, Ferri C, Pampana A et al (2004) Thyroid disorders in chronic hepatitis C. Am J Med 117:10–13 7. Antonelli A, Ferri C, Fallahi P et al (2004) Thyroid involvement in patients with overt HCV-related mixed cryoglobulinaemia. QJM 97:499–506 8. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297:2010–2017 9. Antonelli A, Ferri C, Ferrari SM et al (2009) Endocrine manifestations of hepatitis C virus infection. Nat Clin Pract Endocrinol Metab 5:26–34 10. Antonelli A, Ferri C, Fallahi P (2009) Hepatitis C: thyroid dysfunction in patients with hepatitis C on IFN-alpha therapy. Nat Rev Gastroenterol Hepatol 6:633–635

194 11. Gowans EJ (2000) Distribution of markers of hepatitis C virus infection throughout the body. Semin Liver Dis 20:85–102 12. Bartolomé J, Rodríguez-Iñigo E, Quadros P et al (2008) Detection of hepatitis C virus in thyroid tissue from patients with chronic HCV infection. J Med Virol 80:1588–1594 13. Prummel MF, Laurberg P (2003) Interferon-alpha and autoimmune thyroid disease. Thyroid 13:547–551 14. Apolinario A, Majano PL, Lorente R et al (2005) Gene expression profile of T-cell-specific chemokines in human hepatocyte-derived cells: evidence for a synergistic inducer effect of cytokines and hepatitis C virus proteins. J Viral Hepat 12:27–37 15. Antonelli A, Rotondi M, Fallahi P et al (2004) High levels of circulating CXCL10 are associated with chronic autoimmune thyroiditis and hypothyroidism. J Clin Endocrinol Metab 89:5496–5499 16. Antonelli A, Rotondi M, Fallahi P et al (2005) Increase of interferon-gamma inducible alpha chemokine CXCL10 but not beta chemokine CCL2 serum levels in chronic autoimmune thyroiditis. Eur J Endocrinol 152:171–177 17. Antonelli A, Rotondi M, Ferrari SM et al (2006) Interferongamma-inducible alpha-chemokine CXCL10 involvement in Graves’ ophthalmopathy: modulation by peroxisome proliferator-activated receptor-gamma agonists. J Clin Endocrinol Metab 9:614–620 18. Antonelli A, Ferri C, Fallahi P et al (2008) High values of CXCL10 serum levels in patients with hepatitis C associated mixed cryoglobulinemia in presence or absence of autoimmune thyroiditis. Cytokine 42:137–143 19. Antonelli A, Ferri C, Fallahi P et al (2008) Alpha-chemokine CXCL10 and beta-chemokine CCL2 serum levels in patients with hepatitis C-associated cryoglobulinemia in the presence or absence of autoimmune thyroiditis. Metabolism 57:1270–1277 20. Antonelli A, Ferri C, Fallahi P (1999) Thyroid cancer in patients with hepatitis C infection. JAMA 281:1588 21. Antonelli A, Ferri C, Fallahi P et al (2002) Thyroid cancer in HCV-related mixed cryoglobulinemia patients. Clin Exp Rheumatol 20:693–696

A. Antonelli et al. 22. Antonelli A, Ferri C, Fallahi P et al (2007) Thyroid cancer in HCV-related chronic hepatitis patients: a case-control study. Thyroid 17:447–451 23. Okayasu I, Fujiwara M, Hara Y et al (1995) Association of chronic lymphocytic thyroiditis and thyroid papillary carcinoma: a study of surgical cases among Japanese, and white and African Americans. Cancer 76:2313–2318 24. Noto H, Raskin P (2006) Hepatitis C infection and diabetes. J Diabetes Complications 20:113–120 25. Antonelli A, Ferri C, Fallahi P et al (2005) Hepatitis C virus infection: evidence for an association with type 2 diabetes. Diabetes Care 28:2548–2550 26. Antonelli A, Ferri C, Fallahi P et al (2004) Type 2 diabetes in hepatitis C-related mixed cryoglobulinaemia patients. Rheumatology (Oxford) 43:238–240 27. Mehta SH, Brancati FL, Sulkowski MS et al (2000) Prevalence of type 2 diabetes mellitus among persons with hepatitis C virus infection in the United States. Ann Intern Med 133:592–599 28. Mehta SH, Brancati FL, Strathdee SA et al (2003) Hepatitis C virus infection and incident type 2 diabetes. Hepatology 38:50–56 29. Tanaka H, Shiota G, Kawasaki H (1997) Changes in glucose tolerance after interferon-alpha therapy in patients with chronic hepatitis C. J Med 28:335–346 30. Giordanino C, Bugianesi E, Smedile A et al (2008) Incidence of type 2 diabetes mellitus and glucose abnormalities in patients with chronic hepatitis c infection by response to treatment: results of a cohort study. Am J Gastroenterol 103: 2481–2487 31. Masini M, Campani D, Boggi U et al (2005) Hepatitis C virus infection and human pancreatic beta-cell dysfunction. Diabetes Care 28:940–941 32. Skowronski M, Zozulinska D, Juszczyk J et al (2006) Hepatitis C virus infection: evidence for an association with type 2 diabetes. Diabetes Care 29:750

Cutaneous Cryoglobulinemic Vasculitis

25

Konstantinos Linos, Bernard Cribier, and J. Andrew Carlson

25.1

Introduction

The modern era of cryoglobulinemia started in 1966, when Meltzer et al. described nine patients with serum cryoglobulins [1]. Cryoglobulins are coldprecipitating immunoglobulins that persist in the serum and resolubilize when rewarmed [2, 3]. Mixed cryoglobulins, composed of different immunoglobulins, with a monoclonal IgM rheumatoid factor component and polyclonal IgG in type II, and polyclonal immunoglobulins IgM (with rheumatoid factor activity) and IgG in type III, are associated with connective tissue disease, hematologic malignancies, and/or infectious diseases, chiefly hepatitis C infection virus (HCV) [4–6]. Type I cryoglobulins are monoclonal and typically produce hyaline thrombi rather than vasculitis. Mixed cryoglobulinemia (MC) is associated with a broad range of clinical manifestations, ranging from asymptomatic MC to life-threatening vasculitis. The commonest manifestation of MC is cutaneous vasculitis, denoted by palpable purpura [7, 8]. Few diseases cause as much diagnostic and therapeutic consternation as vasculitis, an inflammatory process directed primarily at the vasculature that results in destruction of the vessel walls, leading to hemorrhage, ischemia, and/or infarction. The skin, in part due to its large vascular bed, exposure to cold temperatures, and frequent circulatory presence of stasis,

J.A. Carlson (*) Division of Dermatology and Dermatopathology, Department of Pathology, Albany Medical College, Albany, NY, USA e-mail: [email protected]

is involved in many distinct as well as un-named vasculitic syndromes, that vary from localized and self-limited to generalized and life-threatening with multi-organ disease [9]. Cryoglobulinemic vasculitis (CV) exemplifies the range of clinical manifestations, degrees of severity, and diverse outcomes for patients affected by vasculitis. CV is defined as “vasculitis, with cryoglobulin immune deposits, affecting small vessels (i.e. capillaries, venules, or arterioles), and associated with cryoglobulins in the serum”according to the Chapel Hill Consensus criteria [10]. However, this pathologic-serologic definition falls short of capturing all cases of CV and does not acknowledge the overlap with thrombotic factors in the pathogenesis of clinical findings. Specifically, many of the cutaneous manifestations of CV are caused by muscular-vessel vasculitis and/or thrombosis, the latter of which can resemble hyaline thrombi of type 1 cryoglobulinemia. This chapter reviews the epidemiology, etiology, pathogenesis, clinical presentations, pathologic and laboratory findings, prognosis, and management of CV, with emphasis on its cutaneous presentations.

25.2

Epidemiology

The incidence of biopsy-proven, cutaneous vasculitis ranges from 39.6 to 59.8 cases per million per year [9]. Cutaneous vasculitis affects people of all ages, slightly fewer males than females, and adults more often than children, with 90% of the latter having HenochSchonlein purpura [9]. For most patients, the onset of cutaneous vasculitis can be associated with exposure to a trigger, such as a drug or infection, and will represent a single, acute, self-limited episode. However, for

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a minority of patients, relapsing or chronic unremitting disease will ensue, and in this population of patients, MC is a common etiologic factor [9]. The prevalence of CV in patients presenting with cutaneous vasculitis ranges from 2.5% to 8% [9, 11], a range that mirrors the seropositivity rate for HCV in the population studied [9, 12]. For example, CV is more common in Southern Europe (4.8 per million in Lugo, Spain [13]) than in Northern Europe or North America (0.6 per million, Capital District of New York (personal observation, [9]) [14]. In underdeveloped countries, where HCV infection is becoming increasingly prevalent, the incidence of HCV-related CV is expected to rise [15]. Notably, females are more frequently affected than males, by a ratio of 3:1.

25.3

Etiology and Pathogenesis

Table 25.1 lists the disorders associated with MC/CV, which include infections (mostly hepatotropic viruses), connective tissue diseases (commonly Sjögren’s syndrome), and hematolymphoid proliferations (mostly B-cell proliferations). The term “essential” cryoglobulinemia is reserved for the small number of cases with no overt infectious, neoplastic, or immuno-rheumatologic disorders [19]. The causative role of hepatotropic viruses in MC/CV has long been hypothesized based on the frequent association of liver involvement in patients with CV [20, 21]. Since Pascual et al. [22] noted the strong association between HCV infection and what was previously called “essential cryoglobulinemia,” the causative role of HCV infection in CV has been widely acknowledged, based on many studies [23–26]. Depending on the region of the world, HCV accounts for 42–98% of MC/CV cases [16] whereas hepatitis-B-related CV represents only a minority (~2%) [25]. HCV infection not only causes chronic hepatic inflammation, but also an array of extrahepatic manifestations, which include MC/CV, diverse systemic autoimmune diseases, such as Sjögren’s syndrome, rheumatoid arthritis, systemic lupus erythematosus, and polyarteritis nodosa, and non-Hodgkin lymphomas [27, 28]. Immunologic problems common to these patients are reflected in the presence of anti-nuclear antibody (ANA), rheumatoid factor (RF), and cryoglobulins. Indeed, the frequency of cryoglobulins increases with the duration of HCV infection, and type

Table 25.1 Disorders and infections associated with cyroglobulinemia Association Disorders Infections Viral infections Hepatitis C virus, 42–98% of cases [16] Hepatitis A and B viruses, human immunodeficiency virus, Herpes viridae, Epstein-Barr virus, varicella zoster virus, cytomegalovirus, parvovirus B19, human T cell lymphotropic virus type I, influenza virus; rubella virus; hanta virus Bacterial Bacterial endocarditis, syphilis, rickettsial infections infections, Q fever, leprosy, Lyme disease, post streptococcal nephritis Fungal Coccidioidomycosis infections Parasitic Toxoplasmosis, echinococciasis, malaria, infections leishmaniasis (kala-azar), schistosomiasis Autoimmune Sjögren’s syndrome diseases Systemic lupus erythematosus Rheumatoid arthritis Dermatomyositis/polymyositis Systemic sclerosis, endomyocardial fibrosis, pulmonary fibrosis Inflammatory bowel disease Systemic vasculitis: giant cell (temporal) arteritis, Henoch-Schönlein purpura Sarcoidosis Autoimmune thyroiditis Primary antiphospholipid syndrome Pemphigus vulgaris Biliary cirrhosis Myelo/ B cell non-Hodgkin lymphoma lymphoprolifChronic lymphocytic leukemia erative diseases Multiple myeloma Waldenström’s macroglobulinemia Angioimmunoblastic lymphoma Chronic myeloid leukemia Castleman disease Cold agglutinin disease Myelodysplasia and chronic myeloid leukemia Thrombocytopenic thrombotic purpura Adapted from [7, 17, 18]

III and oligoclonal MC are considered an intermediate stage in the progression to type II MC [8, 29, 30]. Up to 70% of patients with HCV have MC; however, CV develops only in a minority of them [28, 30–35], with a prevalence of 2–13% [36, 37]. Factors associated with the development of MC include female sex, alcohol consumption above 50 g/day, HCV genotype 2 or

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3, and extensive liver fibrosis [38]. Persistent HCV infection is believed to act as a chronic stimulator of the immune system, resulting in clonal B-lymphocyte proliferations and the production of cryoglobulins, which lead to circulating immune complexes (onethird of which become insoluble when exposed to cold temperatures) [3, 8, 39]. Indeed, the major difference between HCV patients with and without MC is the presence of functional derangements of B cells and restrictions of the humoral response promoted by the expansion of clonal B-cell proliferations [40, 41]. Expansion of RF-synthesizing B lymphocytes (the hallmark of MC) in combination with RF activity and cryoprecipitability is believed to be responsible for the pathogenesis of CV, an immune complex (antigen-antibody) mediated systemic vasculitis (Coombs Gel type III reaction) [8, 9]. In HCV-related CV, cold-insoluble immune complexes are formed by IgM with RF activity linked to IgG, which also bind HCV particles and non-enveloped nucleocapsid proteins, in which the viral proteins confer peculiar physical and chemical properties on cryoimmunoglobulins [5]. Indeed, in support of the immune complex pathogenesis of CV are electron microscopy findings of the typical crystalline structures of cryoglobulins in vessels [42–44]. In addition, IgM and IgG vascular deposits are detected by direct immunofluorescence exam, immunohistochemistry, and in situ hybridization localizing HCV to vessel endothelium affected by vasculitis [45–47]. The deposition of immune complexes (IgM-RF-IgG-HCV) induced by cold exposure, stasis, or another trigger, such as local inflammation, activates complement, which in turn attracts neutrophils, setting off an inflammatory cascade that disrupts the integrity of the vessel wall resulting in the clinical manifestations of vasculitis [9, 45, 48]. While HCV is the principal antigen driving immune-complex formation, in some patients either a sustained anti-viral response does not lead to remission of CV or CV relapses shortly afterwards [49, 50]. These phenomena implicate other, unknown antigens and progression of the aberrant immune response by epitope spreading [51] and/or the development of autonomous B-cell proliferations [5]. Cellmediated cytotoxicity may also play a role in the pathogenesis of CV, as there may be numerous CD8+ lymphocytes in cutaneous lesions of HCV-related CV [46], and lymphocytic vasculitis has been reported in rare cases of CV [47, 52].

197

Lastly, non-inflammatory small- and/or muscularvessel thrombosis is an under-recognized pathogenic mechanism contributing to the cutaneous manifestations of MC/CV, mostly ulceration. About a third of CV patients exhibit thrombosis [6, 37, 42, 47, 53–62]. These CV-related vaso-occlusive thrombi can be found either concurrently with vasculitis or independently [42, 53, 54, 59, 61, 63]. In general, abnormal coagulation, blood flow (stasis), chronic inflammation, and endothelial cell activation all contribute to the development of individual lesions of vasculitis [9]. Moreover, hypercoagulable states (e.g., factor V Leiden, protein C or S deficiency) are significantly more frequent in patients with ulcerative cutaneous vasculitis [64, 65]. In connective tissue disease vasculitis, the coexistence of prothombotic antiphospholipid antibodies contributes to the rapid evolution of vascular insufficiency, with progressive tissue ischemia and infarction [66, 67]. Recurrent episodes of CV and its corresponding tissue damage likely lead to abnormal blood flow, producing a prothrombotic environment and the subsequent formation of vaso-occlusive thrombi, in some cases type I cryoglobulin thrombi.

25.4

Clinical Findings

Table 25.2 lists the demographics and clinical manifestations found in MC/CV. Most of these clinical symptoms, signs, and complications of CV significantly increase with disease duration [25] The disease expression is variable, ranging from mild clinical symptoms to life-threatening complications [8], but this is dependent on disease activity and duration [25] The most frequent target organs in patients with MC are the skin, joints, nerves, and kidney. Up to 80% of patients present with the typical clinical CV triad of purpura, arthralgias, and weakness [25]. Palpable purpura, the clinical hallmark of cutaneous vasculitis, is found in almost all patients at some point in their disease and usually affects the lower extremities, as venous stasis and environmental exposure favor the precipitation of cryoglobulins [24, 52, 69]. Extension of palpable purpura above the waistline is a clue to the presence of systemic disease in patients presenting with cutaneous vasculitis [9] and is a correspondingly frequent occurrence in CV [4]. In decreasing order of frequency, the lower extremities, the upper extremities, trunk, head and neck, oral and nasal mucosa are involved by CV [61]. Typically, cutaneous CV

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Table 25.2 Evolution of clinical findings in mixed cryoglobulinemia/cryoglobulinemic vasculitis Clinical findings Female/male ratio Mean age at onset Mean duration of disease Cutaneous manifestations (any) Arthralgias Arthritis Weakness (asthenia) Raynaud’s phenomenon Neurologic involvement (peripheral and CNS) Peripheral neuropathy Central nervous system Renal involvement Sicca syndrome Skin ulcers Lung disease Liver disease (abnormal liver functions, hepatomegaly) Hyperviscosity syndrome Systemic (diffuse) vasculitis Cancer (B-cell lymphoma, hepatocellular carcinoma, thyroid cancer) Gastrointestinal disease

Onset/beginning of follow-upa 3:1 53 ± 12 years

End of follow-upa

11 ± 8 years 56%, (9–100%)b

98% (purpura)

39%, (17–72%)b 8% 72% (9–80%)b 24% (5–50%)b

91% 7% 98% 48%

24% (8–52%)b

58%

81%

2%c

2% c

20% (2–40%)b 13% (2–29%)b 11% 0 35% (0–58%)b

30% 53% 22% 2% 77%

0

0.5%

0

6.2%

0.5%

16%

0.2%c

1%c

digital gangrene (Table 25.3 and Figs. 25.1–25.3). The ulcers of CV are distinctive. They are located distally on the legs, are painful and bilateral, show a fibrinous or necrotic bed, and have well circumscribed margins surrounded by purpuric and pigmented skin [55] (Fig. 25.3). As many of these clinical findings can be due to nonvasculitic disorders, skin biopsy is crucial in documenting the presence of vasculitis. This histologic finding coupled with direct immunofluorescence data, serologic studies, and review of the body’s organ systems is the most effective method at arriving at a specific diagnosis of a vasculitic syndrome and excluding vasculitis mimics [9, 89, 90]. Notably, cryocrit levels correlate with cutaneous findings of CV: purpuric outbreaks are frequently observed in the late afternoon, when the cryocrit levels are highest [25, 91]. In patients with CV, palpable purpura is accompanied less frequently by systemic symptoms, such as fever (>70%), arthralgias (70%), membranoproliferative glomerulonephritis type I (55%), neuropathy (<40%), and/or pulmonary symptoms of hemoptysis and dyspnea (5%) [4, 61]. B cell non-Hodgkin’s lymphoma represents the most common neoplastic complication of CV [27, 92, 93]. Inflammatory complications of HCV infection, with or without MC, comprise a broad spectrum of systemic autoimmune diseases, including primary systemic vasculitic syndromes, such as polyarteritis nodosa, giant cell arteritis, Wegener’s granulomatosis, Churg Strauss syndrome, microscopic polyangiitis, Henoch-Schönlein purpura, and Takayasu’s arteritis [28]. In some instances, these systemic vasculitis syndromes may simply represent the progression of CV.

a

25.5

occurs as crops of non-pruritic, purpuric papules that range in size from 3 to 10 mm in diameter and persist 3–10 days, leaving residual pigmentation [70]. Other cutaneous manifestations of CV include urticaria, petechiae, leg edema, ulceration, nodules, plaques, livedo racemosa, Raynaud’s phenomenon, acral cyanosis, and

Table 25.3 and Figs. 25.1–25.4 list and illustrate the wide spectrum of clinicopathologic findings that characterize CV. In particular, the pathologic changes of CV include vaso-occlusive thrombi, urticarial vasculitis, muscular-vessel vasculitis with or without small-vessel vasculitis, lymphocytic or granulomatous vasculitis, endarteritis obliterans, and reactive endothelial hyperplasia (angiomatosis). These varied findings highlight the complexity of the vessel involvement, the multiple pathogenic mechanisms, and the recurrent vascular injury that occur in CV. By and large, the majority of skin biopsies of CV’s palpable purpura show

Most of the data reported are from the case series of Ferri et al. [25], consisting of 231 MC patients: beginning and end of follow-up data b Mean of means (range) from case series review of onset of cryoglobulinemia by Tedeschi et al. [7] c Data from [68]

Histologic Findings

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199

Table 25.3 Cutaneous clinicopathologic manifestations of cryoglobulinemic vasculitisa Clinical findings (Palpable) purpura Leg ulcers Post-inflammatory hyperpigmentation Lower limb edema (Cold) Urticaria Livedo reticularis Raynaud’s phenomenon Petechiae Digital gangrene Acral cyanosis Infrequent/rare findings Bare white spots (alternating anemic and cyanotic regions) Hemorrhagic bullae Erythematosus macular/papular rash (exanthem) Follicular pustules Nodule(s) Violaceous plaque(s) Scarring Telangiectases Hemorrhagic crusts Digital erythema (red finger) Histologic findings Small vessel neutrophilic vasculitis (LCV) Small vessel lymphocytic vasculitis Muscular vessel neutrophilic vasculitis Granulomatous vasculitis Electron microscopy Thrombi, not otherwise specified Hyaline (type I) thrombi Reactive angioendotheliomatosis Direct immunofluorescence findings IgM vascular deposits IgG vascular deposits IgA vascular deposits Complement (C3 >> C4) vascular deposits Basement membrane zone immunoreactants (Lupus band) Cutaneous HCV studies Immunohistochemistry anti-HCV In situ hybridization HCV RNA

Mean frequencya 74% 34% 34% 15% 11% 10% 8% 7% 4% 3% <3%

References a a a a a a a a a a

[71] [4, 72] [4, 50, 58, 59, 73] [74] [50, 60, 74, 75] [57–60] [61] [61] [61] [76]

75% (11/13 reports document deep dermal/subcutis LCV) <2% 18% <1% Crystalline (cryoglobulins) vascular deposits 33% 21% 10%

a

72% 47% 12% 70%

a

9%

[79, 83, 84]

40%; intraluminal or endothelial, both normal and vasculitic 60–100%, virion associated with IgG, IgM; found in vasculitic endothelium, keratinocytes and adnexae

[47]

[1, 4, 6, 42, 43, 53, 61–63, 75, 77–79]

[47, 52] [1, 36, 42, 43, 52, 53, 80, 81] [75, 82] [42–44] [6, 37, 53–56] [42, 47, 53, 57–62] [4, 57–60, 62]

a a a

[45, 46]

LCV leukocytoclastic vasculitis Mean frequency derived from a summation of case reports and case series reporting 343 skin biopsy results in 498 patients with CV: [1, 4, 6, 36, 37, 42–47, 49, 52–61, 63, 75–88]

a

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Fig. 25.1 Urticarial vasculitis as a presenting sign of hepatitis C infection. A 38-year-old female intravenous drug abuser presented with severe, recurrent painful urticarial papules that lasted >24 h (left). Biopsy demonstrated a sparse perivascular infiltrate

of neutrophils (top right) associated with nuclear debris and a few extravasated red blood cells, diagnostic of CV (bottom right)

a small-vessel neutrophilic vasculitis that is pan-dermal and mediated by immune complex deposition, as evidenced by direct immunofluorescence, which shows, in decreasing frequency, IgM, complement (C3), IgG, and IgA vascular deposits [4, 61, 80]. As CV typically occurs intermittently, as crops of petechiae or palpable purpura, biopsies negative for vasculitis are likely due to timing, since sampling a lesion of neutrophilic (leukocytoclastic) vasculitis >48-h-old will show the features of resolving vasculitis in the form of perivascular lymphocytic infiltrates, extravasated red blood cells, and no immunoglobulin deposits [4, 51]. The diversity of cutaneous lesions in CV correlates with the size of vessel affected by vasculitis [89]. Urticaria in CV is

associated with superficial perivascular neutrophilic infiltrates together with nuclear debris and extravasated red blood cells, i.e., the features of urticarial vasculitis (Fig. 25.1). Palpable purpura, vesiculobullous lesions and superficial ulcers correlate with pan-dermal smallvessel neutrophilic vasculitis. Deep ulcers with sharp borders, nodules, pitted scars, livedo racemosa, or digital gangrene are associated with arterial-muscular vessel vasculitis, typically located at the dermal-subcutis interface or within the subcutis. Ulcers in CV can also be associated with underlying vaso-occlusive thrombi, typical or hyalinized as seen in type I cryoglobulinemia, and can co-exist with vasculitis [6, 42, 61]. In HCV-related CV, HCV proteins have been detected

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201

Fig. 25.2 Hepatitis C infection and recurrent cryoglobulinemic vasculitis. A 50-year-old man with a 20-year history of hepatitis C infection presented with malaise, fever, chills, cough, petechial lower extremity rash (top left), splinter hemorrhages (top right), and palmar and digital erythema. Examination revealed diffuse

petechiae and hyperpigmentation and black, punctate scarring over the lower leg, evidence of healed episodes of vasculitis. Direct immunofluorescence showed predominate IgM vascular deposits (bottom left). Biopsy revealed both vaso-occlusive thrombi and small vessel neutrophilic vasculitis

in vessels and keratinocytes [36, 45, 47] and even in apparently normal skin [5], while HCV RNA is present in endothelium [75].

HCV viremia and anti-HCV antibodies are found in the majority of MC patients [25]. However cryoprecipitates may deplete HCV antibody and HCV antigens from serum, leading to a false-negative hepatitis serology. Paired testing from both serum and cryoprecipitate may overcome this problem [17]. Other laboratory abnormalities that can be used as surrogate markers for the presence of MC are low C4, depressed total hemolytic complement levels, RF activity, or monoclonal proteinemia. Hypocomplementemia is an important and sensitive finding, as it is seen in 60–90% of MC patients [8] and its presence aids in distinguishing CV from anti-neutrophilic cytoplasmic antibody (ANCA)-associated vasculitis. In addition, the levels of the early complement components C1, C4, and C2 are decreased in HCV + MC patients, whereas

25.6

Laboratory Findings

Table 25.4 lists the most frequent laboratory findings. The first diagnostic step is the detection of serum cryoglobulins, which requires that blood samples be maintained at 37°C on the way to the laboratory [94]. Cryoglobulins are readily detectable in HCV-infected patients (40–60%) [38] using standard detection methods such as immunoelectrophoresis and immunofixation [17]. Different serum antibodies can be present in over 50% of CV patients [95, 112]. As expected,

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Fig. 25.3 Cryoglobulinemic vasculitis with livedo racemosa and ulceration. A middle-aged female presented with extensive livedo racemosa (top left) and geographic ulcers with steep, violaceous edges over the lower extremities (top right). Deep

punch biopsy demonstrated pan-dermal and subcutaneous smallvessel vasculitis (bottom left) and muscular-vessel thrombosis (bottom right)

C3 levels fluctuate with disease course. RF activity is observed in up to 70% of patients [8]. Finally, autoantibodies such as ANA, ANCA, anti-phospholipid, antismooth muscle cell, and anti-thyroglobulin antibodies can be detected in MC [31, 96].

by malignancy. Prognostic predictive factors for HCV-related CV include duration of HCV infection, peripheral nerve disease, Raynaud’s disease, cutaneous ulcers, immunosuppressant treatment (leading to secondary infection, the most common cause of death at this time), and renal function as measured by serum creatinine [85]. Survival data from older studies do not take into account recent therapeutic advances. Antiviral treatment may cure or control HCV-associated CV and possibly improve prognosis [34].

25.7

Prognosis

The 10-year survival of patients with MC/CV is much lower than in an age and sex-matched population [25]. The natural history of this disease can be divided into three clinical patterns [25]: (1) a mild, slow-progressive disorder with relatively good prognosis and survival (approximately 50% of all cases); (2) a moderatesevere clinical course that is impacted by the severity of renal and/or liver disease; and (3) MC/CV complicated

25.8

Treatment

Table 25.5 lists a therapeutic ladder for CV. In summary, the therapeutic options are: (1) antiviral therapy, (2) immunosuppressive agents and steroids,

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203

Fig. 25.4 The broad histologic spectrum of cutaneous cryoglobulinemic vasculitis. Most biopsies of cryoglobulinemic vasculitis show small-vessel neutrophilic vasculitis (leukocytoclastic vasculitis) that affects both the superficial and deep vascular plexus (top left). In a substantial minority of patients, a muscular-vessel neutrophilic vasculitis (arrow, top right) can be identified. A few patients presenting with HCV-related cutaneous

vasculitis will have hyaline, PAS-positive thrombi similar to those found in type 1 cryoglobulinenemia instead of frank vasculitis (bottom left). Due to the recurring episodes of vascular injury and/or occlusion, neoangiogenesis, reactive angioendotheliomatosis and/or endarteritis obliterans can develop. Elastic tissue stain highlights recanalization of an occluded artery (endarteritis obliterans) at the bottom right

(3) rituximab (a monoclonal anti-CD20 antibody), and (4) plasmapheresis. HCV infection, as the most common possible underlying condition, has to be excluded in every case. Subsequently, HCV eradication should be attempted in all patients with HCV-associated CV, using pegylated interferon (IFN)-a and ribavirin [102, 103]. IFN-a has been shown to be effective in HCV-negative CV patients as well, possibly due to its immunomodulating effects [104]. Patients with mild manifestations, such as purpura and arthralgias, may be treated with low doses of steroids and a lowantigen-content (LAC) diet [97] or colchicine, whose use is limited by its toxicity [105] Antidepressants

and anticonvulsants have been used for the treatment of neuropathic pain [106]. Cyclophosphamide, high dose of corticosteroids, and plasma exchange are typically reserved for the most severe, life threatening complications of CV, such as glomerulonephritis, sensory-motor neuropathy, and multi-organ vasculitis [107]. Recently, rituximab, which targets the B cells involved in cryoglobulin formation, has been used successfully in CV patients [99, 100, 108–110]. For HCV-related MC/CV, the combination of pegylated IFN-a, ribavirin, and rituximab has been shown to be more effective and produce more durable responses than the standard of care pegylated IFN-a and ribavirin [99].

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Table 25.4 Serological findings in patients with cryoglobulinemic vasculitis Test Anti-HCV antibodies Detectable HCV RNA Rheumatoid factor Type II cryoglobulins Low C4 complement levels Hypocomplementemia Elevated transaminases Antinuclear antibodies (ANA) Anti-smooth muscle antibodies Antimitochondrial antibodies Antithyroid antibodies Antiphospholipid antibodies ANCA

Frequency (%) 90 85 70–80 70–80 60–80 60–90 25–40 15–40 10–40 10 10 5–20 <5

Adapted from [8, 17, 18] ANCA anti-neutrophil cytoplasmic antibody Table 25.5 Treatment of cryoglobulinemic vasculitis Disease severity Asymptomatic

Mild-moderate (mild neuropathy, purpura, weakness) Moderate to severe (active chronic hepatitis, MPGN, skin vasculitis) Severe, rapidly progressive (Glomerulonephritis, sensory-motor neuropathy, widespread vasculitis)

Treatment Monitoring General measures: (a) Avoid cold exposure and tight fitting clothing (b) Wear supportive stockings (c) Avoid standing or sitting in the same position (for prolonged periods [70] Peg-IFN-a + Riba, low-medium dose corticosteroids +/− lowantigen diet [17, 97] Peg-IFN-a + Riba [73, 98] Peg-IFN-a + RIBA + rituximab [99] Plasma exchange + corticosteroids + Cyclophosphamide or rituximab [100, 101]

Adapted and modified from Ferri et al. [34] and Chen and Carlson [89] Peg-IFN-a pegylated interferon-a, Riba ribavirin

25.9

Conclusion

MC/CV is a multifaceted disease, characterized clinically by vasculitic, thrombotic, autoimmune, and lymphoproliferative manifestations [28, 40, 41, 85].

Considerable progress has been made in the last two decades elucidating the pathogenesis of HCV infection and MC. However, key questions, such as why CV develops only in a subset of HCV-positive patients, have yet to be answered. For HCV-related CV, eradication of the virus infection represents the gold standard of therapy, but the added effectiveness of anti-B cell therapy clearly demonstrates the role of B-cell proliferation in MC/CV. In the future, a vaccine-based therapy with recombinant HCV protein may prevent the appearance of extrahepatic complications or interrupt the self-perpetuating autoimmune mechanism of CV [111].

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14. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 3:25 15. Herrine SK (2002) Approach to the patient with chronic hepatitis C virus infection. Ann Intern Med 136(10): 747–757 16. Dispenzieri A, Gorevic PD (1999) Cryoglobulinemia. Hematol Oncol Clin North Am 13(6):1315–1349 17. Lamprecht P, Gause A, Gross WL (1999) Cryoglobulinemic vasculitis. Arthritis Rheum 42(12):2507–2516 18. Braun GS, Horster S, Wagner K et al (2007) Cryoglobulinaemic vasculitis: classification and clinical and therapeutic aspects. Postgrad Med J 83(976):87–94 19. Gumber SC, Chopra S (1995) Hepatitis C: a multifaceted disease. Review of extrahepatic manifestations. Ann Intern Med 123(8):615–620 20. Bombardieri S, Ferri C, Migliorini P et al (1986) Cryoglobulins and immune complexes in essential mixed cryoglobulinemia. Ric Clin Lab 16(2):281–288 21. Levo Y, Gorevic PD, Kassab HJ et al (1977) Association between hepatitis B virus and essential mixed cryoglobulinemia. N Engl J Med 296(26):1501–1504 22. Pascual M, Perrin L, Giostra E, Schifferli JA (1990) Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 162(2):569–570 23. Abel G, Zhang QX, Agnello V (1993) Hepatitis C virus infection in type II mixed cryoglobulinemia. Arthritis Rheum 36(10):1341–1349 24. Monti G, Galli M, Invernizzi F et al (1995) Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC. Italian group for the study of cryoglobulinaemias. QJM 88(2):115–126 25. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33(6):355–374 26. Ferri C, La Civita L, Longombardo G et al (1993) Hepatitis C virus and mixed cryoglobulinaemia. Eur J Clin Invest 23(7):399–405 27. Monti G, Pioltelli P, Saccardo F et al (2005) Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 165(1): 101–105 28. Ramos-Casals M, Munoz S, Medina F et al (2009) Systemic autoimmune diseases in patients with hepatitis C virus infection: characterization of 1020 cases (the HISPAMEC registry). J Rheumatol 36(7):1442–1448 29. Santagostino E, Colombo M, Cultraro D et al (1998) High prevalence of serum cryoglobulins in multitransfused hemophilic patients with chronic hepatitis C. Blood 92(2): 516–519 30. Lunel F, Musset L, Cacoub P et al (1994) Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 106(5):1291–1300 31. Pawlotsky JM, Ben Yahia M, Andre C et al (1994) Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. Hepatology 19(4):841–848 32. Ramos-Casals M, Font J (2005) Extrahepatic manifestations in patients with chronic hepatitis C virus infection. Curr Opin Rheumatol 17(4):447–455

205 33. Agnello V (1998) Mixed cryoglobulinaemia after hepatitis C virus: more and less ambiguity. Ann Rheum Dis 57(12):701–702 34. Ferri C, Mascia MT (2006) Cryoglobulinemic vasculitis. Curr Opin Rheumatol 18(1):54–63 35. Kayali Z, Buckwold VE, Zimmerman B, Schmidt WN (2002) Hepatitis C, cryoglobulinemia, and cirrhosis: a metaanalysis. Hepatology 36(4 Pt 1):978–985 36. Karlsberg PL, Lee WM, Casey DL et al (1995) Cutaneous vasculitis and rheumatoid factor positivity as presenting signs of hepatitis C virus-induced mixed cryoglobulinemia. Arch Dermatol 131(10):1119–1123 37. Levey JM, Bjornsson B, Banner B et al (1994) Mixed cryoglobulinemia in chronic hepatitis C infection. A clinicopathologic analysis of 10 cases and review of recent literature. Medicine (Baltimore) 73(1):53–67 38. Cacoub P, Poynard T, Ghillani P et al (1999) Extrahepatic manifestations of chronic hepatitis C. MULTIVIRC group. Multidepartment virus C. Arthritis Rheum 42(10):2204–2212 39. Vallat L, Benhamou Y, Gutierrez M et al (2004) Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection. Arthritis Rheum 50(11): 3668–3678 40. Sansonno D, De Vita S, Iacobelli AR et al (1998) Clonal analysis of intrahepatic B cells from HCV-infected patients with and without mixed cryoglobulinemia. J Immunol 160(7):3594–3601 41. Racanelli V, Sansonno D, Piccoli C et al (2001) Molecular characterization of B cell clonal expansions in the liver of chronically hepatitis C virus-infected patients. J Immunol 167(1):21–29 42. Berliner S, Weinberger A, Ben-Bassat M et al (1982) Small skin blood vessel occlusions by cryoglobulin aggregates in ulcerative lesions in IgM-IgG cryoglobulinemia. J Cutan Pathol 9(2):96–103 43. Feiner HD (1983) Relationship of tissue deposits of cryoglobulin to clinical features of mixed cryoglobulinemia. Hum Pathol 14(8):710–715 44. Szymanski IO, Pullman JM, Underwood JM (1994) Electron microscopic and immunochemical studies in a patient with hepatitis C virus infection and mixed cryoglobulinemia type II. Am J Clin Pathol 102(3):278–283 45. Agnello V, Abel G (1997) Localization of hepatitis C virus in cutaneous vasculitic lesions in patients with type II cryoglobulinemia. Arthritis Rheum 40(11):2007–2015 46. Bernacchi E, Civita LL, Caproni M et al (1999) Hepatitis C virus (HCV) in cryoglobulinaemic leukocytoclastic vasculitis (LCV): could the presence of HCV in skin lesions be related to T CD8+ lymphocytes, HLA-DR and ICAM-1 expression? Exp Dermatol 8(6):480–486 47. Sansonno D, Cornacchiulo V, Iacobelli AR et al (1995) Localization of hepatitis C virus antigens in liver and skin tissues of chronic hepatitis C virus-infected patients with mixed cryoglobulinemia. Hepatology 21(2):305–312 48. Wei G, Yano S, Kuroiwa T et al (1997) Hepatitis C virus (HCV)-induced IgG-IgM rheumatoid factor (RF) complex may be the main causal factor for cold-dependent activation of complement in patients with rheumatic disease. Clin Exp Immunol 107(1):83–88 49. Levine JW, Gota C, Fessler BJ et al (2005) Persistent cryoglobulinemic vasculitis following successful treatment of hepatitis C virus. J Rheumatol 32(6):1164–1167

206 50. Landau DA, Saadoun D, Halfon P et al (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58(2):604–611 51. Chan LS, Vanderlugt CJ, Hashimoto T et al (1998) Epitope spreading: lessons from autoimmune skin diseases. J Invest Dermatol 110(2):103–109 52. Dupin N, Chosidow O, Lunel F et al (1995) Essential mixed cryoglobulinemia. A comparative study of dermatologic manifestations in patients infected or noninfected with hepatitis C virus [see comments]. Arch Dermatol 131(10):1124–1127 53. Cattaneo R, Fenini MG, Facchetti F (1986) The cryoglobulinemic vasculitis. Ric Clin Lab 16(2):327–333 54. Buezo GF, Garcia-Buey M, Rios-Buceta L et al (1996) Cryoglobulinemia and cutaneous leukocytoclastic vasculitis with hepatitis C virus infection. Int J Dermatol 35(2):112–115 55. Auzerie V, Chiali A, Bussel A et al (2003) Leg ulcers associated with cryoglobulinemia: clinical study of 15 patients and response to treatment. Arch Dermatol 139(3):391–393 56. Krunic AL, Medenica MM, Laumann AE, Shaw JC (2003) Cryoglobulinaemic vasculitis, cryofibrinogenaemia and lowgrade B-cell lymphoma. Br J Dermatol 148(5):1079–1081 57. Baughman RD, Sommer RG (1966) Cryoglobulinemia presenting as “factitial ulceration”. Arch Dermatol 94(6): 725–731 58. Ellis FA (1964) The cutaneous manifestation of cryoglobulinemia. Arch Dermatol 89:690–697 59. Harper JI, Gray W, Wilson-Jones E (1983) Cryoglobulinaemia and angiomatosis. Br J Dermatol 109(4):453–458 60. Resnik KS (2009) Intravascular eosinophilic deposits-when common knowledge is insufficient to render a diagnosis. Am J Dermatopathol 31(3):211–217 61. Cohen SJ, Pittelkow MR, Su WP (1991) Cutaneous manifestations of cryoglobulinemia: clinical and histopathologic study of seventy-two patients. J Am Acad Dermatol 25(1 Pt 1):21–27 62. Buchsbaum M, Werth V (1994) Erythema nodosum-like nodules associated with vasculitis resulting from mixed cryoglobulinemia. J Am Acad Dermatol 31(3 Pt 1): 493–495 63. Boom BW, Brand A, Bavinck JN et al (1988) Severe leukocytoclastic vasculitis of the skin in a patient with essential mixed cryoglobulinemia treated with high-dose gammaglobulin intravenously. Arch Dermatol 124(10):1550–1553 64. Mekkes JR, Loots MA, van der Wal AC, Bos JD (2004) Increased incidence of hypercoagulability in patients with leg ulcers caused by leukocytoclastic vasculitis. J Am Acad Dermatol 50(1):104–107 65. Claudy A (1999) Coagulation and fibrinolysis in cutaneous vasculitis. Clin Dermatol 17(6):615–618 66. Tomizawa K, Sato-Matsumura KC, Kajii N (2003) The coexistence of cutaneous vasculitis and thrombosis in childhood-onset systemic lupus erythematosus with antiphospholipid antibodies. Br J Dermatol 149(2):439–441 67. Rocca PV, Siegel LB, Cupps TR (1994) The concomitant expression of vasculitis and coagulopathy: synergy for marked tissue ischemia. J Rheumatol 21(3):556–560 68. Trejo O, Ramos-Casals M, Garcia-Carrasco M et al (2001) Cryoglobulinemia: study of etiologic factors and clinical and immunologic features in 443 patients from a single center. Medicine (Baltimore) 80(4):252–262 69. Bryce AH, Kyle RA, Dispenzieri A, Gertz MA (2006) Natural history and therapy of 66 patients with mixed cryoglobulinemia. Am J Hematol 81(7):511–518

K. Linos et al. 70. Iannuzzella F, Vaglio A, Garini G (2010) Management of hepatitis C virus-related mixed cryoglobulinemia. Am J Med 123(5):400–408 71. Bessis D, Dereure O, Rivire S et al (2002) Diffuse Bier white spots revealing cryoglobulinaemia. Br J Dermatol 146(5):921–922 72. Wager O, Mustakallio KK, Rasanen JA (1968) Mixed IgAIgG cryoglobulinemia. Immunological studies and case reports of three patients. Am J Med 44(2):179–187 73. Bruchfeld A, Lindahl K, Stahle L et al (2003) Interferon and ribavirin treatment in patients with hepatitis C-associated renal disease and renal insufficiency. Nephrol Dial Transplant 18(8):1573–1580 74. Nir MA, Pick AI, Schreibman S et al (1974) Mixed IgGIgM cryoglobulinemia with follicular pustular purpura. Arch Dermatol 109(4):539–542 75. Crowson AN, Nuovo G, Ferri C, Magro CM (2003) The dermatopathologic manifestations of hepatitis C infection: a clinical, histological, and molecular assessment of 35 cases. Hum Pathol 34(6):573–579 76. Abajo P, Porras-Luque JI, Buezo GF et al (1998) Red finger syndrome associated with necrotizing vasculitis in an HIV-infected patient with hepatitis B. Br J Dermatol 139(1):154–155 77. Koda H, Kanaide A, Asahi M, Urabe H (1978) Essential IgG cryoglobulinemia with purpura and cold urticaria. Arch Dermatol 114(5):784–786 78. Lin RY, Caren CB, Menikoff H (1995) Hypocomplementaemic urticarial vasculitis, interstitial lung disease and hepatitis C. Br J Dermatol 132(5):821–823 79. Erkek E, Ayaslioglu E, Erdogan S, Bagci Y (2007) Vasculopathic skin lesions following epilation, leading to a discovery of hepatitis C virus infection. Clin Exp Dermatol 32(2):221–222 80. Rieu V, Cohen P, Andre MH et al (2002) Characteristics and outcome of 49 patients with symptomatic cryoglobulinaemia. Rheumatology (Oxford) 41(3):290–300 81. Genereau T, Martin A, Lortholary O et al (1998) Temporal arteritis symptoms in a patient with hepatitis C virus associated type II cryoglobulinemia and small vessel vasculitis. J Rheumatol 25(1):183–185 82. Parodi A, Cozzani E, Sorbara S, Rebora A (2005) Hepatitis C virus-related cutaneous vasculitis in the absence of specific antibodies. Clin Exp Dermatol 30(2):188 83. Popp JW Jr, Harrist TJ, Dienstag JL et al (1981) Cutaneous vasculitis associated with acute and chronic hepatitis. Arch Intern Med 141(5):623–629 84. Daoud MS, El-Azhary RA, Gibson LE et al (1996) Chronic hepatitis C, cryoglobulinemia, and cutaneous necrotizing vasculitis. Clinical, pathologic, and immunopathologic study of twelve patients. J Am Acad Dermatol 34(2 Pt 1): 219–223 85. Landau DA, Scerra S, Sene D et al (2010) Causes and predictive factors of mortality in a cohort of patients with hepatitis C virus-related cryoglobulinemic vasculitis treated with antiviral therapy. J Rheumatol 28:2010 86. Pakula AS, Garden JM, Roth SI (1993) Cryoglobulinemia and cutaneous leukocytoclastic vasculitis associated with hepatitis C virus infection. J Am Acad Dermatol 28(5 Pt 2): 850–853 87. Sepp NT, Umlauft F, Illersperger B et al (1995) Necrotizing vasculitis associated with hepatitis C virus infection: successful treatment of vasculitis with interferon-alpha despite persistence of mixed cryoglobulinemia. Dermatology 191(1):43–45

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88. von Kobyletzki G, Stucker M, Hoffmann K et al (1998) Severe therapy-resistant necrotizing vasculitis associated with hepatitis C virus infection: successful treatment of the vasculitis with extracorporeal immunoadsorption. Br J Dermatol 138(4):718–719 89. Chen KR, Carlson JA (2008) Clinical approach to cutaneous vasculitis. Am J Clin Dermatol 9(2):71–92 90. Carlson JA, Chen KR (2007) Cutaneous pseudovasculitis. Am J Dermatopathol 29(1):44–55 91. Ferri C, Mannini L, Bartoli V et al (1990) Blood viscosity and filtration abnormalities in mixed cryoglobulinemia patients. Clin Exp Rheumatol 8(3):271–281 92. Monteverde A, Rivano MT, Allegra GC et al (1988) Essential mixed cryoglobulinemia, type II: a manifestation of a lowgrade malignant lymphoma? Clinical-morphological study of 12 cases with special reference to immunohistochemical findings in liver frozen sections. Acta Haematol 79(1):20–25 93. Saadoun D, Suarez F, Lefrere F et al (2005) Splenic lymphoma with villous lymphocytes, associated with type II cryoglobulinemia and HCV infection: a new entity? Blood 105(1):74–76 94. Andre M, Mahammedi H, Aumaitre O et al (2000) A “missed” cryoglobulin: the importance of in vitro calcium concentration. Ann Rheum Dis 59(6):490–492 95. Lamprecht P, Gutzeit O, Csernok E et al (2003) Prevalence of ANCA in mixed cryoglobulinemia and chronic hepatitis C virus infection. Clin Exp Rheumatol 21(6 Suppl 32): S89–S94 96. Cacoub P, Renou C, Rosenthal E et al (2000) Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d’Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l’Hepatite C. Medicine (Baltimore) 79(1):47–56 97. Ferri C, Pietrogrande M, Cecchetti R et al (1989) Lowantigen-content diet in the treatment of patients with mixed cryoglobulinemia. Am J Med 87(5):519–524 98. Sabry AA, Sobh MA, Sheaashaa HA et al (2002) Effect of combination therapy (ribavirin and interferon) in HCVrelated glomerulopathy. Nephrol Dial Transplant 17(11): 1924–1930 99. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a long-term study. Blood 116(3):343–353

207 100. Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis. Nephrol Dial Transplant 19(12):3054–3061 101. Cacoub P, Lidove O, Maisonobe T et al (2002) Interferonalpha and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 46(12): 3317–3326 102. Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 52(3):911–915 103. Mazzaro C, Zorat F, Caizzi M et al (2005) Treatment with peg-interferon alfa-2b and ribavirin of hepatitis C virusassociated mixed cryoglobulinemia: a pilot study. J Hepatol 42(5):632–638 104. Casato M, Lagana B, Pucillo LP, Quinti I (1998) Interferon for hepatitis C virus-negative type II mixed cryoglobulinemia. N Engl J Med 338(19):1386–1387 105. Monti G, Saccardo F, Rinaldi G et al (1995) Colchicine in the treatment of mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl 13):S197–S199 106. Attal N, Cruccu G, Haanpaa M et al (2006) EFNS guidelines on pharmacological treatment of neuropathic pain. Eur J Neurol 13(11):1153–1169 107. Ferri C, Giuggioli D, Cazzato M et al (2003) HCV-related cryoglobulinemic vasculitis: an update on its etiopathogenesis and therapeutic strategies. Clin Exp Rheumatol 21(6 Suppl 32):S78–S84 108. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101(10):3827–3834 109. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101(10): 3818–3826 110. Cacoub P, Delluc A, Saadoun D et al (2008) Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand? Ann Rheum Dis 67(3):283–287 111. Bhopale GM, Nanda RK (2005) Emerging drugs for chronic hepatitis C. Hepatol Res 32(3):146–153 112. Ferri C, Longombardo G, La Civita L et al (1994) Hepatitis C virus chronic infection as a common cause of mixed cryoglobulinaemia and autoimmune liver disease. J Intern Med 236(1):31–36

Peripheral Neuropathy and Central Nervous System Involvement in Cryoglobulinemia

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Salvatore Monaco, Sara Mariotto, and Sergio Ferrari

26.1

Introduction

Cold-induced physicochemical changes in circulating immunoglobulins and fibrinogen, or thermal-mediated binding of autoreactive monoclonal IgM cold agglutinins, may be associated with central and peripheral nervous system dysfunction. Cryoglobulin (CG) and cryofibrinogen are cold-precipitable molecules that are usually found in autoimmune diseases, chronic infections, and hematologic cancers; in the absence of underlying disorders, they are referred to as “essential” [1, 2]. Both cryoproteins may cause occlusion of small and medium-size vessels in different target organs, including the brain and peripheral nerves, and are frequently found, or even coexist, in hepatitis C virus (HCV)infected patients. By contrast, in cold agglutinin disease (CAD), erythrocyte agglutination by monoclonal autoantibodies with high-thermal amplitude, or cryoagglutinins, causes chronic anemia and cold-induced Raynaud phenomenon, acrocyanosis, and hematuria. CAD can be associated with monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia (WM), or lymphoma. Patients with CAD may develop multiple ischemic/vasculitic changes of peripheral nerves or a chronic ataxic neuropathy with ophthalmoplegia, which is secondary to GD1b disialosyl-reactive agglutinins [3]. CAD with monoclonal IgM but no overt lymphoma is currently classified as “IgM-related disorder,” a group that also includes cryoglobulinemia and peripheral neuropathy with S. Monaco (*) Department of Neuroscience, University of Verona, Verona, Italy e-mail: [email protected]

monoclonal IgM protein [4]. The link between CAD and cryoglobulinemia, and, in turn, with cryofibrinogemia, is further highlighted by the occurrence of antibodies with both cryoglobulinemic and cold agglutinin properties.

26.2

Cryoglobulins and Peripheral Nerve Vessels

Three types of CGs are recognized, either essential or secondary to chronic infections, autoimmune diseases, and lymphoproliferative disorders. Type I CG, accounting for about 10–15% of all CGs, consists of single monoclonal Ig or free light chain, and is usually associated with lymphoproliferative diseases. Type II CG (mixture of polyclonal IgG and monoclonal Ig rheumatoid factor) accounts for 50–60% of the cases and mostly occurs in patients with chronic HCV infection or primary Sjögren syndrome. Type III CG (polyclonal IgG and polyclonal IgM rheumatoid factor) is encountered in lymphoproliferative disorders and infections [5]. The prevalence of essential mixed cryoglobulinemia (MC) is about 1 per 100,000, whereas data on secondary MC are more heterogeneous, depending on the geographic area. Secondary MCs are mostly found in association with chronic infections, especially HCV and HIV (73–95%), autoimmune disease such as primary Sjögren syndrome, systemic lupus erythematosus, and panarteritis nodosa (up to 24%), or hematologic disease [6]. Despite the presence of circulating CGs in about 40% of patients with HCV infection, vasculitic changes in target organs, including skin, kidney, and peripheral nerves, are seen only in a small number of patients [7]. In addition to MC, more than 30 extrahepatic

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diseases have been associated with HCV, and many are immunologic in nature [8]. The management of these extrahepatic manifestations, including MC and its neurological complications, often presents the complexity of treating autoimmune manifestations against a background of an infectious disease, and this may further induce distinctive forms of neurological dysfunction. CGs and/or HCV mostly involve the peripheral nervous system (PNS), inducing important vascular pathology. Peripheral nerves are vascularized by anastomosing epineurial and perineurial networks of arterioles, capillaries, and venules. The main nutrient vessel consists of an artery, 150–300 mm, which gives origin to epineurial and transperineurial arterioles, 15–30 mm in diameter. In turn, these small arterioles feed an endoneurial plexus of longitudinally oriented capillaries [9]. Two types of vasculitis are encountered in CG neuropathy: (i) necrotizing vasculitis, characterized by transmural fibrinoid necrosis of the vessel wall, thrombotic lumen occlusion, and polymorphonuclear infiltration and (ii) microvasculitis, a non-necrotizing lymphocytic form affecting small-size arteries. In addition, the presence of perivascular infiltrates can be indicative of “probable vasculitis” if associated with regenerating small vessels, endoneurial purpura, asymmetric fiber loss, or asymmetric acute axonal degeneration [10]. Vulnerable sites for the main nerve trunks are watershed areas of nutrient arteries at proximal limb locations, whereas at a fascicular level vascular axonal loss is most notable in central locations.

26.3

Peripheral Neuropathy

The prevalence of PNS involvement in cryoglobulinemia is variable, largely depending on the type of CG, the presence of chronic HCV infection, other co-morbidities, and iatrogenic factors. In type I CG, Raynaud syndrome, acrocyanosis, peripheral gangrene, and livedo reticularis are accompanied by peripheral nerve targeting in approximately 6% of cases [6]. The pathogenesis of nerve involvement is poorly understood. Pathological studies in selected cases favor an ischemic non-inflammatory pathogenesis, due to interstitial endoneurial cryoglobulin deposition [11, 12]. However, in three cases with axonal polyneuropathy, we observed perivascular infiltrates, endoneurial purpura, and microangiopathy [13]. Ultrastructural investigation of peripheral nerve biopsies in patients with

axonal polyneuropathy and WM or multiple myeloma revealed the presence of cryoprecipitate around endoneurial capillaries and/or at intracapillary locations [12]. In addition to endoneurial vessels, occlusive microangiopathy may also affect epineurial small vessels. Vallat et al. reported one case of WM-associated IgMk CG with anti-myelin associated glycoprotein (MAG) reactivity, pathologically characterized by IgM deposition in the endoneurium and immunologically mediated demyelination with typical widely spaced myelin [14]; we observed two similar cases (Fig. 26.1). One patient with mixed demyelinating and axonal features was also reported by Lippa [15]. The occurrence of chronic inflammatory demyelinating polyneuropathy (CIDP) in association with type I CG has been reported, as well as the unusual association between sensory neuropathy and IgMk monoclonal cryoglobulin in the setting of the POEMS (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) syndrome [16]. The foregoing reports enlarge the spectrum of associated disorders and further suggest the pathogenic role of autoantibody production in CG. In MC, clinical manifestations are characterized by palpable purpura, arthralgias, peripheral neuropathy, and kidney disease. The reported prevalence of peripheral nerve involvement is highly variable, mostly depending on the clinical criteria and electrophysiological protocol used for neuropathy ascertainment. In some studies, peripheral neuropathy is reported in 26% of patients and is frequently the presenting manifestation of CG [6]. However, in larger series of MC, the incidence of peripheral neuropathy was up to 86% at disease progression. Between 50% and 80% of MC is associated with HCV infection, and among these patients symptomatic vasculitis manifests in 1–10% of cases [17–19]. At variance with type I CG, lymphocytic vasculitis of small vessels is the commonest pathological change encountered in nerve biopsies, whereas necrotizing arteritis of medium-sized vessels is less frequently encountered [10]. Peripheral neuropathy in MC may occur under different clinical patterns, and subclinical PNS involvement has also been shown in HCV-infected patients, when appropriated electrophysiological investigations were performed [17]. According to some authors, sensorimotor axonal polyneuropathy represents the most frequent complication and is the most common form encountered at presentation, or at the time of first

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211

Fig. 26.1 (a) Electron micrograph showing multiple abnormal deposits of cryoglobulin (arrows) in the sural nerve of a patient with type I cryoglobulinemia and neuropathy (6,300×).

(b) Peripheral widenings of myelin lamellae in a patient with type I cryoglobulinemia and IgM anti-MAG antibodies (1,500×)

neurological evaluation. Salient clinical features are those of a distal, symmetrical neuropathy, characterized by sensory loss and motor weakness. Similar to other forms of small-vessel vasculitis, the distal regions of all four limbs are more affected than proximal sites, a pattern reflecting a “length-dependent” disease process; however, it is not unusual to observe patients with arms more affected than legs. Signs and symptoms of neuropathy usually begin acutely in the feet and later extend proximally. This presentation is observed in more aggressive forms of CG, with active syndrome, i.e., intensive recurrent purpura and cryocrit >5%, and reflects a diffuse involvement of the PNS [6]. Pathological changes include marked fiber loss with ongoing wallerian-type degeneration and vasculitic changes (Fig. 26.2). In other instances, the clinical pattern is that of a “stocking-glove” asymmetric polyneuropathy, as the result of step-wise deficits leading to overlapping multiple mononeuropathies. There are no systematic studies regarding the frequency of individual nerve involvement in MC-associated multiple mononeuropathies. However, in large series of necrotizing vasculitis of different etiologies, the peroneal nerve was the most affected, followed by the posterior tibial, ulnar, and median nerves [20], whereas the radial and femoral nerves were usually spared. Regardless of the involved nerve, a stereotypical pattern has been observed at presentation in patients with mononeuropathy, clinically characterized by deep aching limb pain followed over hours to days by burning and severe weakness [21].

In several series of patients diagnosed with MC-associated neuropathy, symmetric or asymmetric forms of sensory neuropathy are reported as the most prevalent. Symmetric forms have been found in CG with or without HCV infection, as well as in HCV without CG [22], and in a recent study, the overall prevalence of sensory neuropathy in CG-negative, chronic HCV infection was 43.5% [23]. Asymmetric sensory neuropathy may occur under two forms: (i) largefiber sensory neuropathy (LFSN), and (ii) small-fiber sensory polyneuropathy (SFSN) [24]. LFSN is characterized by predominant sensory loss and paresthesias, in the absence of muscle weakness. This form has frequently been associated with CG and/or chronic HCV infection [22] as well as with primary Sjögren syndrome with positive markers of oligoclonal B-cell proliferation, i.e., rheumatoid factor or cryoglobulins. In some patients, LFSM may coexist with small-fiber involvement. SFSN is a sensory painful form that targets the thinly myelinated and unmyelinated sensory nerve fibers. According to Gemignani et al. [24], this type of neuropathy is the most frequent, and mainly affects women in their sixth and seventh decades, being the initial manifestation of MC in about 50% of patients. Burning feet, tingling, and restless legs syndrome were the main manifestations. As pointed out by these authors, a finding of interest was that several patients had SFSN, since CG is not usually listed among the causes of SFSN and vasculitis is rarely encountered. Intriguingly, one case of non-length dependent SFSN, highly suggestive of ganglionopathy

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Fig. 26.2 Pathological features of sural nerves from patients with MC as shown in plastic semithin sections. (a) Nerve fascicles show marked depletion of myelinated fibers with ongoing wallerian-type axonal degeneration and (b) endoneurial purpura with erythrocyte extravasation. (c) Diffuse small-size vessel

inflammatory changes and (d) necrotizing arteritis with severe transmural mononuclear inflammation and fibrinoid necrosis affecting epineurial arteries. (a, 80×; b, 230×; toluidine blue stain; c, 65×; d, 70× paraffin H&E stain)

but not peripheral neuropathy, has been reported in CG-negative HCV infection [25]. The clinical diagnosis of SFSN may be corroborated by laboratory tests, in particular skin biopsy and quantitative sensory tests. In addition to the foregoing forms, rare patterns of PNS involvement in HCV-MC also include autonomic dysfunction, with altered sympathetic skin response [26], and pure motor polyneuropathies [27]. Electroneurographic investigations, including pain related-evoked potentials, represent an important tool in assessing PNS involvement, especially in subclinical cases. Testing side-to-side motor and sensory responses in symmetric nerve trunks may help in identifying features suggestive of vasculitic neuropathy, if asymmetry is detected. Using such an approach, a 50% difference in amplitudes of symmetric nerves is considered

significant, whereas lesser degrees of asymmetric involvement may be indicative, provided the appropriate clinical context. The detection of disproportionate abnormalities in the amplitudes of neighboring intralimb nerves, i.e., low ulnar sensory amplitudes with normal median and radial responses, is considered diagnostically more important than the magnitude of the disparity. Additional features to be taken in consideration include the detection of pseudoconduction blocks, or an inverted pattern with abnormalities of the upper-extremity nerves that are out of proportion to the lower-extremity nerves. It is assumed that the pathogenesis of CG neuropathy is linked to ischemia, as a consequence of the occlusion of epineurial arterioles and small vessels. We also suggested the important role of microangiopathic

26

Peripheral Neuropathy and Central Nervous System Involvement in Cryoglobulinemia

changes, following immunoglobulin deposition and activation of cytolytic complement in endoneurial capillaries [13]. In addition, we detected HCV RNA in epineurial perivascular cells surrounded by infiltrates of lymphocytes and monocytes; this pattern is highly reminiscent of the lobular lesion in HCV hepatitis [28]. Authier et al. found positive-strand genomic HCV RNA in nerve and muscle tissues with necrotizing and lymphocytic vasculitis, in addition to detecting immune complexes in small-vessel vasculitis but not in perivascular infiltrates [29]. Taken together, the foregoing studies suggest a major role for HCV as a T-cell mediated cause of nerve damage, in addition to an antigendriven B-cell response with immune complex formation. The occurrence of HCV-triggered dysimmune mechanisms is also supported by the observation of antiMAG neuropathy and acute/chronic inflammatory demyelinating polyradiculoneuropathies [29].

26.4

Central Nervous System Disorders

In contrast to the PNS, the CNS is rarely involved in the course of essential and secondary CG; therefore, evidence concerning the pathogenesis and the treatment of different conditions is scarce. In addition, numerous clinical and pathological observations were reported before 1991, and these patients were not tested for HCV infection. In patients with CG and/or HCV infection, neurological manifestations are highly variegated and include cerebrovascular events, such as stroke and stroke-like syndromes, acute encephalopathy, cranial neuropathies, meningitis, myelopathy, and demyelinating syndromes. In recent years, the study of patients with chronic HCV infection, either CG-positive or CG-negative, has also revealed a number of emerging neuropsychiatric conditions.

26.4.1 Cognitive Changes and Behavioral/ Psychological Symptoms Several studies have reported an increased prevalence of fatigue, depression, memory deficit, attention disturbances, and reduction in verbal learning ability in HCV-infected subjects. Intriguingly, most of these symptoms do not correlate with the severity of liver involvement [30]. Using batteries of cognitive tests it has been demonstrated that HCV-infected patients

213

have impairments in executive function and attention [30, 31]. On the other hand, there are also studies disclosing no relationship between HCV and cognitive or behavioral dysfunction [32]. Using DMSV-IV criteria it has been shown that depression has a prevalence of 28% in chronically HCV-infected patients, and its presence is a major limitation in initiating or continuing specific antiviral treatment. Depression might have a dual origin, as a phenomenon secondary to the psychological burden of HCV infection or through altered serotoninergic and dopaminergic neurotransmission [33]. Fatigue is another common and highly disabling symptom among patients with chronic HCV infection, with an overall prevalence of 50–67%; its extent has been correlated with metabolic brain abnormalities at magnetic resonance spectroscopy (MRS). In addition, the earlier observation that ondansetron, a competitive antagonist at serotonin receptors, ameliorated chronic fatigue in HCV-infected subjects, has spurred a number of studies on serotoninergic transmission. By single photon emission computed tomography, a significant decrease in mesencephalic/hypothalamic serotonin and striatal dopamine transporter binding has been found in patients with disabling fatigue and cognitive dysfunction, thus providing a biochemical support for these HCV-related disturbances [33]. Moreover, positron emission tomography studies with 18F-fluorodeoxyglucose have shown decreased glucose metabolism in the limbic association cortex of HCVinfected patients with attention deficits and memory disturbances. In chronic HCV infection, cognitive impairment is variably reported, according to the early- or end-stage of liver disease. However, it is still unproved whether this impairment is a consequence of the infection itself or the cumulative effect of one or more co-morbidities [34]. At a molecular level, brain MRS investigations have detected altered myoinositol/creatine ratio in the frontal white matter of patients with decreased performance at working memory tests. Moreover, an increased choline/creatine ratio in the basal ganglia and white matter has been correlated with mild cognitive dysfunction in HCV-replicating patients [35]. Overall MRS studies demonstrate an increased turnover of cell membrane and decreased neuronal function. In addition, neurophysiological tests of cognitive processing, such as the P300 event-related potentials, have disclosed delayed peak

214

latencies and reduced amplitudes in P300 in HCVinfected patients with cognitive impairment [36]. Taken together, several lines of evidence point to HCV infection as the exclusive cause of cognitive and neuropsychiatric symptoms in patients without relevant liver disease or psychiatric disorders. The link between HCV infection and cognitive impairment is further suggested by observations that HCV eradication improves cognitive function. While the detection of HCV genetic sequences in brain tissues of infected patients has been reported, confirmation of behavioral and psychological effects awaits further studies.

26.4.2 Neurological Manifestations Neuroinvasion and direct brain infection by HCV is also supported by anecdotal reports. HCV-RNA was found in brain tissues of a young woman with cognitive and aphasic dysfunction, who transiently improved after anti-viral treatment. Neuropathological examination was consistent with HCV leukoencephalitis and showed microglial nodules and perivascular-T cell infiltration [37]. There are reports of HCV-RNA in the cerebrospinal fluid (CSF) of patients with chronic HCV infection and of the detection of negative-strand HCV-RNA in brain tissues. Bolay et al. reported an intriguing case of progressive encephalomyelitis, pathologically characterized by perivascular lymphocyte inflammation in cervical spinal cord and brainstem; in this patient, HCV genome was detected in brain tissues but not in the CSF [38]. These studies support HCV neuroinvasion, although it cannot be excluded that the detected HCV sequences were an effect of mononuclear blood cell contamination. In line with the foregoing reports, relapsing episodes of CNS and PNS demyelination have been associated with concomitant replicative HCV infection, in the absence of vasculitis or CG detection [39]. In some instances, HCV infection may trigger immune-mediated demyelination with simultaneous or sequential involvement of the CNS and PNS. One such patient worsened with interferon (IFN)-a treatment, suggesting an exacerbation of a pre-existing autoimmune disorder or de novo drug-induced autoimmunity [40]. Further examples of HCV-triggered CNS demyelinating syndromes have been described in patients with recurrent myelitis. Spinal cord involvement may occur under different clinical forms,

S. Monaco et al.

encompassing acute transverse myelopathy, acute partial transverse myelopathy, or spastic paraplegia. In one patient with spastic paraplegia, neuropathological studies disclosed demyelination of corticospinal tracts with scarce perivascular lymphocyte infiltration. Biopsy-proven HCV-related recurrent demyelinating myelitis has been reported also in the absence of vasculitis or necrotizing pathology [41]; the immune-mediated pathogenesis in this case was supported by the detection of CSF anti-HCV antibodies but not genomic HCV RNA or viral antigens. There are reports of transverse myelitis in patients with chronic HCV infection, and we observed one patient with partial myelitis (Fig. 26.3). Based on the foregoing observations, several authors have suggested that HCV infection should be taken in due consideration in the differential diagnosis of transverse myelitis. Sacconi et al. [42] described a patient with acute disseminated encephalomyelitis (ADEM) and, at MRI, multifocal hyperintense, contrast-enhancing areas involving the gray and white matter; ADEM developed a few weeks after HCV infection from contaminated blood transfusion; treatment with high-dose steroids led to clinical and radiological improvement. Cerebrovascular involvement has been rarely reported in patients with HCV infection and/or CG. The most common clinical presentations include ischemic stroke and diffuse encephalopathic syndromes. There is little documented evidence of diffuse or segmental vasculitis; instead, there are indirect signs, such as focal narrowing or occlusive changes of the affected vessel at cerebral angiography. Lacunar syndromes may also occur as ischemic attacks or pure motor, sensory, or, less frequently, sensorimotor deficits. There may be subcortical lobar hematoma as well as diffuse petechial involvement of the white matter. Ischemic and hemorrhagic stroke may also occur in the setting of HCV-related anticardiolipin syndrome. Recently, evidence has been provided that HCV increases the risk of arterial plaque formation, especially in patients with elevated HCV RNA levels, representing an independent risk predictor of cerebrovascular deaths [43]. In a multicenter case–control study combining MRI and neuropsychological tests, Casato et al. reported an increased number of whitematter high-intensity signals in periventricular areas, corona radiata, and centrum semiovale in patients with HCV-MC vasculitis and cognitive dysfunction [44]. In unselected patients with chronic HCV and

26

Peripheral Neuropathy and Central Nervous System Involvement in Cryoglobulinemia

215

Fig. 26.3 (a) Sagittal and (b) axial T2-weighted MRI shows intramedullary hyperintensity with mild spinal cord swelling at the T2-T3 level (arrow) in an HCV-infected patient with partial transverse myelitis. (c, d) Axial MRI FLAIR showing different

degrees of subcortical and periventricular hyperintense areas in two patients with chronic HCV infection and mild cognitive changes

cognitive impairment, we have observed similar MRI changes (Fig. 26.3). Diffuse vasculitis with involvement of small and medium-size cerebral arteries has been pathologically verified in fatal cases of encephalopathic syndromes

with PNS vasculitis [45]. In other reported cases, acute/ subacute CNS manifestations ranged from dementia with walking difficulties to mental impairment, somnolence, confusion, stupor, dysarthria, dysphagia, and incontinence.

216

S. Monaco et al.

Ince et al. reported fatal cases of relapsing “encephaloenteropathy,” presenting with transient weakness, chorea, psychiatric disturbances, and ischemic enteropathy. At autopsy, many cerebral and enteral vessels were occluded, as the combined effect of wall abnormalities, plugging by red cells/cryoglobulins, and intravascular Russell bodies [46], thus fulfilling the Virchow triad.

26.5

Treatment of Neurological Complications

Treatment of cryoglobulinemia can be etiological, pathogenic, or symptomatic. In HCV-associated MC, virus eradication represents the primary goal of etiological therapy, whereas pathogenic treatment is aimed at reducing B-cell expansion, removing circulating CGs, and suppressing vasculitis. Depending on disease activity and severity, different therapeutic approaches can be taken in due consideration. Pegylated IFN-a (Peg-IFN-a) in combination with ribavirin (RBV) is the standard therapy for chronic HCV infection. HCV genotype1 shows rates of sustained virological responses (SVR) of less than 50%, but the addition of boceprevir has increased the SVR [47]. Occurrence or worsening of peripheral neuropathy has been reported during Peg-IFN-a treatment [48, 49], but was not confirmed in other studies [50]. Rituximab (RTX), a chimeric monoclonal antibody against CD20 antigen on B-cells, which interferes with monoclonal IgM production, cryoglobulin synthesis, and deposition of immune complexes, has been tested in a few studies. RTX has been reported to improve fatigue and peripheral neuropathy in up to 75% of treated patients, with amelioration of neurophysiological parameters [51]. RTX is generally well tolerated, although a modest increase in HCV viral load has been observed in some patients [52]; rarely, severe infections, but not progressive multifocal leukoencephalopathy, have been reported in immunocompromised patients. Apheretic procedures, alone or in combination with cyclophosphamide, are currently recommended in hyperviscosity syndrome [53]; however, Scarpato et al. reported marked remission of peripheral neuropathy in a small group of patients [54]. It has been reported that high-dose pulsed corticosteroids are useful in treating active vasculitis, but they are contraindicated for chronic immunosuppression [53]. Finally, antidepres-

sant molecules, such as duloxetine, and the anticonvulsivant pregabalin can be efficacious for the treatment of neuropathic pain [55].

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Long-Term Course of Patients with Mixed Cryoglobulinemia

27

Damien Sene and Patrice P. Cacoub

27.1

Introduction

Since 1974, cryoglobulins have been classified according to Brouet’s system, which recognizes monoclonal cryoglobulins (type I) and the two types of mixed cryoglobulins(MC), types II and III [1]. Type II MC are composed of a monoclonal component and polyclonal immunoglobulins; they account for 20–65% of the cases of mixed cryoglobulinemia, while type III MC consist of an association of polyclonal immunoglobulins and represent 35–80% of the cases. Another type of MC, the oligoclonal or microheterogeneous type, in which there are more than two heterogeneous bands of heavy chains as detected on immunoblotting, has been characterized but seldom reported. The oligoclonal type may represent 10–34% of the cases of hepatitis C virus (HCV)-related mixed cryoglobulinemia [2, 3]. The clinical and biological features of mixed cryoglobulinemia are now well established, from the first report, in 1966, by Meltzer et al. [4] to the most recent data, which describe the spectrum of causal conditions, dominated by HCV infection, and the therapeutic management of the disease. Indeed, HCV infection may account for more than 70–80% of the cases of mixed cryoglobulinemia, followed by connective tissue diseases (mainly Sjögren syndrome and systemic lupus erythematosus) and malignant B-cell proliferation. The therapeutic management of MC is based on eradiP.P. Cacoub (*) Department of Internal Medicine, Hôpital La Pitié Salpêtrière, Paris, France UMR 7211 (UPMC/CNRS), U 959 (INSERM), Université Pierre Marie Curie, Paris, France e-mail: [email protected]

cation of the causal agent. In patients with HCV infection, treatment consists of pegylated interferon (IFN)-a and ribavirin, both of which target the virus, and drugs to inhibit malignant B-cell proliferation. Rituximab, a monoclonal antibody targeting CD20, expressed on B cells, can be used alone or in association with another etiological treatment, yielding a high rate of clinical remission [5]. In this review, we analyze data dealing with HCVrelated mixed cryoglobulinemia, focusing on: (1) the spontaneous long-term course of the disease with respect to the immunochemical types of MC, (2) the outcome of mixed cryoglobulinemia and associated symptoms under treatment, and (3) the reappearance of the disease despite successful treatment of HCV infection. The relationship between MC and B-cell non-Hodgkin lymphoma (BNHL) is analyzed elsewhere in this volume.

27.2

Long Term Course of the Immunochemical Types of MC During HCV Infection

Although HCV is known to be frequently associated with MC, the mechanisms underlying this relationship remain poorly understood. Schifferli hypothesized a transitional process between immunochemical types of MC during HCV infection, in which the presence of type III MC are followed by an evolution to type II MC in response to chronic HCV antigenic stimulation [6]. However, thus far, only one study has searched for evidence of a transitional process between the different immunochemical types of MC during HCV infection [3]. In that retrospective study, data from HCV-infected

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_27, © Springer-Verlag Italia 2012

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27.2.1 Impact of MC Immunochemical Type on Vasculitis Symptoms The presence of MC-vasculitis-related symptoms was found to be tightly associated with the HCV immunochemical type. Type II MC were more often symptomatic (68%) than either type III (30%) or the oligoclonal type (25%) (p < 10−4). Compared to type III and oligoclonal MC, type II MC were more frequently associated with peripheral neuropathy (46.7% vs. 17% and 16.7%, respectively; p = 0.002), arthralgia (43.3% vs. 17% and 16.7%; p = 0.005), cutaneous vasculitis (40% vs. 9.4% and 0%; p < 10−4), renal involvement (10% vs. 0.23% for both type III and oligoclonal type; p = 0.03), and BNHL (15% vs. 0%; p = 0.004). From a biological point of view, MC immunochemical types were defined by peculiar features. For example, in type II mixed cryoglobulinemia, serum MC

80 n = 47 (78%)

60 % of patients

patients who were MC-positive between January 1989 and December 2000, as determined in the same immunochemical laboratory, were analyzed. Additional inclusion criteria were chronic HCV infection (positive HCV viral load), MC positivity, and two MC immunochemical typings performed during a 24-month minimum interval and carried out using a validated immunoblotting method [7] able to distinguish between type III and type II MC and the intermediary oligoclonal type. Patients with another cause of MC were excluded. In the 125 patients (62 women and 63 men) with persistent mixed cryoglobulinemia the mean age at diagnosis was 51.9 ± 13.2 years (without distinction according to sex) and the mean duration of HCV infection 18.4 ± 10.1 years (range: 3–43 years). The mean fibrosis METAVIR score was 1.97 ± 1.3, and 19% of patients had cirrhosis. HCV genotype 1 was present in 56% of patients, genotypes 2 and 3 in 33%, and genotypes 4 and 5 in 11%. Sixty patients (48%) presented with MC-vasculitis-related symptoms, which included peripheral neuropathy (39/125 = 31%), arthralgia (29.6%), cutaneous vasculitis with purpura (23%), recent-onset hypertension (8.8%), renal involvement (5.6%), and cerebral vasculitis (2.4%). BNHL was diagnosed in 7% of patients. The baseline immunochemical type distribution was type II MC in 60 patients (48%), type III in 53 patients (42%), and oligoclonal type in 12 patients (10%).

D. Sene and P.P. Cacoub

n = 31 (59%)

n=6 (50%)

40 n = 19 (36%)

n=4 (33%)

20 n=9 (15%) n = 4 (7%)

0 A

n=2 (17%)

n=3 (6%)

B

C

Fig. 27.1 Course of each immunochemical type of mixed cryoglobulins (MC) within a 45 month-interval time. Evolution of mixed cryoglobulinemia in disease originally consisting of (a) type II MC, (b) III MC, and (c) oligoclonal type MC. Black columns represent the proportion of MC that remained or converted to type II MC; gray columns that remained or converted to type III MC, and white columns the proportion that remained or converted to the oligoclonal type, as determined in each case at the second immunochemical typing (From [3], with permission)

levels (0.54 ± 1.11 g/L) were higher than in type III disease (0.28 ± 0.15 g/L) or in disease involving oligoclonal type MC (0.24 ± 0.25 g/L). In addition, C4 levels were lower in a larger proportion of type II than type III and oligoclonal type patients (78% vs. 30%; p = 0.015).

27.2.2 The Long-Term Course of the Immunochemical Types of MC The immunochemical type of MC at the first and second determinations, performed at a mean interval of 45 ± 20 months was analyzed in each patient (Fig. 27.1). The results were as follows: In patients with type II mixed cryoglobulinemia, the disease in 78% (47/60) remained as such whereas evolution to the oligoclonal and type III forms was seen in 15% (9/60) and 7% (4/60) of the patients, respectively. In patients with type III mixed cryoglobulinemia, the disease in 58.5% (31/53) remained type III whereas in 36% (19/53) evolution to type II and in 6% (3/53) to the oligoclonal type was determined. In patients with oligoclonal mixed cryoglobulinemia, evolution to another type occurred in 83% of patients (10/12), with six patients developing type II

27

Long-Term Course of Patients with Mixed Cryoglobulinemia

and four type III, whereas the disease remained unchanged in only two patients (17%) (p = 0.001). Overall, patients with type II mixed cryoglobulinemia had more stable (78%) disease than patients with type III (58.5%) or oligoclonal type (17%) disease (p = 10−4), as assessed within a 45 month-interval. The transition towards another MC type was more frequently directed to type II (55.5%) than to type III (29%) or oligoclonal type (15.5%) (p = 0.0002). These findings support the existence of disease evolution with respect to immunochemical types in HCVinfected patients. To our knowledge, there are no reports of bone marrow or liver immunohistopathologic longterm follow-up of HCV-infected patients at each stage of the immunological disorder; however, we can hypothesize that, during chronic HCV infection, the natural history of MC development may start with the appearance of type III MC, gradually evolving to the oligoclonal type. As previously reported by Musset et al. [7], oligoclonal type MC are characterized by multiple clonal bands, previously detected as type II MC, and with possible expansion of multiple B cell clones [8]. At some later point in time, oligoclonal MC disease may evolve towards type II mixed cryoglobulinemia, accompanied by a trend to oligoclonal and/or monoclonal selection of B lymphocytes in bone marrow and/or in liver tissue. In type II disease, immunohistopathologic analyses of bone marrow and/or liver biopsies often highlight a clonal/oligoclonal B cell expansion [8, 9]. At this final stage, careful follow-up is recommended, as patients with type II mixed cryoglobulinemia are at pronounced risk of developing systemic vasculitis, particularly those with IgMk type II MC.

27.2.3 Summary Among MC immunochemical types, type II MC are the most stable (78%), followed by type III (58.5%) and oligoclonal type (17%). Disease evolution is more frequently towards type II (55.5%) rather than towards type III (29%) or oligoclonal type MC (15.5%). As seen in 83% of the cases examined, the oligoclonal type MC appear to be an intermediary stage between type III and type II. Patients with type II mixed cryoglobulinemia, particularly those with an IgMk monoclonal component, are more frequently symptomatic than patients with type III or oligoclonal type MC disease.

27.3

221

Outcome of Treatment for Mixed Cryoglobulinemia

Mixed cryoglobulinemia is considered to be one of the consequences of chronic B-cell activation induced by HCV antigenic stimulation. Accordingly, withdrawal of this stimulant, by viral clearance, should induce the disappearance of both mixed cryoglobulinemia and the symptoms caused by the associated vasculitis. This hypothesis was first evaluated using the best-available antiviral treatment for HCV-infected patients presenting with symptomatic or asymptomatic mixed cryoglobulinemia. More recently, the physiopathological relationship between B-cell proliferation and the development of mixed cryoglobulinemia has awakened interest in the efficacy of monoclonal anti-CD20 (rituximab) therapy in patients with HCV-MC vasculitis.

27.3.1 Long-Term Course of Mixed Cryoglobulinemia After HCV Treatment It is now well established that HCV-related mixed cryoglobulinemia and its clinical manifestations are mainly due to persistence of the virus. Thus, effective antiviral therapy should induce a significant clinical and biological improvement, particularly in those patients achieving a sustained virological response defined as the absence of viral replication 6 months after the withdrawal of antiviral therapy. Monotherapy with standard IFN-a was the first antiviral treatment for HCV-related mixed cryoglobulinemia and its associated symptoms. The efficacy of this approach was poor because of the very low rate of sustained virological response after a 6- or 12-month course of treatment (14–35%). However, clinical improvement was associated with the disappearance of serum MC, which was shown to be tightly linked to viral clearance (11–40%). The proportions of clinical improvement (complete or partial) varied from 50–100% for cutaneous manifestations (purpura), to 0–60% for kidney involvement (renal insufficiency, proteinuria) and 0–45% for peripheral neuropathies [10–18]. Combination therapy with IFN-a and ribavirin achieved better results in terms of sustained virological response, MC disappearance, and MC vasculitis remission. Colleja et al. reported that, among 13

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HCV-infected patients with symptomatic MC who were non-responders or relapsers to a 12-month course of IFN-a, seven patients (54%) showed a sustained virological response and all of them had a complete clinical response [16]. These data were confirmed by larger studies of a combination course of IFN-a and ribavirin in HCV-infected patients with symptomatic mixed cryoglobulinemia [19–23]. A sustained virological response was associated with MC disappearance and MC vasculitis improvement in 60–100% of patients with cutaneous manifestations, 35–62% with kidney involvement, and 25–80% with peripheral neuropathies. An improved form of HCV treatment is the standard of care combination of PEGylated IFN-a (PEGIFN-a) and ribavirin, which has achieved sustained virological response rates of 50–60%, and up to 80% for HCV genotypes 2 and 3 [24, 25]. The efficacy of this combination was also evidenced in HCV-related mixed cryoglobulinemia, with sustained virological and complete immunological (sustained disappearance of MC) responses in up to 60% of patients. The lack of immunological response was in most of the cases associated with viral persistence [26, 27]. New antiviral regimen with PegINFa and ribavirin plus a protease inhibitor (boceprevir, telaprevir) has shown better efficacy on sustained HCV clearance and should increase the efficacy of MC vasculitis treatments.

27.3.2 Long-Term Course of MC Treatment with Rituximab Two Italian groups initially reported the efficacy of rituximab treatment in patients with HCV-related mixed cryoglobulinemia vasculitis resistant or intolerant to IFN-a monotherapy [28, 29]. Other studies also evidenced the potential of rituximab in the treatment of HCV-associated MC vasculitis [30–35]. Rituximab was mostly used at a dose of 375 mg/m2/week for four consecutive weeks. The overall findings of these studies, reviewed in [5], showed that, when used alone, rituximab induced complete and partial immunological responses in 73% and 13% of patients, respectively, paralleling a complete MC vasculitis clinical response in 40–70% of patients according to the involved organ. The main drawback was the high rate of relapse, which occurred in up to 40% of patients after a mean delay of 7 months. Thus, rituximab may not be a curative treat-

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ment as long as the viral antigenic trigger of the vasculitis remains. A sequential treatment strategy was therefore developed, in which rituximab was used first, to block B-cell activation and proliferation, followed by PEG-IFN-a plus ribavirin, which targeted HCV, specifically by clearing the antigenic trigger and blocking the causative agent. This approach was found to achieve greater efficacy, with lower rates of relapse after treatment [5]. A recent evaluation of this sequential strategy [36] showed satisfying results, with a complete immunological response in 67% of patients and a partial response in 33%. The immunological responses were associated with a complete MC vasculitis clinical response in up to 80% of patients, while a sustained virological response was reached in 55%. Immunological relapse occurred in 30% of patients. All clinical and immunological relapses were associated with the absence of virological clearance. These findings underline the importance of targeting the viral trigger (HCV) and not only B cells, thereby supporting the use of combined rituximab/antiviral therapy to also achieve viral eradication, except in patients in whom antiviral therapy is contraindicated. The proposed therapeutic regimen, consisting of four consecutive weekly infusions of rituximab at a dose of 375 mg/m2 followed by a course of PEG-IFN-a-2b + ribavirin for at least 12 months, appears to be very effective for achieving this objective. The efficacy of combined rituximab/PEG-IFN-a + ribavirin was recently compared with that of PEGIFN-a and ribavirin only, the gold-standard treatment for HCV-related MC vasculitis, in two multicenter prospective studies [37, 38]. Both studies showed that PEGylated IFN-a + ribavirin and rituximab combined therapy was more effective than the gold-standard treatment. A sustained virological response was achieved in 77% of patients who received PEGylated IFN-a + ribavirin + rituximab compared to 67% of those who received PEG-IFN-a + ribavirin. Using a composite definition of complete response, combining the clinical, immunological, virological and molecular (disappearance of blood B cell clonalities) responses, the authors showed that 54.5% of patients who received PEGylated IFN-a + ribavirin + rituximab achieved a complete response compared to 33% of those who received PEG-IFN-a + ribavirin [38]. The efficacy of PEGylated IFN-a + ribavirin + rituximab was even greater in patients exhibiting kidney involvement of MC vasculitis [37]. After a 36-month-follow-

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Long-Term Course of Patients with Mixed Cryoglobulinemia

a

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b

Fig. 27.2 (a) Flare-up of lower-limb skin purpura after rituximab infusion. (b) Digestive vasculitis with intestinal-wall thickening (arrows) after rituximab infusion, as seen on CT scan (From [39], with permission)

up, 83% of PEGylated IFN-a + ribavirin + Rituximab responders still had a complete response compared to 40% of those who had received the PEG-IFN-a + ribavirin. Overall tolerance of Rituximab was good except for patients aged > 70 years with renal failure and receiving corticosteroids who may develop severe infections [43].

27.3.3 Summary Both HCV-related mixed cryoglobulinemia and the associated symptoms can be alleviated following appropriate treatment. A complete immunological response, defined as the disappearance of MC and associated symptoms, can be achieved using: (1) the best antiviral treatment combination, i.e., PEG-IFN-a + ribavirin, which induces viral clearance in up to 60% of patients; (2) B-cell depleting rituximab, shown to achieve immunological and clinical responses in up to 70% of patients; or (3) both strategies, particularly in patients with severe forms of the disease that include kidney involvement.

27.3.4 Role of IgMk MC in RituximabAssociated Systemic Reactions We recently reported six cases of severe, often lifethreatening, systemic side effects after rituximab infu-

sions in patients with HCV-related MC vasculitis, demonstrating an important relationship between IgMk type II MC and the dose of rituximab infusion [39]. These side effects included four life-threatening flares of MC vasculitis and two cases of typical serum sickness syndrome. In this study, systemic side effects occurring after rituximab infusions in a cohort of 22 prospectively followed HCV-infected patients with biopsy-proven MC vasculitis were analyzed. Eighteen patients received the low-dose rituximab protocol, consisting of 375 mg/m2/week (RTX-375) for 4 consecutive weeks, and four patients the high-dose protocol of 1,000 mg rituximab on days 1 and 15 (RTX-1000). Six out of the 22 (27.8%) patients experienced severe systemic drug reactions after rituximab infusions. Four patients with type II IgMk MC developed a severe flare-up of MC vasculitis, including cardiac (n = 2), renal (2), digestive (3), and neurological (1) involvement, 1–2 days after rituximab infusion (Fig. 27.2). These patients successfully responded to methylprednisolone pulses (n = 4) and plasma exchanges (n = 2). Two other patients developed typical serum sickness syndrome 7 and 9 days after the first RTX-1000 infusion, with a spontaneous recovery. Compared to the absence of rituximab-associated drug reactions, the most striking finding was an association between the occurrence of rituximab-associated drug reactions and higher MC (1.4 ± 0.8 vs. 0.71 ± 0.77 g/L; p = 0.0475) and lower C4 (0.02 ± 0.006 vs. 0.073 ± 0.07 g/L;

224 Fig. 27.3 (a) In vitro cryoprecipitation experiments after rituximab addition (RTX+) to serum containing type II MC with an IgMk component. Within <30 min, rituximab (RTX+, right tube) induced the appearance of a visible cryoprecipitate. (b) The remaining “pancakelike” deposit (arrow) after a 7 day-incubation of type II IgMk MC serum with rituximab at +4°C followed by rewarming (RTX+, right tube) (From [39], with permission)

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a

b

RTX–

p = 0.02) baseline serum levels and with the RTX1000 protocol (50% vs. 6.25%; p = 0.046). In five out of the six patients, a dramatic decrease of MC serum levels was evidenced 24 h after rituximab infusion (0.93 ± 0.7 vs. 0.18 ± 0.15 g/L; p = 0.04), suggesting rapid immune complex formation between MC and rituximab. Immunochemical tests showed that the addition of rituximab to patient serum containing type II IgMk MC, which exhibits high rheumatoid factor activity, was associated with visible, accelerated cryoprecipitation, a sharp decrease in MC serum levels, and the appearance of a solid “pancake-like” deposit at the bottom of the tube after 8 days at 4°C (Fig. 27.3). These phenomena were closely related to rituximab dose and baseline MC levels. The main conclusion to be drawn from this report is the need for careful use of rituximab in treating HCVrelated MC vasculitis in patients with high MC serum levels (>1.00 g/L) and C4 levels low (£0.03 g/L). In these situations, rituximab is better administered at low doses (375 mg/m2 weekly for four consecutive weeks or even lower doses [40]), preferentially preceded by plasma exchanges to lower MC serum levels.

RTX+

RTX–

RTX+

27.3.5 Summary Up to one-fourth of HCV-infected patients with MC vasculitis may develop severe systemic reactions after rituximab infusion. These reactions are related to the “accelerated” formation of immune complexes between rituximab and type II IgMk MC exhibiting rheumatoid factor activity, and they occur in a rituximab dose- and serum-MC-level-dependent manner. Accordingly, rituximab should be prescribed cautiously in HCV-related MC vasculitis, with the low-dose rituximab protocol (375 mg/m2/week) and, in patients with high baseline MC levels, plasma exchanges prior to rituximab infusion as the recommended therapeutic strategy.

27.4

Mixed Cryoglobulinemia Relapse After a Sustained Virological Response

The relapse of mixed cryoglobulinemia and MC vasculitis is frequent in the absence of a sustained virological response after HCV treatment. Conversely, relapse of the disease, including its associated clinical

27

Long-Term Course of Patients with Mixed Cryoglobulinemia

manifestations, is rarely reported after successful HCV treatment. Levine et al. presented four patients in whom MC vasculitis relapse occurred during the first year after withdrawal of antiviral treatment, accompanied by rising MC levels and decreasing C4 levels. An exhaustive work-up excluded the presence of a BNHL [41]. Our group reported on eight patients who experienced a relapse of HCV-related MC vasculitis despite having achieved a sustained viral response after HCV treatment [42]. Relapse appeared early after the end of antiviral treatment (2.5 ± 3.5 months) and was associated with an increase in serum MC levels. In most patients, the relapse was brief and the MC vasculitis manifestations subsided. A search for HCV RNA by transcription-mediated amplification was negative, both in sera and in cryoprecipitates, in seven out of the eight patients tested. In three patients, MC vasculitis symptoms persisted and were associated with high serum MC levels. BNHL was finally diagnosed in two of these three patients. In summary, a relapse of MC vasculitis occurs in only a few patients with HCV-related MC vasculitis who have achieved a sustained viral response. In such patients, different underlying conditions should be considered, with a special emphasis on malignant B-cell lymphoproliferative disease.

27.5

Conclusion

The long-term course of mixed cryoglobulinemia during HCV infection depends on chronic HCV antigenic stimulation of B cells. As seen in many patients, the natural tendency of the disease is the evolution of type III MC to the oligoclonal type and finally to type II MC, which mostly exhibit rheumatoid factor activity and are frequently associated with vasculitis. The course of mixed cryoglobulinemia after treatment depends on the ability to achieve viral clearance. Clinical studies have shown that PEG-IFN-a + ribavirin is the most efficient treatment, inducing a complete immunological response in most patients, always associated with viral clearance. Clinical and immunological responses were achieved in most patients treated with rituximab, which specifically targets B cells and thus MC production; however, there was a high rate of post-treatment relapse due to viral persistence. More recently, a combined approach, consisting of rituximab

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and antiviral treatment, was shown as the best current therapeutic strategy in patients with HCV-related MC vasculitis, since both HCV clearance and B-cell depletion are targeted. However, rituximab must be used cautiously, preferably at a low dose and, in those patients with high serum MC levels, preceded by plasma exchanges. After a sustained virological response, MC and symptoms related to MC vasculitis may relapse in a very few patients, in which case an underlying BNHL should be ruled out.

References 1. Brouet JC, Clauvel JP, Danon F et al (1974) Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 57(5):775–788 2. Tridon A, Abergel A, Kuder P et al (1997) Mixed cryoglobulins and autoimmunity in hepatitis C. Pathol Biol (Paris) 45(4):291–297 3. Sène D, Ghillani-Dalbin P, Thibault V et al (2004) Longterm course of mixed cryoglobulinemia in patients infected with hepatitis C virus. J Rheumatol 31(11):2199–2206 4. Meltzer M, Franklin EC, Elias K et al (1966) Cryoglobulinemia – a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med 40(6):837–856 5. Cacoub P, Delluc A, Saadoun D et al (2008) Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand? Ann Rheum Dis 67(3):283–287 6. Schifferli JA, French LE, Tissot JD (1995) Hepatitis C virus infection, cryoglobulinemia, and glomerulonephritis. Adv Nephrol Necker Hosp 24:107–129 7. Musset L, Diemert MC, Taibi F et al (1992) Characterization of cryoglobulins by immunoblotting. Clin Chem 38(6):798–802 8. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 43(1):94–102 9. Sansonno D, De Vita S, Iacobelli AR et al (1998) Clonal analysis of intrahepatic B cells from HCV-infected patients with and without mixed cryoglobulinemia. J Immunol 160(7):3594–3601 10. Migliaresi S, Tirri G (1995) Interferon in the treatment of mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl 13):S175–S180 11. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa2a therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 330(11):751–756 12. Cohen P, Nguyen QT, Deny P et al (1996) Treatment of mixed cryoglobulinemia with recombinant interferon alpha and adjuvant therapies. A prospective study on 20 patients. Ann Med Interne (Paris) 147(2):81–86 13. Adinolfi LE, Utili R, Zampino R et al (1997) Effects of longterm course of alpha-interferon in patients with chronic hepatitis C associated to mixed cryoglobulinaemia. Eur J Gastroenterol Hepatol 9(11):1067–1072

226 14. Polzien F, Schott P, Mihm S et al (1997) Interferon-alpha treatment of hepatitis C virus-associated mixed cryoglobulinemia. J Hepatol 27(1):63–71 15. Casato M, Agnello V, Pucillo LP et al (1997) Predictors of long-term response to high-dose interferon therapy in type II cryoglobulinemia associated with hepatitis C virus infection. Blood 90(10):3865–3873 16. Calleja JL, Albillos A, Moreno-Otero R et al (1999) Sustained response to interferon-alpha or to interferon-alpha plus ribavirin in hepatitis C virus-associated symptomatic mixed cryoglobulinaemia. Aliment Pharmacol Ther 13(9): 1179–1186 17. Cresta P, Musset L, Cacoub P et al (1999) Response to interferon alpha treatment and disappearance of cryoglobulinaemia in patients infected by hepatitis C virus. Gut 45(1):122–128 18. Naarendorp M, Kallemuchikkal U, Nuovo GJ et al (2001) Longterm efficacy of interferon-alpha for extrahepatic disease associated with hepatitis C virus infection. J Rheumatol 28(11):2466–2473 19. Cacoub P, Renou C, Kerr G et al (2001) Influence of HLA-DR phenotype on the risk of hepatitis C virus-associated mixed cryoglobulinemia. Arthritis Rheum 44(9):2118–2124 20. Cacoub P, Lidove O, Maisonobe T et al (2002) Interferonalpha and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 46(12): 3317–3326 21. Cacoub P, Ratziu V, Myers RP et al (2002) Impact of treatment on extra hepatic manifestations in patients with chronic hepatitis C. J Hepatol 36(6):812–818 22. Zuckerman E, Keren D, Slobodin G et al (2000) Treatment of refractory, symptomatic, hepatitis C virus related mixed cryoglobulinemia with ribavirin and interferon-alpha. J Rheumatol 27(9):2172–2178 23. Mazzaro C, Zorat F, Comar C et al (2003) Interferon plus ribavirin in patients with hepatitis C virus positive mixed cryoglobulinemia resistant to interferon. J Rheumatol 30(8):1775–1781 24. Manns MP, McHutchison JG, Gordon SC et al (2001) Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 358(9286):958–965 25. Fried MW, Shiffman ML, Reddy KR et al (2002) Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347(13):975–982 26. Saadoun D, Resche-Rigon M, Thibault V et al (2006) Antiviral therapy for hepatitis C virus–associated mixed cryoglobulinemia vasculitis: a long-term followup study. Arthritis Rheum 54(11):3696–3706 27. Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 52(3):911–915 28. Zaja F, Russo D, Fuga G et al (1999) Rituximab for the treatment of type II mixed cryoglobulinemia. Haematologica 84(12):1157–1158

D. Sene and P.P. Cacoub 29. Zaja F, De Vita S, Russo D et al (2002) Rituximab for the treatment of type II mixed cryoglobulinemia. Arthritis Rheum 46(8):2252–2254, author reply 2254–2255 30. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101(10):3827–3834 31. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101(10):3818–3826 32. Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis. Nephrol Dial Transplant 19(12):3054–3061 33. Lamprecht P, Lerin-Lozano C, Merz H et al (2003) Rituximab induces remission in refractory HCV associated cryoglobulinaemic vasculitis. Ann Rheum Dis 62(12):1230–1233 34. Catuogno M, Rezai S, Priori R et al (2005) Serum sickness associated with rituximab in a patient with hepatitis C virusrelated mixed cryoglobulinaemia. Rheumatology (Oxford) 44(3):406 35. Basse G, Ribes D, Kamar N et al (2005) Rituximab therapy for de novo mixed cryoglobulinemia in renal transplant patients. Transplantation 80(11):1560–1564 36. Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with peg-interferon-ribavirin in refractory HCVassociated cryoglobulinemia vasculitis. Ann Rheum Dis 67(10):1431–1436 37. Saadoun D, Resche Rigon M, Sene D et al (2010) Rituximab plus Peg-interferon-alpha/ribavirin compared with Peginterferon-alpha/ribavirin in hepatitis C-related mixed cryoglobulinemia. Blood 116(3):326–334 38. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a longterm study. Blood 116(3):343–353 39. Sène D, Ghillani-Dalbin P, Amoura Z et al (2009) Rituximab may form a complex with IgMkappa mixed cryoglobulin and induce severe systemic reactions in patients with hepatitis C virus-induced vasculitis. Arthritis Rheum 60(12): 3848–3855 40. Visentini M, Granata M, Veneziano ML et al (2007) Efficacy of low-dose rituximab for mixed cryoglobulinemia. Clin Immunol 125(1):30–33 41. Levine JW, Gota C, Fessler BJ et al (2005) Persistent cryoglobulinemic vasculitis following successful treatment of hepatitis C virus. J Rheumatol 32(6):1164–1167 42. Landau DA, Saadoun D, Halfon P et al (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58(2):604–611 43. Terrier B, Saadoun D, Sene D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 60(8):2531–2540

HBV/HCV Co-infection and Mixed Cryoglobulinemia

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Massimo Galli and Salvatore Sollima

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are major public health problems. It is estimated that there are more than 350 million chronic carriers of HBV, and 170 million people are chronically infected with HCV. Since the two viruses share routes of transmission, dual infections are a common occurrence, particularly in highly endemic areas. Co-infection is also frequently seen in individuals at risk for parenterally transmissible infections, such as injection drug users, patients on hemodialysis or undergoing organ transplantation, and patients with b-thalassemia, as well as in HIV-positive individuals [1–8]. Due to the lack of large-scale population-based studies, the worldwide prevalence of dual chronic infection with HBV and HCV remains undefined [1]. Among HBV-infected patients, anti-HCV positivity is estimated to be 10–15%, with wide geographic variation [6]. Moreover, limited data are available on the correlates of risk of co-infection. In a multicenter prospective study performed in Italy, age over 42 years, history of injection drug use and/or blood transfusion, and residence in the south of the country were independently related to HBV/HCV co-infection [9]. Compared with mono-infections, HBV/HCV coinfections are accompanied by more severe liver injury, with a higher probability of liver cirrhosis and hepatic failure, and a higher incidence of hepatocellular carcinoma [1, 4, 6, 10–15].

M. Galli (*) Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, Università di Milano, Milan, Italy e-mail: [email protected]

Clinical and laboratory studies have shown that HBV and HCV interact in chronically co-infected patients [5, 15]. In particular, in vitro studies showed that HCV core protein can exert an inhibitory effect on HBV replication [16–19]. In most co-infected patients, HCV viremia is detectable whereas serum HBV DNA is low, suggesting an interplay between the two viruses in which HBV replication is inhibited [3, 12, 20–22]. Nevertheless, clinical studies do not uniformly report a dominant role of HCV in HBV/ HCV co-infections; instead, some findings suggest a reciprocal interference, or even a dominant effect of HBV [2, 4, 6, 7, 23–26]. Zarski et al. found that HCV RNA levels were significantly lower in HBV/HCV coinfected patients with positive serum HBV DNA than in HBV DNA-negative patients [6]. Furthermore, in a multicenter longitudinal follow-up study, Raimondo et al. demonstrated wide fluctuations of HBV and HCV viremia levels over time, with both viruses showing alternating phases of activation and suppression [7]. These findings suggest that each virus exerts its own pathogenetic role. The resulting cumulative effects on the liver may explain the high grade of disease severity frequently observed in HBV/HCV coinfected patients [1, 7]. A further intriguing but poorly investigated issue regarding HBV/HCV interactions is the so-called occult HBV infection, defined as the presence of HBV genomes in the liver tissue and sometimes, at very low levels, in the serum of hepatitis B surface antigen (HBsAg)-negative individuals. Suspected since the early 1980s, this peculiar form of chronic viral infection has been demonstrated in the last 15 years, as highly sensitive molecular biology techniques have become available [27].

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Occult HBV status is due to the long-lasting persistence of HBV DNA as covalently closed circular DNA (cccDNA) in hepatocyte nuclei [28, 29]. It may be caused by mutant viruses either producing a modified HBV S protein undetectable by HBsAg assays [30–32] or unable to express the S gene [33]. More frequently, it is due to the strong suppression of viral replication and gene expression exerted by the host’s immune system or co-infecting agents, particularly HCV [27, 34–36]. In fact, the highest prevalence (up to 50% in some series) of occult HBV is seen in patients with HCV infection [27, 37], in whom HBV replication is often inhibited by HCV, as discussed above. Occult HBV infection is usually associated with the presence of anti-HBV antibodies (namely, anti-HBc and antiHBs), although more than 20% of occult carriers are negative for all serum markers of HBV infection [38]. This condition may impact transmission of the infection by blood transfusion or organ transplantation [39–41], with the potential for acute reactivation occurring against a background of immunosuppression [42–45]. Moreover, there is abundant evidence that occult HBV infection favors the progression of liver fibrosis and the development of cirrhosis and hepatocellular carcinoma [24, 38, 46–52]. These complex interactions between the two viruses raise many questions about their roles in causing cryoglobulinemias. It is well known that HBV can induce immuno-pathogenetic processes that are responsible for extrahepatic manifestations, such as polyarteritis nodosa (PAN) and glomerulonephritis [53, 54]. Even before the discovery of HCV, an association between liver disease and cryoglobulin production was recognized [55, 56]. Initially, HBV was investigated as the possible causative agent of so-called essential cryoglobulinemia [57]. However, the attribution of a causative role to HBV was confuted within a short time on the basis of epidemiological and serological data. In particular, HBsAg and HBV DNA were detected only in a small minority of patients with essential cryoglobulinemia, and the prevalence of antiHBV antibodies widely varied among different patient populations with mixed cryoglobulinemia (MC) and did not significantly differ from the prevalence found in age and sex-matched individuals living in the same areas and sharing the same degree of risk of parenteral exposure as cryoglobulinemic patients [58–61]. Nevertheless, an etiologic role of HBV in MC can be postulated in some patients, in the absence of other

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conditions known to be capable of inducing cryoglobulin production. In a study performed in the first half of the 1990s, we found that a role for HBV in causing MC could be supported in <5% of the patients [62]. More recently, in a series of 125 patients with symptomatic HCV-negative cryoglobulinemias who were recruited by the Italian Group for the Study of Cryoglobulinemias (GISC), eight patients (6.5%) were found to be HBsAg-positive; in five of them, no other underlying condition known to cause cryoglobulinemia could be determined (Galli et al., manuscript in preparation). It can thus be concluded that HBV has at least a marginal role as a causative factor of cryoglobulinemic syndromes. The reasons for their development in a small minority of HBV chronically infected patients are as yet poorly understood. Moreover, studies aimed at identifying occult HCV infection in these patients have not been performed. Apart from anecdotal observations, there are no data regarding MC in individuals with dual chronic infection by HCV and overt or occult HBV. Della Rossa et al. [63] described the case of an HBV/HCV co-infected patient who developed PAN (abdominal pain, fever, lower-limb paresthesias and digital necrosis, hypertension, mononeuritis, and angiographic evidence of multiple mesenteric and renal arterial aneurysms) and, after a few years, MC syndrome (lower-limb purpura, proteinuria, positive rheumatoid factor, low complement fraction C4, and mixed cryoglobulins in the serum). The authors, on the basis of serological and virological findings, attributed the PAN to HBV and the MC to HCV, after HCV inhibition of HBV activity. Garcia de La Peña Lefebvre et al. [64] described a case of systemic vasculitis in a patient with HBV (HBe antigen-positive) and HCV (HCV RNA-positive) dual infection The clinical (arthralgias, myalgias, weakness, upper- and lower-limb paresthesias, abdominal pain, and weight loss) and laboratory (low C4 levels, type II cryoglobulinemia with a monoclonal IgMk component, axonal neuropathy on electromyography, multiple microaneurysms and arterial stenoses on abdominal angiography, leukocytoclastic vasculitis of small-and medium-sized vessels on muscle biopsy) findings supported the diagnosis of both PAN and MC. The patient recovered from all symptoms after a few months of combined and sequential treatment with corticosteroids, plasma exchanges, and interferon (IFN)-a, which also led to the disappearance of cryoglobulinemia, seroconversion to anti-HBe

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antibodies, and a decrease in HCV RNA to an undetectable level. Both cases are emblematic of the clinical, diagnostic, and therapeutic complexities that systemic vasculitides may display in HBV/HCV co-infected patients. Furthermore, they suggest that in patients with HBV/ HCV co-infection the extrahepatic manifestation can be attributed to one or the other virus during its dominant phase. This dominance can be lost over time after therapy or as a consequence of the interactions of the co-infecting viruses. However, a synergistic relationship between HBV and HCV in triggering the production of cryoglobulins in some cases of overt or occult co-infection cannot be excluded. Viral eradication or sustained suppression of viral replication should be attempted in patients with cryoglobulinemic syndrome and HBV/HCV co-infection. Antiviral therapy should be the first-line treatment in patients with mild-moderate manifestations of HCVrelated MC syndrome [65, 66]. Data on the treatment of HBV/HCV co-infected patients are limited [67–69], and established guidelines are not available. However, after the dominant virus has been determined by repeated serological and virological testing, treatment guidelines in existence for chronic HBV [70–73] and chronic HCV [74, 75, 76] infection can be considered for co-infected patients [1]. When HCV infection is dominant and HBV viremia is low (<2,000 IU/mL), pegylated IFN-a plus ribavirin can achieve a sustained virological response comparable to that achieved in HCV monoinfected patients [68, 69]. Nevertheless, reactivation of HBV replication after clearance of HCV has been reported. For patients with active HBV and inactive HCV, treatment with IFN-a (standard or pegylated) or oral nucleos(t)ide analogs is recommended according to current guidelines [70–73]. Finally, definition of the optimal regimen when both infections are active is not supported by sufficient clinical data. However, a suggested reasonable empirical option is to add oral nucleos(t)ide analogs to pegylated IFN and ribavirin [1].

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38. Torbenson M, Thomas DL (2002) Occult hepatitis B. Lancet Infect Dis 2:479–486 39. Liu CJ, Lo SC, Kao JH et al (2006) Transmission of occult hepatitis B virus by transfusion to adult and pediatric recipients in Taiwan. J Hepatol 44:39–46 40. Chazouilleres O, Mamish D, Kim M et al (1994) Occult hepatitis B virus as source of infection in liver transplant recipients. Lancet 343:142–146 41. Wachs ME, Amend WJ, Ascher NL et al (1995) The risk of transmission of hepatitis B from HBsAg(−), HBcAb(+), HBIgM(−) organ donors. Transplantation 59:230–234 42. Lok ASF, Liang RHS, Chiu EKW et al (1991) Reactivation of hepatitis B virus replication in patients receiving cytotoxic therapy. Gastroenterology 100:182–188 43. Dhedin N, Douvin C, Kuentz M et al (1998) Reverse seroconversion of hepatitis B after allogeneic bone marrow transplantation: a retrospective study of 37 patients with pretransplant anti-HBs and anti-HBc. Transplantation 66: 616–619 44. Abdelmalek MF, Pasha TM, Zein NN et al (2003) Subclinical reactivation of hepatitis B virus in liver transplant recipients with past exposure. Liver Transpl 9:1253–1257 45. Onozawa M, Hashino S, Izumiyama K et al (2005) Progressive disappearance of anti-hepatitis B surface antigen antibody and reverse seroconversion after allogeneic hematopoietic stem cell transplantation in patients with previous hepatitis B virus infection. Transplantation 79:616–619 46. Zhang YY, Hansson BG, Kuo LS et al (1993) Hepatitis B virus DNA in serum and liver is commonly found in Chinese patients with chronic liver disease despite the presence of antibodies to HBsAg. Hepatology 17:538–544 47. Sagnelli E, Coppola N, Scolastico C et al (2001) HCV genotype and “silent” HBV coinfection: two main risk factors for a more severe liver disease. J Med Virol 64:350–355 48. Chemin I, Trepo C (2005) Clinical impact of occult HBV infections. J Clin Virol 34(Suppl 1):S15–S21 49. Yu MC, Yuan JM, Ross RK, Govindarajan S (1997) Presence of antibodies to the hepatitis B surface antigen is associated with an excess risk for hepatocellular carcinoma among non-Asians in Los Angeles County, California. Hepatology 25:226–228 50. Brechot C (2004) Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 127(Suppl 1):S56–S61 51. Pollicino T, Squadrito G, Cerenzia G et al (2004) Hepatitis B virus maintains its pro-oncogenic properties in the case of occult HBV infection. Gastroenterology 126:102–110 52. Squadrito G, Pollicino T, Cacciola I et al (2006) Occult hepatitis B virus infection is associated with the development of hepatocellular carcinoma in chronic hepatitis C patients. Cancer 106:1326–1330 53. Gocke DJ, Hsu K, Morgan C et al (1970) Association between polyarteritis and Australia antigen. Lancet 296:1149–1153 54. Han SH (2004) Extrahepatic manifestations of chronic hepatitis B. Clin Liver Dis 8:403–418 55. Jori GP, Buonanno G (1972) Chronic hepatitis and cirrhosis of the liver in cryoglobulinemia. Gut 13:610–613 56. Florin-Christensen A, Roux MEB, Arana RM (1974) Cryoglobulins in acute and chronic liver diseases. Clin Exp Immunol 16:599–605

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231 68. Potthoff A, Wedemeyer H, Boecher WO et al (2008) The HEP-NET B/C co-infection trial: a prospective multicenter study to investigate the efficacy of pegylated interferonalpha2b and ribavirin in patients with HBV/HCV coinfection. J Hepatol 49:688–694 69. Liu CJ, Chuang WL, Lee CM et al (2009) Peginterferon alfa-2a plus ribavirin for the treatment of dual chronic infection with hepatitis B and C viruses. Gastroenterology 136: 496–504 70. Lok AS, McMahon BJ (2007) Chronic hepatitis B. Hepatology 45:507–539 71. Carosi G, Rizzetto M (2008) Treatment of chronic hepatitis B: recommendations from an Italian workshop. Dig Liver Dis 40:603–617 72. European Association for the Study of the Liver (2009) EASL clinical practice guidelines: management of chronic hepatitis B. J Hepatol 50:227–242 73. Lok AS, McMahon BJ (2009) Chronic hepatitis B: update 2009. Hepatology 50:661–662 74. Ghany MG, Strader DB, Thomas DL, Seeff LB (2009) Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 49:1335–1374 75. Italian Association for the Study of the Liver; Italian Society of Infectious, Tropical Diseases; Italian Society for the Study of Sexually Transmitted Diseases (2009) Practice guidelines for the treatment of hepatitis C: recommendations from an AISF/SIMIT/SIMAST expert opinion meeting. Dig Liver Dis 42:81–91 76. European Association for the Study of the Liver (2011) EASL clinical practice guidelines: management of hepatitis C infection. J Hepatol 55:245–64

Clinical and Immunological Features of HCV/HIV Co-infected Patients with Mixed Cryoglobulinemia

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David Saadoun and Patrice P. Cacoub

29.1

Introduction

Mixed cryoglobulinemia (MC) is a systemic vasculitis characterized by the proliferation of B-cell clones producing pathogenic IgM with rheumatoid factor (RF) activity. MC leads to clinical manifestations ranging from MC syndrome (purpura, arthralgia, asthenia) to more serious lesions with neurologic and renal involvement [1]. Hepatitis C virus (HCV) infection is associated with most cases of MC. Indeed, 60–80% of patients with MC are infected with the virus. The primary role of HCV in the mechanism of cryoprecipitation is mainly suggested by its selective concentration in cryoglobulins [2]. Many other viral infections have been reported to cause MC vasculitis [1] but most of these reports are anecdotal. Cryoglobulins are detected in 17–26% of human immunodeficiency virus (HIV)-infected patients [3, 4] but are rarely associated with MC vasculitis [5, 6]. However, it is not clear whether HIV and MC vasculitis are causally or coincidentally related. Co-infection with HIV and HCV is common because these viruses share similar transmission routes. It has been estimated that up to 30% of all HIV-infected persons are co-infected with HCV [7]. Several reports have found a higher cryoglobulin prevalence in HIV/ HCV co-infected patients than in HCV patients [3, 8, 9] whereas there are only a few case reports of MC vasculitis in HIV/HCV co-infected patients [10, 11].

D. Saadoun (*) Department of Internal Medicine, Hôpital La Pitié-Salpêtrière, Paris, France e-mail: [email protected]

A wide range of vasculitides can be encountered during HIV infection, ranging from infective vasculitides (cytomegalovirus, herpes simplex virus, toxoplasmosis, pneumocystis, salmonella, or Mycobacterium tuberculosis), hypersensitivity vasculitis, polyarteritisnodosa-like syndrome, angiocentric immunoproliferative lesions, primary angiitis of the central nervous system, large vessel vasculopathy, and miscellaneous vasculitides [12]. The association between HIV and vasculitis, although well described, remains rare (<1%), with only a few cases of cryoglobulinemic vasculitis reported in HIV infected patients [5, 6, 13, 14]. Therapy for HIV-associated vasculitis is controversial and problematic, with the largest reported experience that of Gisselbrecht et al., who advocated a combination of glucocorticoids, plasmapheresis, and antiviral therapy [15].

29.2

Immunologic Manifestations in HCV Patients with or Without HIV Co-infection

From January 1996 to January 1997, 321 patients with an average age of 46 ± 16 years and chronically infected with HCV were prospectively enrolled in a study designed to determine the prevalence of extrahepatic manifestations associated with HCV infection in a large cohort of HCV patients, to identify associations between clinical and biologic manifestations, and to compare the results obtained in HIV-positive versus HIV-negative subsets [8]. In a cross-sectional study, clinical extrahepatic manifestations, viral co-infections with HIV and/or HBV, connective tissue diseases, and a wide panel of autoantibodies were assessed. At least

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_29, © Springer-Verlag Italia 2012

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one clinical extrahepatic manifestation was found in 38% (122/321) of the patients, including arthralgia (60/321, 19%), skin manifestations (55/321, 17%), xerostomia (40/321, 12%), xerophthalmia (32/321, 10%), and sensory neuropathy (28/321, 9%). The main biologic abnormalities were mixed cryoglobulins (110/196, 56%), thrombocytopenia (50/291, 17%), and the presence of the following autoantibodies: antinuclear (123/302, 41%), rheumatoid factor (107/280, 38%), anti-cardiolipin (79/298, 27%), anti-thyroglobulin (36/287, 13%), and anti-smooth muscle cell (27/288, 9%). At least one autoantibody was present in 210/302 (70%) sera. Multivariate logistic regression analysis identified four parameters that were significantly associated with cryoglobulin positivity: systemic vasculitis (p = 0.01, OR = 17.3), HIV positivity (p = 0.0006, OR = 10.2), rheumatoid factor positivity (p = 0.01, OR = 2.8), and sicca syndrome (p = 0.03, OR = 0.27). A definite connective tissue disease was noted in 44 patients (14%), mainly symptomatic MC and systemic vasculitis. HIV co-infection (23%) was associated with three parameters: anti-cardiolipin (p = 0.003, OR = 4.18), thrombocytopenia (p = 0.01, OR = 3.56), and arthralgia or myalgia (p = 0.017, OR = 0.23). HIV-positive patients presented with more severe liver histologic lesions (p = 0.0004). Although co-infections of HCV and HIV are frequent (ranging from 20% to 50%), few studies have attempted to evaluate the differences between HCV patients who are HIV-positive and those who are HIV-negative. In this series, multivariate analysis showed several significant differences between the 242 HIV-negative and 74 HIVpositive patients. The lower prevalence of arthralgia and myalgia in HIV-negative patients might have been related to an underestimation of these symptoms, considering the numerous, more severe complications associated with HIV infection. The relatively high prevalence in HIV-positive patients of anti-phospholipid antibodies and thrombocytopenia has already been reported, and appears to be amplified due to the co-infection of HCV and HIV. Subsequent to the discovery of anti-HIV triple therapies and the increased survival rates of HIV patients, an evaluation of the severity of HCV infection in such cases appears particularly relevant. The study also found that the hepatic effects of HCV were more severe in HIV-positive patients, who had higher Knodell (p = 0.0004) and METAVIR scores (p = 0.0003) [8]. However, the small number of HIV-positive patients who underwent liver

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biopsies limits the scope of this conclusion. HIVpositive patients reportedly have more severe HCV infections, implying that they might benefit from early management of HCV [8].

29.3

Cryoglobulinemia and the Level of Immunosuppression in HIV/HCV Co-infection

Reported prevalences of serum cryoglobulin vary from 40% to 56% in HCV infection and from 34.5% to 81% in HIV/HCV co-infection, but the association with the CD4 cell count is unclear. The study by Aaron et al. [16] consisted of 57 patients co-infected with HIV and HCV, most of whom had been infected with the two viruses via exposure to contaminated blood. HCV genotype 1 was detected in 53.2% of infected patients, genotype 3 in 29.8%, genotype 4 in 17%, and genotype 2 in 4.3%. Only a few patients were infected with virus of two different genotypes. The levels of HCV viremia and the prevalence of cirrhosis were significantly greater among patients with a CD4 cell count <200 cells/mL than among those with a CD4 cell count ³200 cells/mL. In accordance with the findings of previous reports, cryoglobulins were detected in 18 (31.6%) of 57 patients, but the prevalence of cryoglobulins was significantly higher among patients with a CD4 cell count ³200 cells/mL than among those with a CD4 cell count <200/mL [17 (44.7%) of 38 vs. 1 (5.3%) of 19; p = 0.0064]. In other words, HCV-associated cryoglobulins were virtually absent among patients with a CD4 cell count <200 cells/mL (cryoglobulins were detected in only one patient, who had a CD4 cell count of 189 cells/mL), whereas, among patients with a CD4 cell count ³200 cells/mL, the prevalence was similar to that observed among patients not infected with HIV. As expected on the basis of other studies, only three patients—all with a CD4 count ³200/mL— presented with cryoglobulin-related complications (one had nephropathy and two had peripheral neuropathy). Fourteen patients underwent repeated testing for cryoglobulinemia: the presence or absence of cryoglobulinemia was consistent in most cases, but mixed cryoglobulins appeared in one of the three patients during immune restoration while receiving highly active anti-retroviral therapy (HAART). Cryoglobulins were detected in only 4.9% of HIV-positive, HCVnegative patients. In our study, co-infected patients had

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Clinical and Immunological Features of HCV/HIV Co-infected Patients with Mixed Cryoglobulinemia

a CD4 T cell count >250/mm3 at the time of MC vasculitis diagnosis [17]. HIV infection leads to a profound immune dysregulation involving CD4 cells, CD8 cells, and B cells [18]. The susceptibility of B cells to apoptosis is increased such that—due to CD4 cell defects—they respond poorly to mitogenic and antigenic stimuli in vitro and exhibit poor antibody responses to T-cell-dependent and -independent antigens in vivo. Antinori et al. reported the remission of symptoms and the disappearance of HCV-MC, despite detectable HCV viremia, in a patient who became infected with HIV and thus developed immunosuppression (decrease in CD4 cell counts from 337 to 21/ mm3) [11]. Conversely, Monsuez et al. described the appearance of cryoglobulin-associated polyarthalgia in one HIV/HCV co-infected patient at the time of immune restoration, while the patient was receiving HAART (increase in the CD4 cell count from 70 to 567/mm3) [10]. These results strengthen the role of cell-mediated immunity in MC vasculitis and are in agreement with previous studies showing T-cell involvement in the pathogenesis of HCV-related MC vasculitis [19, 20].

29.4

Cryoglobulinemia Vasculitis in HIV/HCV Co-infection

We recently determined the characteristics and outcome of MC vasculitis in patients with HIV/HCV coinfection and compared the results with comparable data from HCV-mono-infected patients [17]. Specifically, we conducted a retrospective multicenter study through the GERMIVIC database, analyzing 6,168 HIV/HCV co-infected patients [17]. Eleven HIV/HCV MC patients, nine men and two women, mean age 46 ± 14 years (range 39–55), were included. The mode of contamination with HIV and HCV was blood transfusion in two patients and intravenous drug use in nine. The distribution of HCV genotypes was genotype 1 (n = 6), genotype 2 (n = 1), and genotype 3 (n = 4). Four patients (36%) had an AIDS-defining illness. The presenting manifestations of MC vasculitis were polyneuropathy (n = 5, 45%), purpura (n = 4, 36%), renal insufficiency (n = 1, 9%), and glomerular proteinuria (n = 1, 9%). Clinical manifestations of MC included polyneuropathy in seven patients (64%), general symptoms (i.e., asthenia, recent weight loss) in six (54%), purpura in four (36%), arthralgia in four (36%),

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myalgia in five (45%), and kidney involvement in three (27%). Glomerular proteinuria was observed in three patients (27%), with renal insufficiency in two (18%). One patient had Raynaud’s syndrome and another had digital necrosis. Uveitis was noted in two patients (18%). All 11 patients had a cryoglobulinemia, with a mean cryoglobulin level of 0.21 g/L (range 0.08–0.4). MC was type II in ten patients and type III in one patient. C4 and CH50 serum levels were low in 64% of the patients. RF activity was observed in five patients (45%). The mean HCV RNA level was 6.1 ± 0.6 Log. The mean HIV-1 RNA level was 5,519 ± 7,782 copies/mL with a mean CD4 cell count of 407 ± 173/mm3 (range 252–846). On liver biopsy, all patients had signs of chronic active hepatitis, with a mean METAVIR activity score of 1.8 ± 0.9 and a mean fibrosis score of 2.2 ± 0.6. Three patients (27%) had cirrhosis. Renal biopsy specimens showed membranoproliferative glomerulonephritis in two patients. Skin biopsy specimens revealed leukocytoclastic vasculitis in one patient. Neuromuscular biopsy specimens showed severe axonal degeneration and an inflammatory process involving nerve in one patient. After a mean follow-up of 44.4 months (range 24–85), nine patients (82%) were still alive. The two deaths were due to hepatocellular failure (n = 1) and drug overdose (n = 1). Six patients received standard interferon (IFN)-a, three million IU × 3/weeks, subcutaneously plus oral ribavirin, 800–1,000 mg/day, for at least 6 months. The mean duration of the IFN-a/ribavirin combination was 9.6 ± 2.6 months (range 6–12). Three patients (50%) had a sustained HCV virologic response and were complete clinical responders. One HIV/HCV co-infected patient who exhibited a significant reduction of HCV viral load (>2 Log) had a complete clinical response. In another patient with a complete clinical and virological response, a relapse of HCV-MC coincided with the reappearance of serum HCV RNA 2 months after the end of HCV therapy. Corticosteroids were administered to four patients (prednisone 1 mg/kg/day initially, with a progressive decrease to 5–10 mg/day within 3 months), plus six pulses of cyclophosphamide (0.5 g/m2/3 weeks) in one case and intravenous immunoglobulins (2 g/kg/month) in another. The mean duration of corticosteroids was 4.2 ± 2.5 months. A partial clinical response was noted in two of these patients (50%). The patient who received cyclophosphamide had skin improvement but

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there was no effect on the polyneuropathy. Intravenous immunoglobulins were added to corticosteroids in another case but without efficacy. No side effects were observed following immunosuppressive therapy. Seven patients were under HAART, which consisted initially of two nucleoside reverse transcripase inhibitors plus at least one protease inhibitor or a non-nucleotide reverse transcriptase inhibitor. Four HIV/HCV co-infected patients were on HAART at the time of MC vasculitis diagnosis and three others started antiretroviral therapy following the diagnosis of vasculitis. The mean duration of HAART was 37.5 ± 13.5 months. All patients were HIV virologic (HIV-1 RNA <50 copies/mL) and immunologic responders (CD4 T cells count >300/mm3). None had a clinical improvement of MC vasculitis under antiretroviral therapy. At the end of follow-up, serum cryoglobulins disappeared in two patients (18%) who cleared HCV. Anti-HCV therapy therefore seems to be the main factor influencing the clinical course of vasculitis. In agreement with the observations in HCV-MC vasculitis patients, complete clinical response correlated with the eradication of HCV in HIV/HCV co-infected patients. We recently reported that treatment with IFN or PegIFN-a-2b and ribavirin achieved, respectively, a sustained virological response in 59% [21] and 77% [22] of patients with HCV-related MC vasculitis. A sustained virologic response was recorded in 50% of HIV/HCV co-infected patients receiving IFN-a plus ribavirin; these patients were also complete clinical responders. A clinical relapse of MC vasculitis occurred in one patient, associated with the reappearance of HCV RNA. One HIV/HCV co-infected patient with a significant decrease (>2 Log) of HCV viral load also achieved a complete clinical response. The symptoms partially improved following treatment with corticosteroids in two patients, with no side effects. Conversely, although all HIV/HCV co-infected patients were virologic responders under HAART, there was no correlation with MC vasculitis improvement. The discrepant results in terms of clinical improvement obtained with HCV and HIV therapy are probably explained by the fact that HCV infection is curable, unlike HIV infection. In conclusion, we showed a beneficial effect of anti-HCV therapy for HIV/HCV coinfected patients with MC vasculitis. A role for cellular immunity in the pathogenesis of MC vasculitis is supported by initial CD4 cell counts >250/mm3 in all

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co-infected patients. Taken together, these data suggest an indirect role for HIV itself in MC vasculitis of patients HIV/HCV co-infected [17].

29.5

Comparison of MC Vasculitis in HCV and HIV/HCV Patients

Among certain subgroups of HIV infected patients, such as injecting drug users, the prevalence of coinfection with HCV approaches 70–90% [7]. We therefore compared the characteristics of MC vasculitis in 11 HIV/HCV co-infected patients with those of 118 HCV patients [17]. The HIV/HCV-MC patients were younger (46 ± 14 vs. 67 ± 13 years, p < 0.001), more frequently of male gender (82% vs. 47%, p = 0.03) and intravenous drug users (82% vs. 11%, p < 0.001), had a higher HCV RNA level (6.1 ± 0.6 vs. 5.6 ± 0.8 Log, p = 0.004), a higher liver necroinflammation score (METAVIR activity 1.87 ± 0.99 vs. 1.14 ± 0.85, p = 0.03), a lower cryoglobulin level (0.21 ± 0.13 vs. 0.64 ± 1.2 g/L, p = 0.03), and a higher level of gammaglobulins (25.6 ± 13.2 vs. 12.1 ± 3.6 g/L, p < 0.001). There was no significant difference regarding the clinical manifestations of MC vasculitis between HIV/ HCV co-infected patients and HCV-infected patients. Despite the high frequency of circulating cryoglobulins, the clinical consequences of cryoglobulinemia are less commonly encountered in HIV/HCV coinfected patients than in HCV mono-infected patients [8, 10, 11, 15]. In our study, the presenting manifestation of HIV/HCV MC vasculitis was most frequently polyneuropathy, followed by skin purpura and renal involvement. There was a trend toward a higher frequency of polyneuropathy in co-infected patients compared with HCV mono-infected patients. However, the clinical manifestations of MC vasculitis in co-infected patients did not differ significantly from those in HCV mono-infected patients. The close association between intravenous drug use and male gender may explain the predominance of male in our HIV/HCV MC patients. In accordance with our findings, Cohen et al. found that co-infected patients with cryoglobulinemia were younger, had higher gammaglobulinemia and lower cryoglobulin concentration than HCV mono-infected patients [9]. In conclusion, the clinical manifestations of MC vasculitis in co-infected patients did not differ significantly from those in HCV mono-infected patients.

29

Clinical and Immunological Features of HCV/HIV Co-infected Patients with Mixed Cryoglobulinemia

References 1. Gorevic PD, Kassab HJ, Levo Y et al (1980) Mixed cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients. Am J Med 69:287–308 2. Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327:1490–1495 3. Bonnet F, Pineau JJ, Taupin JL et al (2003) Prevalence of cryoglobulinemia and serological markers of autoimmunity in human immunodeficiency virus infected individuals: a cross-sectional study of 97 patients. J Rheumatol 30: 2005–2010 4. Dimitrakopoulos AN, Kordossis T, Hatzakis A, Moutsopoulos HM (1999) Mixed cryoglobulinemia in HIV-1 infection: the role of HIV-1. Ann Intern Med 130: 226–230 5. Stricker RB, Sanders KA, Owen WF et al (1992) Mononeuritis multiplex associated with cryoglobulinemia in HIV infection. Neurology 42:2103–2105 6. Castillo JR, Kirchner E, Farver C, Calabrese LH (2005) Cryoglobulinemic vasculitis and lymphocytic interstitial pneumonitis in a person with HIV infection. AIDS Read 15:252–255 7. Sulkowski MS, Mast EE, Seeff LB, Thomas DL (2000) Hepatitis C virus infection as an opportunistic disease in persons infected with human immunodeficiency virus. Clin Infect Dis 30(Suppl 1):S77–S84 8. Cacoub P, Renou C, Rosenthal E et al (2000) Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d’Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l’Hepatite C. Medicine (Baltimore) 79:47–56 9. Cohen P, Roulot D, Ferriere F et al (1997) Prevalence of cryoglobulins and hepatitis C virus infection in HIV-infected patients. Clin Exp Rheumatol 15:523–527 10. Monsuez JJ, Vittecoq D, Musset L et al (1998) Arthralgias and cryoglobulinemia during protease inhibitor therapy in a patient infected with human immunodeficiency virus and hepatitis C virus. Arthritis Rheum 41:740–743 11. Antinori S, Galimberti L, Rusconi S et al (1995) Disappearance of cryoglobulins and remission of symptoms in a

12.

13.

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17.

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patient with HCV-associated type II mixed cryoglobulinemia after HIV-1 infection. Clin Exp Rheumatol 13(Suppl 13): S157–S159 Calabrese LH (2004) Infection with the human immunodeficiency virus type 1 and vascular inflammatory disease. Clin Exp Rheumatol 22:S87–S93 Gherardi R, Belec L, Mhiri C et al (1993) The spectrum of vasculitis in human immunodeficiency virus-infected patients. A clinicopathologic evaluation. Arthritis Rheum 36:1164–1174 Le Lostec Z, Fegueux S, Vitale C et al (1994) Peripheral neuropathy associated with cryoglobulinaemia but not related to hepatitis C virus in an HIV-infected patient. AIDS 8:1351–1352 Gisselbrecht M, Cohen P, Lortholary O et al (1998) Human immunodeficiency virus-related vasculitis. Clinical presentation of and therapeutic approach to eight cases. Ann Med Interne (Paris) 149:398–405 Aaron L, Lebray P, Alyanakian MA et al (2005) Prevalence of mixed cryoglobulins in relation to CD4 cell count among patients coinfected with HIV and hepatitis C virus. Clin Infect Dis 40:306–308 Saadoun D, Aaron L, Resche-Rigon M et al (2006) Cryoglobulinaemia vasculitis in patients coinfected with HIV and hepatitis C virus. AIDS 20:871–877 Mackay F, Ambrose C (2003) The TNF family members BAFF and APRIL: the growing complexity. Cytokine Growth Factor Rev 14:311–324 Boyer O, Saadoun D, Abriol J et al (2004) CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood 103:3428–3430 Bonetti B, Invernizzi F, Rizzuto N et al (1997) T-cell-mediated epineurial vasculitis and humoral-mediated microangiopathy in cryoglobulinemic neuropathy. J Neuroimmunol 73: 145–154 Cacoub P, Lidove O, Maisonobe T et al (2002) Interferonalpha and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 46: 3317–3326 Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 52: 911–915

HCV-Negative Mixed Cryoglobulinemia: Facts and Fancies

30

Massimo Galli, Salvatore Sollima, and Giuseppe Monti

Although clinico-epidemiological and laboratory studies have identified hepatitis C virus (HCV) as the etiologic agent of mixed cryoglobulinemia (MC) [1–9], in a minority of cases there is no evidence of HCV infection, as assessed either by anti-HCV antibodies or by HCV RNA determination in serum and/or cryoprecipitates [10]. The true number of these cases is an open question, further confused by the fact that there are significant geographical differences, in addition to the lack of large epidemiological studies. In a survey performed in France, HCV-negative cases accounted for about the 9% of MC [10]. Patients with HCV-negative MC are more frequently found in northern Europe and North America, where the overall prevalence of MC, but also of HCV infection, is significantly lower than in the Mediterranean area [11, 12]. In Italy, a rough estimate extrapolated from the data of the GISC (Italian Group for the Study of Cryoglobulins) suggests that HCV-negative symptomatic MC accounts for no more than 5% of the total number of MC patients followed up in the participating centers. Several different conditions can account for the appearance of MC in HCV-negative individuals. In particular, a wide number of acute and chronic infections as well as connective tissue and lympho-proliferative diseases have been shown to play a causative role in some cases of MC [13–18]. MC patients presenting without any of the underlying diseases known to induce cryoglobulin production are few, probably

M. Galli (*) Department of Clinical Sciences “Luigi Sacco”, Section of Infectious and Tropical Diseases, Università di Milano, Milan, Italy e-mail: [email protected]

accounting for <1% of symptomatic cryoglobulinemic syndromes. In these cases, MC is still classified as “essential” [14, 15, 18]. In addition, in many cases reported in the literature as non-HCV-related cryoglobulinemias, circulating cryoglobulins represent a mere by-product of immune activation and are without clinical significance. This clearly represents a confusing element in determining the actual number of real non-HCV-related cryoglobulinemic syndromes. Circulating cryoglobulins have been found to occur in more than 20 acute and chronic infections due to a variety of pathogens (Table  30.1) [19, 20]. In most cases, they disappear from the circulation with the regression of symptoms. These cryoglobulinemias are rarely symptomatic and only exceptionally accompanied by a complete MC syndrome (purpura, asthenia, arthralgia, and/or serious vasculitis with nervous system and renal involvement). Based on the frequent association between MC and liver involvement, a role for hepatitis viruses in the etiopathogenesis of the disease was hypothesized beginning in the early 1970s [21, 22]. For example, Levo et al. [23] claimed that most MCs were caused by hepatitis B virus (HBV), but the conclusions of these authors were later refuted. The apparently greater prevalence of anti-HBV antibodies was attributed to confounding variables, such as age, geographical origin, socioeconomic status, and frequent hospitalizations [24, 25]. Moreover, only a slight difference in the prevalence of serological markers of HBV was found when a large series of cryoglobulinemic patients was compared with hospitalized patients or blood donors resident in the same areas [26, 27]. Thus, HBV is currently considered to be an etiologic factor of MC in <5% of individuals [20, 28].

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240 Table 30.1  lnfectious cryoglobulinemia

M. Galli et al. agents

associated

with

mixed

RNA viruses • HIV-l • HTLV-l • Hepatitis A virus • Hepatitis C virus DNA viruses • Adenovirus • Epstein-Barr virus • Cytomegalo virus • Hepatitis B virus Chlamydia and Rickettsia • Chlamydia psittaci • Rickettsia conorii Bacteria • Streptococcus spp. • Proteus mirabilis • Brucella spp. • Leptospira icterohaemorrhagiae • Borrelia burgdorferi • Treponema pallidum • Mycobacterium tuberculosis Protozoa • Toxoplasma gondii • Leishmania donovani • Plasmodium falciparum • Entamoeba histolytica Helminths • Echinococcus granulosus • Schistosoma mansoni Fungi • Coccidioides immitis

A significant prevalence of MC has also been observed in patients with human immunodeficiency virus (HIV) infection, with and without HCV co-infection. In most cases, these cryoglobulinemias are asymptomatic and belong to type III of the Brouet classification. In HIV mono-infected patients, MC prevalence ranges between 3% and 27% [29–32]. In the study by Dimitrakopoulos et  al., HIV RNA sequences were detected in cryoprecipitates, and the mean HIV viral load in cryoglobulin-positive patients was significantly higher than in cryoglobulin-negative individuals. This finding suggests that HIV replication exerts a direct role in inducing cryoglobulinemia, by inducing a continuous antigenic stimulation of B lymphocytes, which can result in the production of cryoglobulins already in the early stages of infection [30].

Indirect evidence of the etiologic role of HIV in the pathogenesis of cryoglobulins is also provided by the finding that highly active antiretroviral therapy (HAART) seems to decrease the prevalence of cryoglobulinemia in HIV-infected, HCV-negative patients [33]. However, the case of a patient with an HCVrelated cryoglobulinemic syndrome and concomitant HIV infection was reported in which the latter caused the remission of symptoms and the disappearance of circulating cryoglobulins [34]. In this particular case, HIV seemed to have exerted a suppressive rather than deregulatory effect on the immune system, leading to the clinical remission of MC. A similar phenomenon has been described in several patients with systemic lupus erythematosus after HIV infection [35]. In conclusion, HIV is an example of an infectious agent that can chronically activate B lymphocytes, causing the production of cryo-precipitating immune complexes, mostly without pathological significance. Once cases of transient cryoglobulinemias and those of uncertain pathological significance have been excluded, the number of HCV-unrelated MC cases is drastically reduced, which also explains why HCVnegative MC is a neglected topic, specifically examined in only a few studies. In a small series of 57 patients, Trejo et  al. [36] evaluated the relationship between MC and other systemic disorders. MC was associated with HCV in 82% of cases, and with other infections, or connective tissue or hematologic diseases in another 11%; the remaining 7% of cases were classified as “essential.” Missing from that study, however, was a clear distinction between cryoglobulinemic syndromes and asymptomatic forms. In a retrospective study of 1434 MC patients, Saadoun et  al. [10] described 133 HCV-negative patients (9%), 49% with symptomatic MC. HCV-negative MC was associated with connective tissue diseases (mainly systemic lupus erythematosus and Sjögren’s syndrome) in 34% of cases, hematologic disorders (mostly B-cell nonHodgkin lymphoma) in 26%, and infectious diseases (mostly HBV and HIV infections) in 13%. The remaining 27% of cases were considered as “essential” MC. Type II MC was determined in 72% of HCV-negative patients and was closely associated with MC syndrome and hematologic diseases. A cryocrit >0.6  g/L, MC vasculitis, and hypogammaglobulinemia were independently associated with the development of B-cell non-Hodgkin 1ymphoma. In a retrospective analysis of 195 consecutive Italian patients with MC, Mascia

30  HCV-Negative Mixed Cryoglobulinemia: Facts and Fancies

et  al. [37] found a 16% prevalence of HCV-negative MC, ascribing “essential” MC to 25% of a series of 65 HCV-negative MC patients, also including previously observed cases. Interestingly, overt MC syndrome was present in the majority of patients with “essential” MC, Sjögren’s syndrome, or lymphoma, while it was absent or rare in MC patients with other underlying conditions. The recent GISC survey did not find any pathological condition associated with the production of cryoglobulins in 74/125 cases of HCV-unrelated MC (59.2%). Interestingly, type II cryoglobulinemia and a complete Meltzer and Franklin triad were observed in about half the cases and 55.7% had purpura (M. Galli et  al., manuscript in preparation). Differences in the findings of the various studies clearly derive from the different selection criteria and the mixing of symptomatic and asymptomatic cases. Investigations adopting more stringent criteria identified more cases of “essential” MC, which were recognized only by the presence of MC-related symptoms. It must also be considered that, in carriers of other diseases, symptoms attributed to cryoglobulins may not necessarily be due to them and the MC may be an entirely incidental laboratory finding. In all studies examined, however, the classic Meltzer triad, frank vasculitis, and, when MC typing was performed, the presence of type II MC were most frequently found in “essential” forms and in association with lymphomas. By contrast, MC associated with infections or autoimmune disorders tended to have a less characteristic clinical picture and was of the type III variety. Likewise, in several studies, the different criteria for defining and selecting cases make it difficult to compare symptoms and the course of HCV- and nonHCV-related MC. Moreover, series of HCV-related MC may also include varying proportions of patients with asymptomatic cryoglobulinemias. This may account for the conclusion that the clinical manifestations and cryoglobulin levels in MC patients with and without HCV do not differ significantly [9, 36–38], although in a French series [39] of 115 patients both hepatic and cutaneous involvement were more frequent and cryoglobulin levels higher in HCV-positive patients. The typical clinical triad, especially purpura [37] and arthralgia [38], seems to be more common among HCV-positive than HCV-negative patients, although the data are discordant on this issue [36]. There are also conflicting data regarding survival rates of HCV-negative MC patients, which are either lower

241

[10, 13, 40] or similar [9, 37, 38, 41] to those of HCVpositive patients. One possible scenario that may account for essential forms of MC and, more generally, cases of HCVnegative MC is the presence of an occult HCV infection. Indeed, HCV RNA has been detected in liver biopsies and peripheral blood mononuclear cells, despite the absence of anti-HCV antibodies and HCV RNA in the serum of patients with persistently abnormal liver-enzyme levels of unknown etiology [12, 42]. The appearance of HCV RNA in the serum and/or cryoprecipitate of three previously HCV RNA-negative and persistently anti-HCV antibody-negative patients with type II MC [43] suggests a role for latent HCV infection in some apparently “essential” forms of MC. This issue, however, has not yet been studied systematically and awaits further investigation. Given the complexity and poor definition of HCVnegative MC, the lack of available guidelines specifically addressing treatment is not surprising. In general, therapy should aim at improvement or resolution of clinical manifestations and at eliminating the causative factor. The latter goal can be achieved in a few cases, targeting the etiologic agent (including HBV, HIV) and associated conditions (e.g., connective tissue diseases and lymphoproliferative disorders). Nonetheless, there are still many other unknown factors involved in non-HCV-related MC. In these cases, the therapeutic strategy must be based on the activity and severity of the disease and tailored to the individual patient [15]. When the goal of treating the cryoglobulinemic syndrome, its symptoms, and/or etiology is not feasible, therapeutic approaches used in HCV-related cryoglobulinemias may be effective. In asymptomatic patients, careful monitoring is often sufficient; in those with mild-moderate manifestations (such as purpura, weakness, arthralgia, initial neuropathy), low-medium doses of corticosteroids, colchicine, a low-antigen-content diet, and nonsteroidal anti-inflammatory drugs should be considered; in severe or rapidly progressive disease (including glomerulonephritis, peripheral neuropathy, skin ulcers, widespread vasculitis, or hyperviscosity syndrome), more aggressive treatments are needed, such as high-dose corticosteroids, plasma exchange plus cyclophosphamide, or rituximab [9, 11, 12, 28, 44–51]. In conclusion, non-HCV-related cryoglobulinemic syndromes represent probably <5% of the total number of symptomatic cryoglobulinemias, although more

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stringent classification criteria are needed to better define the true prevalence. The number of true cases of essential MC is probably lower than currently reported, since some of these may be attributable to occult HCV infection, although this has not yet been systematically explored. Finally, according to an assumption not discussed in this chapter, the ability of different triggers to cause similar clinical and immunopathological responses may reflect the particular genetic background of the host.

References 1. Pascual M, Perrin L, Giostra E, Schifferli JA (1990) Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 162:569–570 2. Casato M, TaIiani G, Pucillo LP et  al (1991) Cryoglo­ bulinaemia and epatitis C virus. Lancet 337:1047–1048 3. Ferri C, Greco F, Longombardo G et al (1991) Association between hepatitis C virus and mixed cryoglobulinaemia. Clin Exp Rheumatol 9:621–624 4. Agnello V, Chung RT, Kaplan LM (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327:1490–1495 5. Misiani R, Bellavita P, Fenili D et al (1992) Hepatitis C virus infection in patients with essential mixed cryoglobulinemia. Ann Intern Med 117:573–577 6. Lunel F, Musset L, Cacoub P et al (1994) Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 106:1291–1300 7. Monti G, Galli M, Invenizzi F et al (1995) Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC. Italian Group for the Study of Cryoglobulinaemias. QJM 88:115–126 8. Dammacco F, Sansonno D, Piccoli C et al (2001) The cryoglobulins: an overview. Eur J Clin Invest 31:628–638 9. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33: 355–374 10. Saadoun D, Sellam J, Ghillani-Dalbin P et  al (2006) Increased risks of lymphoma and death among patients with non-hepatitis C virus-related mixed cryoglobulinemia. Arch Intern Med 166:2101–2108 11. Weiner SM, Berg T, Berthold H et al (1998) A clinical and virological study of hepatitis C virus-related cryoglobulinemia in Germany. J Hepatol 29:375–384 12. Ferri C, Mascia MT (2006) Cryoglobulinemic vasculitis. Curr Opin Rheumatol 18:54–63 13. Gorevic PD, Kassab HJ, Levo Y et al (1980) Mixed cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients. Am J Med 69:287–308

M. Galli et al. 14. Lamprecht P, Gause A, Gross WL (1999) Cryoglobulinemic vasculitis. Arthritis Rheum 42:2507–2516 15. Ferri C, Zignego AL, Pileri SA (2002) Cryoglobulins. J Clin Pathol 55:4–13 16. Granel B, Serratrice J, Morange PE et  al (2004) Cryoglobulinemia vasculitis following intravesical instillations of bacillus Calmette-Guerin. Clin Exp Rheumatol 22: 481–482 17. Hermida Lazcano I, Saez Méndez L, Solera Santos J (2005) Mixed cryoglobulinemia with renal failure, cutaneous vasculitis and peritonitis due to Brucella melitensis. J Infect 51:e257–e259 18. Trendelenburg M, Schifferli JA (1998) Cryoglobulins are not essential. Ann Rheum Dis 57:3–5 19. Galli M, Invernizzi F, Chemotti M et al (1986) Cryoglobulins and infectious diseases. Ric Clin Lab 16:301–313 20. Galli M (1995) Viruses and cryoglobulinemia. Clin Exp Rheumatol 13(Suppl 13):S63–S70 21. Iori GP, Buonanno G (1972) Chronic hepatitis and cirrhosis of the liver in cryoglobulinemia. Gut 13:610–613 22. Florin-Christensen A, Roux MEB, Arana RM (1974) Cryoglobulins in acute and chronic liver diseases. Clin Exp Immunol 16:599–605 23. Levo Y, Gorevic PD, Kassab HJ et  al (1977) Association between hepatitis B virus and essential mixed cryoglobulinemia. N Engl J Med 296:1501–1504 24. Dienstag JL, Wands JR, Isselbacher KJ (1977) Hepatitis B and essential mixed cryoglobulinemia. N Engl J Med 297: 946–947 25. Popp JW Jr, Dienstag JL, Wands JR, Bloch KJ (1980) Essential mixed cryoglobulinemia without evidence for hepatitis B virus infection. Ann Intern Med 92:379–383 26. Galli M (1991) Cryoglobulinaemia and serological markers of hepatitis viruses. Lancet 338:758–759 27. Galli M, Monti G, Invernizzi F et  al (1992) Hepatitis B virus-related markers in secondary and in essential mixed cryoglobulinemias: a multicenter study of 596 cases. The Italian Group for the Study of Cryoglobulinemias (GISC). Ann Ital Med Int 7:209–214 28. Ferri C (2008) Mixed cryoglobulinernia. Orphan J Rare Dis 3:25 29. Cohen P, Roulot D, Ferrière F et  al (1997) Prevalence of cryoglobulins and hepatitis C virus infection in HIV-infected patients. Clin Exp Rheumatol 15:523–527 30. Dimitrakopoulos AN, Kordossis T, Hatzakis A, Moutsopoulos HM (1999) Mixed cryoglobulinemia in HIV-l infection: the role of HIV-1. Ann Intem Med 130:226–230 31. Fabris P, Tositti G, Giordani MT et al (2003) Prevalence and clinical significance of circulating cryoglobulins in HIV -positive patients with and without co-infection with hepatitis C virus. J Med Virol 69:339–343 32. Scotto G, Cibelli DC, Saracino A et  al (2006) Cryoglo­ bulinemia in subjects with HCV infection alone, HIV infection and HCV/HIV coinfection. J Infect 52:294–299 33. Kosmas N, Kontos A, Panayiotakopoulos G et  al (2006) Decreased prevalence of mixed cryoglobulinemia in the HAART era among HIV -positive, HCV-negative patients. J Med Virol 78:1257–1261

30  HCV-Negative Mixed Cryoglobulinemia: Facts and Fancies 34. Antinori S, Galimberti L, Rusconi S et al (1995) Disap­pea­ rance of cryoglobulins and remission of symptoms in a patient with HCV-associated type II mixed cryoglobulinemia after HIV-1 infection. Clin Exp Rheumatol 13(Suppl 13): S157–S159 35. Palacios R, Santos J, Valdivielso P, Marquez M (2002) Human immunodeficiency virus infection and systemic lupus erythematosus. An unusual case and a review of the literature. Lupus 11:60–63 36. Trejo O, Ramos-Casals M, Garcia-Carrasco M et al (2001) Cryoglobulinemia: study of etiologic factors and clinical and immunologic features in 443 patients from a single centre. Medicine (Baltimore) 80:252–262 37. Mascia MT, Ferrari D, Campioli D et al (2007) Non HCVrelated mixed cryoglobulinemia. Dig Liver Dis 39(Suppl l): S61–S64 38. Rieu V, Cohen P, André MH et al (2002) Characteristics and outcome of 49 patients with symptomatic cryoglobulinaemia. Rheumatology (Oxford) 41:290–300 39. Cacoub P, Fabiani FL, Musset L et al (1994) Mixed cryoglobulinemia and hepatitis C virus. Am J Med 96:124–132 40. Tarantino A, Campise M, Banfi G et  al (1995) Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis. Kidney Int 47:618–623 41. Della Rossa A, Marchi F, Catarsi E et al (2008) Mixed cryoglobulinemia and mortality: a review of the literature. Clin Exp Rheumatol 26(Suppl 51):S105–S108 42. Castillo I, Pardo M, Bartolomé J et al (2004) Occult hepatitis C virus infection in patients in whom the etiology of persistently abnormal results of liver function tests is unknown. J Infect Dis 189:7–14

243 43. Casato M, Lilli D, Donato G et al (2003) Occult hepatitis C virus infection in type II mixed cryoglobulinaemia. J ViraI Hepat 10:455–459 44. Ferri C, Moriconi L, Gremignai G et al (1986) Treatment of the renal involvement in mixed cryoglobulinemia with prolonged plasma exchange. Nephron 43:246–253 45. Ferri C, Pietrogrande M, Cecchetti C et  al (1989) Lowantigen-content diet in the treatment of patients with mixed cryoglobulinemia. Am J Med 87:519–524 46. Monti G, Saccardo F, Rinaldi G et al (1995) Colchicine in the treatment of mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl 13):S197–S199 47. Tavoni A, Mosca M, Ferri C et al (1995) Guidelines for the management of essential mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl 13):S191–S195 48. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101:3827–3834 49. Sansonno D, De Re V, Lauletta G, et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 50. Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis. Nephrol Dial Transplant 19:3054–3061 51. Cacoub P, Delluc A, Saadoun D et  al (2008) Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemia vasculitis: where do we stand? Ann Rheum Dis 67: 283–287

Cryoglobulinemia in HCV-Positive Renal Transplant and Liver Transplant Patients

31

Lionel Rostaing, Hugo Weclawiak, and Nassim Kamar

31.1

Introduction

Mixed cryoglobulinemia (MC) is a systemic disease that results in the presence of mixed cryoglobulins, defined as plasma proteins composed of immunoglobulins and precipitating reversibly at low temperature. Cryoglobulins are classified into three types, using the classification of Brouet et al. [1]. Type II cryoglobulins consist of a monoclonal immunoglobulin, usually immunoglobulin Mk (IgMk) that has rheumatoid activity against polyclonal IgGs, whereas type III cryoglobulins are characterized by a combination of polyclonal immunoglobulins. In most cases, MCs occur secondary to chronic infections, connective tissue disorders, and lymphoproliferative disorders. When no underlying disease is present, cryoglobulinemia is defined as “essential.” However, in many such cases, hepatitis C virus (HCV) infection has ultimately been determined as the causal factor [2]. Indeed, a strong link is now recognized between HCV infection and MC, based on the high frequency (81–91%) of HCV-RNA in the serum of MC patients, the presence of HCV antibodies, and HCVRNA concentrated in cryoprecipitates [3, 4]. The clinical syndrome of MC was described first by Meltzer et al. in 1966 [5]. MC is characterized by purpura, weakness, arthralgias, and, in some patients, glomerular lesions. In rheumatological surveys, patients

L. Rostaing (*) Department of Nephrology, Dialysis and Organ Transplantation, CHU Rangueil, Toulouse, France INSERM U563, IFR 30, CHU Purpan, Toulouse, France e-mail: [email protected]

with type III mixed cryoglobulins were found to outnumber those with type II mixed cryoglobulins [6]. Conversely, surveys based on renal involvement showed a greater prevalence of type II mixed cryoglobulins, with IgMk being the most common monoclonal immunoglobulin [7]. An oligoclonal non-neoplastic B-cell proliferation that produces cryoglobulins in the course of chronic-infection-related inflammation is the key feature of type II MC [8]. Additionally, an overtly neoplastic evolution is observed in a minority of these patients (<15%) [9]. With respect to kidney involvement in the setting of MC, a multicentric study comprising 146 patients with MC showed a close association between MC and HCV infection (mostly genotype 1b), and between glomerulonephritis and type II cryoglobulins [10]. Significant prognostic variables included age, male gender, creatinine level, and proteinuria at the time of renal biopsy, number of syndromic relapses, and poor blood-pressure control. Moreover, Roccatello et al. showed that the risk of developing MC after HCV infection is greater in patients with DRB1*11, whereas DRB1*15 protects cryoglobulinemic patients from renal involvement [10]. Until recently, the treatment of HCV-related MC was based on interferon (IFN)-a or PEGylated IFNa-2b, either with or without ribavirin [2, 11–15], and leads to an improvement of purpura, an important reduction in serum cryoglobulins and rheumatoid factor, a decrease in proteinuria, and an increase in complement subfractions (C3, C4). However, at the end of antiviral treatment, patients who do not have complete viral clearance are vulnerable to disease relapse. An alternative to antiviral treatment is infusion with the monoclonal anti-CD20 antibody

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rituximab [16, 17], which selectively targets the B-cell compartment, including RF-positive B cells [18]. Recently two randomized studies have demonstrated that in HCV-related MC rituximab therapy in addition to pegylated IFN-a-2b plus ribavirin was significantly more efficient than pegylated IFN-a-2b plus ribavirin [19, 20]. Finally, a group of experts have published recommendations for the management of mixed cryoglobulinemia in hepatitis C virusinfected patients [21]. They advise to “attempt at viral eradication using pegylated interferon-alpha plus ribavirin as the first-line therapeutic option in patients with mild-moderate HCV-related MC. Prolonged treatment (up to 72 weeks) may be considered in the case of virological non-responders showing clinical and laboratory improvements. Rituximab should be considered in patients with severe vasculitis and/or skin ulcers, peripheral neuropathy or glomerulonephritis. High-dose pulsed glucocorticoid therapy is useful in severe conditions and, when necessary, can be considered in combination with rituximab. Apheresis remains the elective treatment for severe, life-threatening hyper-viscosity syndrome; its use should be limited to patients who do not respond to (or who are ineligible for) other treatments, and emergency situations. Cyclophosphamide can be considered in combination with apheresis” [21]. In this chapter, we analyze the published data regarding the prevalence and outcome of MC after solid-organ transplantation, specifically, kidney- and liver-transplant patients.

31.2

Prevalence, Implications, and Outcomes of MC in KidneyTransplant Recipients

Our group was the first to report the prevalence of MC in kidney-transplant (KTx) recipients [22], based on an assessment of the frequency of serological markers of autoimmunity and cryoglobulins in 74 KTx patients presenting with chronic HCV. These results were compared with those obtained in 33 HCVnegative KTx patients and 13 hepatitis B virus (HBV)positive/HCV-negative KTx patients. The groups did not differ significantly according to their mean age, gender ratio, or baseline immunosuppression. Serum specimens from these patients were tested for cryoglobulinemia, complement hemolytic activity (CH50),

C3, C4, and properdin factor B (PFB) components, rheumatoid factor (RF), immunoglobulin patterns, circulating immune complexes, and the following autoantibodies: anti-nuclear (ANA), anti-smooth muscle (ASMA), anti-mitochondrial, anti-thyroid microsomal (ATM), anti-thyroglobulin (ATG), and anti-LKM1. Cryoglobulinemia was a rare event, with type II cryoglobulinemia present in only two HCVpositive KTx patients (2.7%). One of these cases was associated with de novo membranoproliferative glomerulonephritis, which was not found in any of the HCV-negative patients. RF (>40 U/mL) was more frequently observed in HCV-positive (55.4%) than in HCV-negative patients (39%; p = 0.06). There was no significant difference between the prevalence of autoantibodies in HCV-positive and HCV-negative patients, i.e., the frequency of ANA (23 vs. 36.6%) or ASMA (13.5 vs. 23%). In contrast, tissue-specific autoantibodies, i.e., ATM, ATG, and anti-LKM1, were only observed in HCV-positive patients. CH50, C3, C4, and PFB levels were significantly lower in HCVpositive than in HCV-negative patients, although values below the normal ranges were observed only for CH50 and C3. Our conclusions from this study were that, in contrast to HCV-positive immunocompetent patients, HCV-positive KTx patients rarely present with cryoglobulinemia and they have the same frequency of non-organ-specific autoantibodies as HCV-negative KTx patients. Conversely, anti-thyroid autoantibodies are only observed in HCV-positive patients. Interestingly, 2 years after our report, Wu et al. found a higher frequency of cryoglobulinemia in KTx patients [23]. They studied the prevalence and clinical spectrum of cryoglobulinemia amongst 101 maintenance hemodialysis (HD) patients and 148 KTx recipients, with or without chronic HCV infection. Cryoglobulinemia was present in 32% (16 of 50) of HCV-positive HD patients, 5.9% (3 of 51) of HCV-negative HD patients, 37.8% (28 of 74) of HCV-positive KTx recipients, and 27% (20 of 74) of HCV-negative KTx recipients. Cryoprecipitate in 56.3% (9 of 16) of HCV-positive HD patients and 50% (14 of 28) of HCV-positive KTx recipients contained HCV-RNA. Interestingly, cryocrit values among HD and KT patients were much lower than those reported for non-renal-failure patients. In addition, cryoglobulinemic syndrome in HD and KTx patients with cryoglobulinemia was very rare. Wu et al. also were unable to detect a correlation between cryoglobulinemia and age, gender, and liver-function tests. The observation

31

Cryoglobulinemia in HCV-Positive Renal Transplant and Liver Transplant Patients

that >40% of patients with cryoglobulinemia tested negative for HCV raises the question as to whether any other subclinical, viral or non-viral, chronic infection is able to induce cryoglobulinemia in HCV-negative HD or KTx recipients [23]. Anis et al. reported on a series of 208 HD (136) and KTx (72) patients, of whom 103 were HCV-positive [24]. They found a higher prevalence of cryoglobulinemia in HCV-positive patients (57.6%) than in HCVnegative patients (42.4%; p = 0.0001). RF, ANA, and ASMA were also higher in cryoglobulinemia-positive HCV-infected patients, and HCV RNA was present in 84.2% of anti-HCV-positive patients. Also, cryoprecipitable RF activity occurred more frequently in symptomatic patients with HCV genotype 1 than in patients with other HCV genotypes. More recently, Weiner et al. examined whether cryoglobulinemia and complement turnover (CH50 and C3d) were associated with HCV infection in 31 HCV-RNA-positive KTx recipients, and whether this had an adverse effect on graft outcome [25]. These patients were compared with 80 HCV-negative KTx recipients and 72 untreated patients who had chronic HCV and no renal transplant. The authors reported the following results. Cryolobulins were detected in 45% of HCV-positive KTx patients, in 28% of HCVnegative KTx patients, and in 26% of HCV-positive non-transplant patients. Elevated cryocrit (>5%) was only present in HCV-positive non-transplant patients (p < 0.01%). Mean CH50 values were lower and C3d levels higher in HCV-positive patients than in HCV-negative patients (p < 0.0001). Cryoglobulins were not associated with extrahepatic manifestations or graft dysfunction, except in five HCV-positive non-transplant patients who had cryoglobulinemic vasculitis. HCV-positive KTx recipients who had signs of complement activation had significantly higher serum creatinine (0.88 ± 1.14 mg/dL) than determined in patients without complement activation (0.34 ± 0.37 mg/dL; p = 0.035). There was also a tendency towards greater proteinuria in patients with complement activation (1.38 ± 2.17 vs. 0.50 ± 0.77 g/ day; p = 0.25). Weiner et al. concluded that cryoglobulinemia is common in KTx recipients but does not affect graft function. However, complement activation does appear to be involved in chronic allograft dysfunction in HCV-infected recipients [25]. Sens et al. found that cryoglobulinemia affected 74.4% of 39 KTx patients; in those with

247

cryoglobulinemia, HCV was positive in only 37.9% of cases [26]. Cryoglobulinemia did not influence allograft function. In the light of these data and our previous findings regarding the very low prevalence of cryoglobulinemia amongst HCV-positive KTx patients, we recently performed a cross-sectional study to assess the prevalence of cryoglobulinemia and autoimmune markers (ANAs, ANCAs, RF, and anti-cardiolipid antibodies) in 117 maintenance KTx patients whose immunosuppression was based on calcineurin inhibitors (82%). We also sought to determine cryoglobulinemia risk factors and the impact of the disease upon allograft function [27]. Cryoglobulinemia was positive in 47 patients (40.2%), of whom 13 were HCV-positive (27.7%), with characteristics of type II in 21.2% and type III in 78.8% of patients. Moreover, cryoglobulinemia was positive in 13/16 (81.2%) patients who were positive for HCV RNA vs. 34/101 (33.6%) HCV-negative patients (p = 0.0003). The cryoglobulinemia-positive KTx patients had been recipients of a graft for longer than cryoglobulinemia-negative patients (142 vs. 95 months; p = 0.02). Creatinine clearances were similar in the two groups (56 vs. 50 mL/mn, p = 0.5), as were microalbuminuria and albuminemia. There were no differences between cryoglobulinemia-positive or -negative patients in terms of age, gender, HLA mismatch, daily steroid doses, liver and hematological tests, ANAs, anti-cardiolipid antibodies, and serum complement. RF occurred very frequently; i.e., in all cryoglobulinemic-positive KTx patients and in 82.8% of cryoglobulinemic-negative patients, with higher titers in the cryoglobulinemicpositive group (23 vs. 9 UI/mL, p = 0.012). ANCA were found in nine cryoglobulinemic-negative patients but in none of the cryoglobulinemic-positive KTx patients (p = 0.013). Finally, we were not able to identify any independent predictive factors associated with cryoglobulinemia, which is frequent after kidney transplantation and is associated with HCV markers, RF, and an absence of ANCA. To summarize, the prevalence of cryoglobulinemia is quite high in maintenance KTx patients and is comparable to that observed in HD patients. In addition, cryoglobulinemia is often but not always associated with HCV infection. However, cryocrit levels tend to be lower in transplant patients than in non-renal-failure patients, which might explain why developing cryoglobulinemia after kidney transplantation rarely results in clinical symptoms, especially cryoglobulinemic syndrome.

72 KTX 111 KTX

39 KTX 117 KTX

3 LTX 52 LTX

30 LTX

92 LTX

[21] [22]

[23] [24]

[25] [26]

[27]

[28]

0

0

0 0

0 0

136 HD pts 72 non cirrhotic HCV(+) pts

101 HD pts 37.8% in HCV(+) KTx vs.32% in HD patients

ND 13.7%

37.9% 27.7%

49.5% 57.6% 28% of KTx 45% HCV(+) KTx vs. 26% HCV(−) KTx vs. 26% (control)

50%

55.5% in HCV(+) LTx vs. 35.8% in HCV(−) LTx

30% 26%

100%

55.6%

100%

100% 3 100% 19% in HCV(+) LTx 31/52(59%) 100% vs. 0% in HCV(−) LTx

74.4% 40.2%

49.5% 32.4% in KTx vs. 26% in controls

32.4% in HCV(+) KTx vs. 18.8% in HD patients

HCV(+) patients 61.6%

Type II: 27.7% Type III: 61.3%

ND

ND Overall: Type II: 64% Type III: 36% ND Type II: 21.2% Type III: 78.8% Type II: 100% Type II: 60% Type III: 40%

Type II: 48% Type III: 52%

22.2%

44%

100% 50%

24%

ND 0

4.1%

Cryo(+):209.5 ± 70.4 Cryo(−): 12 ± 4.4 Cryo(+): 61.1% Cryo(−): 50%

ND 51 ± 127 (HCV(+) KTx) 39 ± 198 (HCV(−) KTx) 85 ± 246 (controls) ND Cryo(+): 100% Cryo(−): 82.8% ND Increased in 36% HCV(+) and in 19% HCV(−)

ND

Symptomatic cryoglobulinemia Cryoglobulinemia in transplant Rheumatoid factors type patients (% or values) Type II: 100% 50% 55.4% in HCV(+) KTx vs. 39%; p = 0.06 Type III: 0%

ND not done, KTx kidney transplant patients, LTx liver transplant patients, HCV hepatitis C virus, Cryo cryoglobulinemia, HD hemodialysis

148 KTX

Type and number Patients with of patients Control patients (n) cryoglobulinemia 120 KTX 0 2.7% in HCV(+) KTx vs. 0% in HCV(−) KTx

[20]

Study [19]

HCV-positive in cryoglobulinemia patients 100%

Table 31.1 Prevalence, type and clinical manifestations in kidney- or liver-transplant patients

248 L. Rostaing et al.

31

Cryoglobulinemia in HCV-Positive Renal Transplant and Liver Transplant Patients

31.3

Prevalence, Implications, and Outcomes of MC in Liver-Transplant Recipients

Gournay et al. were the first to describe cryoglobulinemia after liver transplant (LTx) [28], in a report on three HCV-positive patients who underwent liver transplantation for HCV-related end-stage liver disease and in whom type II cryoglobulinemia with cutaneous vasculitis developed at 1–17 months after transplantation. In addition, two patients developed membranoproliferative glomerulonephritis, and one patient developed autoimmune hemolytic anemia. Plasmapheresis and the addition of cyclophosphamide reduced the severity of renal disease in one patient, whereas no treatment was able to reverse the renal failure that occurred in the other, who eventually died of multiorgan failure. Abrahamian et al. were the first to address the prevalence of cryoglobulinemia in 31 HCV-positive and 21 HCV-negative LTx recipients [29]. Six patients in the HCV-positive group (19%) had mixed cryoglobulins present at the time of evaluation compared to none in the HCV-negative group (ns). The only parameter associated with cryoglobulins in the HCV-positive group was RF (p < 0.01). Extrarenal signs of cryoglobulinemia, i.e., glomerulonephritis and/or purpura, were present in three HCV-positive patients with cryoglobulins; however, clinical manifestations were reduced after antiviral therapy. More recently, Duvoux et al. evaluated the prevalence of cryoglobulinemia in 30 HCV-positive LTx recipients [30]. The prevalence of cryoglobulinemia was quite high (30% of patients) and was frequently symptomatic, i.e., four of nine cases (glomerulonephritis in one patient and palpable purpura in three patients). RF (209.5 ± 70.4 vs. 12.0 ± 4.4 IU/L, p = 0.004) and IgM levels (3.2 ± 0.5 vs. 1.6 ± 0.9 g/L, p = 0.0001) were significantly higher, and C4 levels (0.16 ± 0.16 vs. 0.30 ± 0.10 g/L, p = 0.009) significantly lower in patients with cryoglobulinemia. One patient died from cryoglobulin-related renal failure. We recently performed a cross-sectional study to assess the prevalence of cryoglobulinemia and autoimmune markers in 92 LTx recipients whose immunosuppression was based on calcineurin inhibitors in 94.6% of the cases [31]. Cryoglobulinemia was found in 18 patients (19.5%), with characteristics of type II in 27.7%, type III in 61.3%, and indeterminate in 11%.

249

Moreover, cryoglobulinemia was present in 55.5% of patients with HCV-positive serology compared to 35.86% of patients with HCV-negative serology (p = 0.06). ANCAs were positive in 23% of patients with cryoglobulinemia but in only 5.4% of patients without cryoglobulinemia (p = 0.006). Albuminemia was significantly lower in patients with cryoglobulinemia than in patients without cryoglobulinemia (38 ± 4.2 vs.40.2 ± 3.4; p = 0.05). Cryoglobulinemia was symptomatic in only four patients (22.2% of all patients). Independent factors associated with cryoglobulinemia were the presence of ANCA, more than four HLA incompatibilities, alanine aminotransferase level ³0.68 microkat/L, and an albuminemia level >38 g/L. From these studies, it appears that cryoglobulinemia occurs relatively frequently in LTx patients, i.e., in 19.5–30% of patients. In addition, cryoglobulinemia is often but not always associated with HCV infection and is more frequently symptomatic in LTx than in KTx patients, i.e., in 22–44% of LTx patients. Table 31.1 summarizes prevalence, type and clinical manifestations in kidney- or liver-transplant patients. In organ transplant patients data necessary to determine which approach, i.e., antiviral therapy or rituximab infusion, is of greatest benefit to treat symptomatic HCV-related cryoglobulinemias are still lacking.

References 1. Brouet JC, Clauvel JP, Danon F et al (1974) Biologic and clinical significance of cryoglobulins: a report of 86 cases. Am J Med 57:775–788 2. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa-2 therapy in cryoglobulinemia associated with hepatitis C virus. N Engl J Med 330:751–756 3. Agnello V, Chung RT, Kaplan LM et al (1992) A role for hepatitis C virus infection in type II cryoglobulinemia. N Engl J Med 327:1490–1495 4. Lunel F, Musset L, Cacoub P et al (1994) Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 106:1291–1300 5. Meltzer M, Franklin EC (1966) Cryoglobulinemia: a study of twenty-nine patients. I. IgG and IgM cryoglobulins and factors affecting cryoprecitability. Am J Med 40:837–856 6. Invernizzi F, Pioltelli P, Cattaneo R et al (1979) A long-term follow-up study in essential cryoglobulinemia. Acta Haematol 61:93–99 7. Tarantino A, Campise M, Banfi G et al (1995) Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis. Kidney Int 47:618–623

250 8. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia. Arthritis Rheum 43:94–102 9. La Civita L, Zignego AL, Monti M et al (1995) Mixed cryoglobulinemia is a possible preneoplastic disorder. Arthritis Rheum 38:1859–1860 10. Roccatello D, Fornasieri A, Giachino O et al (2007) Multicenter study on hepatitis C virus-related cryoglobulinemic glomerulonephritis. Am J Kidney Dis 49:69–82 11. Ferri C, Marzo E, Longombardo G et al (1993) Interferon-a in mixed cryoglobulinemia patients: a randomized, crosscontrolled trial. Blood 81:1132–1136 12. Calleja JL, Albillos A, Moreno-Otero R et al (1999) Sustained response to interferon-a or to interferon-a plus ribavirin in hepatitis C virus-associated symptomatic cryoglobulinaemia. Aliment Pharmacol Ther 13:1179–1186 13. Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis C virus-related systemic vasculitis. Arthritis Rheum 52:911–915 14. Zeman M, Campbell P, Bain VG (2006) Hepatitis C eradication and improvement of cryoglobulinemia-associated rash and membranoproliferative glomerulonephritis with interferon and ribavirin after kidney transplantation. Can J Gastroenterol 20:427–431 15. Kayali Z, LaBrecque DR, Schmidt WN (2006) Treatment of hepatitis C cryoglobulinemia: mission and challenges. Curr Treat Options Gastroenterol 9:497–507 16. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101: 3827–3834 17. Quartuccio L, Soardo G, Romano G et al (2006) Rituximab treatment for glomerulonephritis in HCV-associated mixed cryoglobulinemia: efficacy and safety in the absence of steroids. Rheumatology 45:842–846 18. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 19. Saadoun D, Rescherigon M, Sene D et al (2010) Rituximab plus Peg-interferon-alpha/ribavirin compared with Peg-interferonalpha/ribavirin in hepatitis C-related mixed cryoglobulinemia. Blood 116:326–334

L. Rostaing et al. 20. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia. Blood 116:343–353 21. Pietrogrande M, De Vita S, Zignego AL et al (2011) Recommendations for the management of mixed cryoglobulinemia syndrome in hepatitis C virus-infected patients. Autoimmun Rev 10(8):444–454 22. Rostaing L, Modesto A, Cister JM et al (1998) Serological markers of autoimmunity in renal transplant patients with chronic hepatitis C. Am J Nephrol 18:50–56 23. Wu MJ, Lan JL, Shu KH et al (2000) Prevalence of subclinical cryoglobulinemia in maintenance hemodialysis patients and kidney transplant recipients. Am J Kidney Dis 35:52–57 24. Anis S, Muzaffar R, Ahmed E et al (2007) Cryoglobulinemia and autoimmune markers in hepatitis C virus infected patients on renal replacement therapy. J Pak Med Assoc 57:225–229 25. Weiner SM, Thiel J, Berg T et al (2004) Impact of in vivo complement activation and cryoglobulins on graft outcome of HCV-infected renal allograft recipients. Clin Transplant 18:7–13 26. Sens YAS, Malafronte P, Souza JF et al (2005) Cryoglobulinemia in kidney transplant recipients. Transplant Proc 37:4273–4275 27. Faguer S, Kamar N, Boulestin A et al (2008) Prevalence of cryoglobulinemia and autoimmunity markers in renaltransplant patients. Clin Nephrol 69:239–243 28. Gournay J, Ferrell LD, Roberts JP et al (1996) Cryoglobulinemia after liver transplantation. Gastroenterology 110:265–270 29. Abrahamian GA, Cosimi AB, Farrell ML et al (2000) Prevalence of hepatitis C virus-associated mixed cryoglobulinemia after liver transplantation. Liver Transpl 6:185–190 30. Duvoux C, Tran Ngoc A, Intrator L et al (2002) Hepatitis C virus (HCV)-related cryoglobulinemia after liver transplantation for HCV cirrhosis. Transpl Int 15:3–9 31. Garrouste C, Kamar N, Boulestin A et al (2008) Prevalence of cryoglobulinemia and autoimmune markers in liver transplant patients. Exp Clin Transplant 6:184–189

Part VI HCV Infection, Cryoglobulinemia and Non-Hodgkin’s Lymphomas

Chromosome Abnormalities in HCV-Related Lymphoproliferation

32

Cristina Mecucci, Gianluca Barba, and Caterina Matteucci

32.1 Introduction The host genetic background in patients with hepatitis C virus (HCV)-related lymphoproliferative disorders (LPD) has been intensively explored. This approach has led to the elucidation of a mutator phenotype, in which a “hit-and-run” transformation of B cells alters a previously benign hyper-proliferative condition to a state of malignancy. Figure 32.1 illustrates some of the specific roles played by HCV in LPD pathogenesis. B-cells frequently express an idiotype encoded by the VH1-69 variable gene [1, 2], which is hypothesized to interact with E2, one of the HCV envelope glycoproteins [3, 4]. The binding of HCV E2 to CD81, expressed on the surface of B cells, seems to play a direct role in B-cell hyper-proliferation [5]. The E2-CD81 interaction also enhances the expression of activation-induced cytidine deaminase (AID) and error-prone DNA polymerases, both of which are involved in DNA breaks and in immunoglobulin (VH) gene locus hypermutation [6, 7]. In HCV-infected cell lines as well as in the peripheral blood mononuclear cells of HCV-infected individuals, AID and polymerases zeta and iota overexpression underlie the increased frequency of mutations in tumor suppressor genes or proto-oncogenes (such as TP53, beta-catenin and BCL-6) [7]. The chromosomes of HCV-positive patients show increased aneuploidy [8], i.e., complete or partial chromosome

C. Mecucci (*) Hematology and Clinical Immunology Unit, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy e-mail: [email protected]

loss/gain, indicating ­HCV-associated chromosomal instability. Additional signs of instability are telomere shortening and abnormal nuclear aggregation [9, 10] while enhanced telomerase activity by the HCV core protein contributes to the immortalization of hepatoma cells [11]. This chapter briefly discusses preliminary cytogenetic and molecular results on HCV-related LPD.

32.2 Genome Analysis Due to the poor quality of most chromosome preparations and the low mitotic index of neoplastic B cells, conventional cytogenetic characterizations of HCVrelated LPD are scarce. Instead, most of the information used in the classification of HCV-related cryoglobulinemic syndromes and lymphomas derives from molecular cytogenetic techniques. In fluorescence in situ hybridization (FISH), which detects numerical and/or structural aberrations, a fluorescently labeled DNA probe corresponding to a known human gene/ locus is hybridized to a DNA target on metaphase chromosomes or interphase nuclei (I-FISH). Metaphase comparative genomic hybridization (M-CGH) provides information on the copy-number status of test DNA and is based on the competitive hybridization of different fluorescent DNAs. It detects genomic imbalances by comparing test DNA from a patient’s tumor cells with reference DNA from a healthy donor’s cells and with normal metaphase chromosomes (target), at a maximum resolution corresponding to a single chromosome band (6–10  Mb). Array CGH (aCGH) was developed to increase M-CGH ­sensitivity, using BAC/

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_32, © Springer-Verlag Italia 2012

253

254

C. Mecucci et al.

ORF

5’UTR

Non-structural protein coding genes

Str.prot. coding genes

C AA 1 192

E1 384

E2

3’UTR

P7 747 810

NS2

NS3

1027

4A 1658 1712

4B

5B

5A 1973

1421

3011

-The E2 protein of HCV binds the CD81 of the CD21/CD19/CD81 co-stimulatory complex (B-cell expansion) - E2 -CD81 binding interaction enhances the AID and error-prone DNA polymerases expression causing: a) DSB in the DNA of variable region of Ig (VH) b) increased mutation frequency of tumor suppressors and/or oncogenes. - HCV encoded NS3 protease and helicase have a central roles in the viral replication cycle.

-HCV Core protein (C) enhances telomerase activity contributing to immortalization of hepatoma cells

Fig. 32.1  HCV domains related to specific functions with hypothetical involvement in lymphoproliferation

PAC clones or oligonucleotide molecules spotted on a chip as the target DNA. The resolution of aCGH is in the kb range. In SNP-aCGH, oligonucleotide probes and single-nucleotide polymorphism (SNP) probe sets are used to detect copy-number variations (CNVs) and to profile loss of heterozygosity (LOH).

32.3 Does t(14;18)-IgH/BCL2 Play a Role in HCV-Related Disorders? Chromosomal translocation t(14;18), in which the immunoglobulin heavy chain locus (IgH) is juxtaposed alongside the bcl-2 proto-oncogene, leads to bcl-2 over-expression. The aberration is found in follicular B-cell lymphomas and in salivary gland non-Hodgkin lymphoma (NHL) associated with Sjögren’s syndrome [12], with strong antigenic stimulation possibly underlying its appearance in benign follicular hyperplasia. Reports of the incidence of the bcl-2 rearrangement in HCV-LPD are highly discrepant. The IgH/BCL2

rearrangement was detected in about 28% of HCVpositive NHLs [13–16] and in 16–86% of patients with type II mixed cryoglobulinemia (MC) [15–17]. A dedicated PCR study detected t(14;18) in lymphoma tissues in only 12.5% of patients with HCV-positive MC and NHL [13]. However, I-FISH and/or PCR analysis did not detect t(14;18) in 16 NHL cases or in 19 peripheral blood samples from patients with type II MC [18]. Another PCR study likewise found no evidence of the translocation in peripheral blood mononuclear cells with or without extrahepatic B-cell-related disorders or in liver-biopsy specimens from patients with HCVrelated chronic liver disease [19]. Interestingly, the IgH/BCL2 distribution was substantially different in Asians vs. white Northern Europeans [20], while environmental factors were shown to account for the IgH/BCL2 rearrangement in healthy individuals [21]. According to current evidence from the Italian population, t(14;18) does not correlate with HCV status in LPD and, when identified, may not be considered a predictive marker of virus-induced lymphomagenesis.

32  Chromosome Abnormalities in HCV-Related Lymphoproliferation

255

32.4 Cryoglobulinemia

32.5 HCV-Related NHL

One of the most frequent extrahepatic manifestations of a non-neoplastic LPD in HCV infection is type II MC. With oligo/monoclonal B-cell expansion, MC produces a mixture of polyclonal IgG and monoclonal IgM, the latter with rheumatoid factor activity. Individual host genetic factors have been implicated in the prevalence and distribution of HCV-related MC worldwide, based on the variations observed on different continents. Examples include BAFF gene polymorphisms, which by inhibiting B-cell apoptosis and allowing autoreactive B-cell survival may predispose HCV-positive patients to the development of MC [22]. Compared with HCVnegative controls, a significant association with MC in HCV-positive patients was identified for HLA class II DR5 and DQ3 alleles in addition to other, putative factors [23]. Finally, fibronectin gene polymorphisms reportedly play a role in progression from MC to B-cell NHL [24]. Molecular cytogenetic characterization of genomic imbalances in MC is limited to a series of 19 HCVpositive patients [18]. In I-FISH experiments with centromeric and locus-specific probes mapping at chromosome 3, one patient was found to have a complete chromosome 3 duplication in 34% of the nuclei examined. Three years later, FISH experiments on purified B cells isolated from freshly sampled PBMC from the same patient revealed that approximately 80% of the B cells carried the chromosome 3 duplication. At flow cytometry analysis, 80% of the B cells were IgMk- and VH1-69-positive; blood cell and B-cell counts were normal. The patient did not develop any clinical signs of lymphoma over a 3-year follow-up. Lactate dehydrogenase levels were normal; there was no lymph node or spleen enlargement; and total-body CT and abdominal ultrasound scans were negative. FISH investigation for an IgH/bcl-2 genetic rearrangement was negative. Therefore, in this HCV-positive patient, a clonal population with 3q gain seemed to correlate with a pre-malignant lymphoproliferation rather than with aggressive disease. Interestingly, complete or partial 3q trisomy has been well characterized in persistent, polyclonal B-lymphocytosis [25, 26], suggesting that in itself it does not confer an aggressive profile with respect to lymphocyte proliferation.

Like other infectious agents, particularly Helicobacter pylori, HCV has been implicated in disease manifestations outside the target organ. The extrahepatic manifestations of HCV include at least four different NHLs, from indolent to very aggressive types: lymphoplasmacytic lymphoma, splenic marginal zone lymphoma (SMZL), nodal marginal zone lymphoma, and diffuse large B-cell lymphoma (LBCL). HCV-related lowgrade lymphomas respond to biological therapies such as interferon (IFN) and antivirals while a high-grade large cell lymphoma does not. In the H. pylori model, well characterized cytogenetic changes, such as t(11;18)(q21;q21), t(1;14) (p22;q32), t(14;18)(q32;q21), t(3;14)(p14;q32), and specific trisomies (+3;+18), are related to histopathology and may predict tumor regression after therapy aimed at eradication of the bacterium [27]. In an approach integrating FISH, M- and a-CGH, we recently detected at least one genomic imbalance in 11/14 cases (78%) of NHL in patients with HCV [18]. The most frequent imbalances were gain of the long arm of chromosome 3 (25%), gains of 1q (19%) and 8q (12%), and loss of chromosome 2q (31%). I-FISH analysis identified an additional 3q duplication as a subclone among cells carrying the 8q gain. Despite the few cases in this study, a strong correlation clearly emerged for the first time between genomic lesion and lymphoma subtype. The chromosome 3q duplication was associated with low-grade SMZL, which underwent regression after treatment consisting of antivirals plus IFN. As the 3q duplication is recurrent in other MZLs, with an incidence ranging from 17% to 54% [28–33], and is frequent in low-grade MALT lymphomas secondary to infection with H. pylori, it appears to be recurrent in infection-related lymphoproliferations. Personal observations of 3q trisomy in type II MC without lymphoma development over a 3-year followup and its disappearance with lymphoma regression in two-thirds of cases after antiviral treatment [18] strongly support this hypothesis. At 3q, an unknown gene(s) probably confers B cells with a growth/survival advantage, allowing them to escape the homeostatic mechanisms that control clonal expansion. There is also evidence that 2q loss is involved in HCV-related NHL. This abnormality was found in 31% of our patients and in 80% (4/5) of the cases of

256

LBCL [13]. In three cases, 2q loss was associated with 1q gain, a marker of proliferative advantage also found in HCV-related hepatocellular carcinomas [34]. In another two patients, 2q loss was associated with a 7q deletion, which has previously been described as typical of aggressive MZL, with an un-mutated IgH gene and no 3q gain [31, 35]. In addition, in two patients with 2q loss, LBCL derived from a low-grade lymphoma, as has already been observed in large B-cell tumors associated with H. pylori infection [36]. In fact, in two cases involving a 2q deletion, Barth et al. [37] described a low-grade cellular component presenting as aggressive primary B-cell lymphoma of the gastrointestinal tract. We hypothesize that gene(s) located at the chromosome 2q arm are involved in a subset of aggressive HCV-related NHLs, which include transforming marginal zone B-cell lymphoma. The smallest deleted region at 2q22.3 in our patients was delineated by microarray analysis in one case and the results confirmed by I-FISH in three other patients. Deletion mapping restricted the minimal common region to 850 kb, which contains two candidate genes: (1) a gene encoding human glycosyltransferase-like domain containing 1 (GTDC1), which catalyzes carbohydrate synthesis of glycoproteins, glycolipids, and proteoglycans, and is highly expressed in spleen and blood lymphocytes [38], and (2) a zinc-finger homeobox 1B (ZFHX1B, also named KIAA0569; SIP1; SMAD1P1; ZEB2), haplo-insufficiency that underlies the Mowat-Wilson multiple anomalies congenital syndrome [39]. In epithelial tumor cells, ZFHX1B expression down-regulates E-cadherin and induces tissue invasion [40, 41].

32.6 Towards a Genetic Model Clarification of the pathogenesis of benign and malignant HCV-related LPD awaits a whole-genome approach in a large series of cases. However, currently available immunological and genetic data suggest a three-step model (Fig. 32.2): (1) Chronic HCV infection underlies B cell expansion. (2) Genetic events, such as 3q duplication, provide a growth/survival advantage, allowing B-cells to escape the homeostatic mechanisms that control antigen-driven proliferation. (3) Additional mutations determine the onset of lowand high- grade lymphomas.

C. Mecucci et al. B cells 1st step oligo − monoclonal expansion 3q gain

IgH/BCL2 ?

2nd step

MC+

3rd step

high grade

low grade

2q loss ± additional anomalies (+1q,...)

3q gain ± additional anomalies

Fig. 32.2  A model of HCV-driven lymphomagenesis showing at least three steps. MC mixed cryoglobulinemia

References 1. Agnello V (1997) The etiology and pathophysiology of mixed cryoglobulinemia secondary to hepatitis C virus infection. Springer Semin Immunopathol 19:111–129 2. Ivanovski M, Silvestri F, Pozzato G et  al (1998) Somatic hypermutation, clonal diversity, and preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in hepatitis C virus associated immunocytomas. Blood 91:2433–2442 3. Chan CH, Hadlock KG, Foung SK, Levy S (2001) V(H)1-69 gene is preferentially used by hepatitis C virus-associated B-cell lymphomas and by normal B-cells responding to the E2 viral antigen. Blood 97:1023–1026 4. De Re V, De Vita S, Marzotto A et al (2000) Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 96:3578–3584 5. Rosa D, Saletti G, De Gregorio E et al (2005) Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 102:18544–18549 6. Machida K, Cheng KT-H, Pavio N et al (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79:8079–8089 7. Machida K, Cheng KT-N, Sung VM-H et al (2004) Hepatitis C virus induces a mutator phenotype: enhanced mutations of  immunoglobulins and protooncogenes. PNAS 101: 4262–4267 8. Goldberg-Bittman L, Kitay-Cohen Y, Hadari R et al (2008) Random aneuploidy in chronic hepatitis C patients. Cancer Genet Cytogenet 108:20–23

32  Chromosome Abnormalities in HCV-Related Lymphoproliferation 9. Miura N, Horikawa I, Nishimoto A et al (1997) Progressive telomere shortening and telomerase reactivation during hepatocellular carcinogenesis. Cancer Genet Cytogenet 93:56–62 10. Amiel A, Fejgin MD, Goldberg-Bittman L et  al (2009) Telomere aggregates in hepatitis C patients. Cancer Invest 27:650–654 11. Zhu Z, Wilson AT, Gopalakrishna K et al (2010) Hepatitis C virus core protein enhances telomerase activity in Huh7 cells. J Med Virol 82:239–248 12. Limpens J, de Jong D, van Krieken JH et al (1991) Bcl-2/JH rearrangements in benign lymphoid tissues with follicular hyperplasia. Oncogene 6:2271–2276 13. Libra M, De Re V, De Vita S et al (2003) Low frequency of Bcl-2 rearrangement in HCV-associated non-Hodgkin’s lymphoma tissue. Leukemia 17:1433–1436 14. Libra M, De Re V, Gloghini A et  al (2004) Detection of bcl-2 rearrangement in mucosa-associated lymphoid tissue lymphomas from patients with hepatitis C virus infection. Haematologica 89:873–874 15. Zignego AL, Ferri C, Giannelli F et al (2002) Prevalence of bcl-2 rearrangement in patients with hepatitis C virus-related mixed cryoglobulinemia with or without B-cell lymphomas. Ann Intern Med 137:571–580 16. Zuckerman E, Zuckerman T, Sahar D et al (2001) The effect of antiviral therapy on t(14;18) translocation and immunoglobulin gene rearrangement in patients with chronic hepatitis C virus infection. Blood 97:1555–1559 17. Kitay-Cohen Y, Amiel A, Hilzenrat N et  al (2000) Bcl-2 rearrangement in patients with chronic hepatitis C associated with essential mixed cryoglobulinemia type II. Blood 96:2910–2912 18. Matteucci C, Bracci M, Barba G et  al (2008) Different genomic imbalances in low- and high-grade HCV-related lymphomas. Leukemia 22:219–222 19. Sansonno D, Tucci FA, De Re V et al (2005) HCV-associated B cell clonalities in the liver do not carry the t(14;18) chromosomal translocation. Hepatology 42:1019–1027 20. Yasukawa M, Bando S, Dölken G et  al (2001) Low frequency of BCL-2/J(H) translocation in peripheral blood lymphocytes of healthy Japanese individuals. Blood 98:486–488 21. Roulland S, Lebailly P, Lecluse Y et al (2004) Characterization of the t(14;18) BCL2-IGH translocation in farmers occupationally exposed to pesticides. Cancer Res 64:2264–2269 22. Giannini C, Gragnani L, Piluso A et al (2008) Can BAFF promotor polymorphism be a predisposing condition for HCVrelated mixed cryoglobulinemia? Blood 112:4353–4354 23. De Re V, Caggiari L, De Vita S et al (2007) Genetic insights into the disease mechanisms of type II mixed cryoglobulinemia induced by hepatitis C virus. Dig Liver Dis 39:S65–S71 24. Fabris M, Quartuccio L, Salvin S et  al (2008) Fibronectin gene polymorphisms are associated with the development of B-cell lymphoma in type II mixed cryoglobulinemia. Ann Rheum Dis 67:80–83 25. Agrawal S, Matutes E, Voke J et al (1994) Persistent polyclonal B cell lymphocytosis. Leuk Res 18:791–795

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26. Callet-Bauchu E, Gazzo S, Poncet C et  al (1999) Distinct chromosome 3 abnormalities in persistent polyclonal B cell lymphocytosis. Genes Chromosomes Cancer 26:221–228 27. Inagaki H (2007) Mucosa-associated lymphoid tissue lymphoma: molecular pathogenesis and clinic-pathological significance. Pathol Int 57:474–484 28. Gruszka-Westwood AM, Matutes E, Coignet LJ et al (1999) The incidence of trisomy 3 in splenic lymphoma with villous lymphocytes: a study by FISH. Br J Haematol 104:600–604 29. Brynes RK, Almaguer PD, Leathery KE et al (1996) Numerical cytogenetic abnormalities of chromosomes 3, 7, and 12 in marginal zone B-cell lymphomas. Mod Pathol 9:995–1000 30. Wotherspoon AC, Finn TM, Isaacson PG (1995) Trisomy 3 in low-grade B-cell lymphomas of mucosa associated lymphoid tissue. Blood 85:2000–2004 31. Solé F, Salido M, Espinet B et al (2001) Splenic marginal zone B-cell lymphomas: two cytogenetic subtypes, one with gain of 3q and the other with loss of 7q. Haematologica 86:71–77 32. Dierlamm J, Pittaluga S, Wlodarska I et al (1996) Marginal zone B-cell lymphomas of different sites share similar cytogenetic and morphological features. Blood 87:299–307 33. Novara F, Arcaini L, Merli M et al (2009) High-resolution genome-wide array comparative genomic hybridization in splenic marginal zone B-cell lymphoma. Hum Pathol 40: 1628–1637 34. Tornillo L, Carafa V, Richter J et al (2000) Marked genetic similarities between hepatitis B virus-positive and hepatitis C virus-positive hepatocellular carcinomas. J Pathol 192: 307–312 35. Algara P, Mateo MS, Sanchez-Beato M et al (2002) Analysis of the IgV(H) somatic mutations in splenic marginal zone lymphoma defines a group of unmutated cases with frequent 7q deletion and adverse clinical course. Blood 99: 1299–1304 36. Du MQ, Isaccson PG (2002) Gastric MALT lymphoma: from aetiology to treatment. Lancet Oncol 3:97–104 37. Barth TF, Döhner H, Werner CA et al (1998) Characteristic pattern of chromosomal gains and losses in primary large B-cell lymphomas of the gastro-intestinal tract. Blood 91:4321–4330 38. Zhao E, Li Y, Fu X et al (2004) Cloning and expression of human GTDC1 gene (glycosyltransferase-like domain containing 1) from human fetal library. DNA Cell Biol 23:183–187 39. Zweier C, Albrecht B, Mitulla B et  al (2002) “MowatWilson” syndrome with and without Hirschsprung disease is a distinct, recognizable multiple congenital anomaliesmental retardation syndrome caused by mutation in the zinc finger homeo box 1B gene. Am J Med Genet 108: 177–181 40. Comijn J, Berx G, Vermassen P et al (2001) The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 7:1276–1278 41. Maeda G, Chiba T, Okazaki M et al (2005) Expression of SIP1 in oral squamous cell carcinomas: implications for E-cadherin expression and tumor progression. Int J Oncol 27:1535–1541

Molecular Features of Lymphoproliferation in Mixed Cryoglobulinemia

33

Valli De Re and Maria Paola Simula

33.1

Introduction

The distinctive feature of cryoglobulinemia is an underlying B-cell clonal expansion that mainly involves rheumatoid factor (RF)-secreting cells [1, 2]. HCV-related type II mixed cryoglobulinemia (II-MC) is strictly a benign lymphoproliferative disorder, whose molecular features are primarily determined in the liver, which is also the main target of hepatitis C virus (HCV) and the site of inflammatory events, including the recruitment of inflammatory cells.

33.2

Molecular Characterizations and Pathological Significance of HCV-Related B-Cell Proliferations in Patients with Chronic HCV Infection

33.2.1 Characterization and Significance of Liver Lymphoid Aggregates Ectopic lymphoid follicle formation in the intrahepatic portal area is one of the most characteristic histological features of chronic HCV infection. In adults, the liver is an organ without constitutive lymphoid components. Therefore, intrahepatic B cells can be assumed to have migrated to the liver after HCV infection and subsequent inflammation. The patterns of hepatic V. De Re (*) Clinical and Experimental Pharmacology, Department of Molecular Oncology and Translational Medicine (DOMERT), Centro di Riferimento Oncologico, IRCCS, National Cancer Institute, Aviano, Italy e-mail: [email protected]

lymphoid-cell infiltration include vague lymphoid aggregates; rounded, defined follicles; and well-formed follicles with clearly identifiable germinal centers. Some studies have shown that these follicles mainly consist of B cells, surrounded by zones of T cells at the periphery [3]. Follicular lymphocyte infiltrates are essentially composed of CD10+ immunoglobulin (Ig) M+ B cells, occasionally surrounded by a thin layer of IgD+ cells and by IgG+ cells scattered at the periphery of the germinal center. Moreover, these B cells express the phenotypic markers of B-cell activation (HLA-DR and CD23), proliferation (Ki67), and survival (bcl-2), suggesting that the lymphoid infiltrates are functional follicular structures. The importance of the HCV-altered liver environment in determining clonal B-cell retention and proliferation is evidenced by the presence of repetitive Ig VDJ rearrangements, indicative of antigen-specific monoclonal/oligoclonal B-cell expansions [1], which have been found in 80% and 25% of liver biopsies from HCV-infected patients with and without clinical evidence of cryoglobulinemia, respectively [4].

33.2.2 Recirculating B-Cell Clones and B-Cell Retention in the Liver Although less frequently, clones deriving from expansions of the same founder cell have been detected in the circulation and bone marrow, demonstrating that such B cells have the capacity to recirculate [5, 6]. Interestingly, the regression of B-cell clonality observed in HCV+ patients who have not received other therapies apart from splenectomy for thrombocytopenia suggests that the spleen has a role in the control

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_33, © Springer-Verlag Italia 2012

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of these circulating clonal B cells [7, 8]. Although HCV minus strand RNA can be found in some abnormal B-cell populations [9–11], according to current evidence HCV can bind and enter primary B cells but is unable to replicate efficiently and thus to support a productive infection [12]. By contrast, B-cellassociated virus readily infects hepatoma cells and has an enhanced infectivity compared with extracellular virus. In addition, HCV was shown to promote primary B-cell adhesion to hepatoma cells, thus providing a potential mechanism for B-cell retention in the chronically HCV-infected liver [12].

33.2.3 Molecular Characterization of B-Cell Clones B-cell clones express restricted variable Ig heavy (VH) and light (VL) chain genes, with the VH1-69 and VK3 families being the most frequently represented [1, 13]. Most of these activated B cells have low-to-moderate levels of somatic hypermutations, suggestive of a response to antigenic stimulation associated with HCV infection and compatibility with the presence of a low rate of mutations in the IgM+ memory B-cell phenotype [14]. Why these B-cell expansions occur more readily in chronic HCV infection than in other chronic viral infections, such as hepatitis B virus or human immunodeficiency virus, is as yet unclear. A smaller subset of patients with II-MC develops a non-Hodgkin lymphoma (NHL) that is immunophenotypically and genotypically similar to the expanded B cells seen in II-MC [15, 16]. The antigenic dependence of these B cells is supported by the evidence that almost HCV-related II-MC and NHL resolve after successful treatment of the HCV infection [16–18]. HCV can also localize in the gastric mucosa [19], where a relationship between HCV infection, mucosal dysplasia, and B-cell clonal expansion in gastritis lesions as well as lymphoma of the mucosaassociated lymphoid tissue (MALT) has been noted [20–24]. In addition to the above-reported studies regarding NHL regression after successful treatment of HCV infection, a similar response was shown in MALT in gastric tissue [25]. Molecular analyses of the VH gene repertoire used by a gastric neoplastic clone indicated an antigen-driven expansion of a B-cell population showing the same biased use of VH and VK gene combination sequences as that found in overexpanded B-cell clones from II-MC [19].

V. De Re and M.P. Simula

33.2.4 Changes in Protein Expression that Precede Tissue Damage The identification of changes in protein expression that precede the onset of biochemically detectable tissue damage is useful to better define the etiological pathogenesis of HCV infection. Results from a proteomic study suggest a mechanism involved both in HCV pathogenesis and immune evasion, including a general down-regulation of the antigen processing and presentation machinery as well as an up-regulation of proteins involved in oxidative stress responses [26]. Ubiquinol-cytochrome-C-reductase (UQCRFS1), part of the mitochondrial respiratory chain complexIII, was identified as the most up-regulated protein in the HCV-infected gastric mucosa. Studies from human hepatopathies and animal models suggest that oxidative stress has an important role in steatohepatitis contributing to carcinogenesis and evidenced the increase of lipid peroxidation, a marker of oxidative stress, in association with hepatopathy [27, 28]. The mechanisms causing oxidative stress in HCV-positive individuals have not been fully elucidated but recent studies, including those reported above, support the direct involvement of HCV proteins in mitochondrial dysfunction and oxidative stress [29–31]. These data are the first to indicate the pathways modulated by HCV infection directly in a tissue other than its natural target; moreover, they provide insight into the mechanisms favoring the development of gastric lymphoproliferations in HCV-positive patients [26].

33.2.5 Relationship Between Cryo-IgM, B-Cell Receptor Sequence, and Memory B-Cells Lymphoid infiltrates are found also in the bone marrow of MC patients. They are multifocal, frequently paratrabecular in their location, display a B-cell phenotype, present monotypic immunoglobulin restriction, and are essentially indolent (high bcl-2, very low MIB-1/Ki-67 labelling) [32]. As deduced from the integration of histologic/immunophenotypic findings with molecular and proteomic data, most MC-associated bone marrow proliferations are oligoclonal rather than monoclonal [5, 33]. The oligoclonal nature of the lymphoproliferation is indicative of a still non-malignant process, as clinically

33

Molecular Features of Lymphoproliferation in Mixed Cryoglobulinemia

confirmed by the generally indolent course of the disease. However, among non-treated patients followed for a long period time, there is progression to overt lymphoma in about 10% of cases [32]. This evolution is associated with a frank monoclonal component, although the presence of such clones may also be found in patients with benign II-MC. It has, in fact, been demonstrated that in most patients in whom an overt NHL develops during follow-up, the malignant clone originates from one of the dominant B-cell clones over-expanded in II-MC [5, 6]. To better define the conclusive relationship between the combinatory region of the B-cell receptor (BCR) and the cryo IgM RF sequence, we compared purified serum IgM cryoglobulins either with the BCRs of the monoclonal B-cell clones identified in the bone marrow of patients with a frank NHL associated with HCV infection and II-MC, or with the restricted oligo B-cell clones identified in the bone marrow of several patients with clinical II-MC. In all patients with a monoclonal BCR pattern, and in the majority of the selected oligoclonal conditions, the combinatory region of IgM cryoglobulins correlated with at least one of the B-cell clones. This finding demonstrated that the over-expanded B-cell clones found in the bone marrow of HCV+ patients are either the RF-IgM-producing B cells causing II-MC syndrome or their consequential subclones [33]. Moreover, it allows a clear immunophenotypic identification of the B-cell clones producing RF with cryoprecipitating properties. Since monoclonal RF+ IgM from II-MC patients expresses the features of a natural antibody, it has long been hypothesized that a major source of these autoantibodies is the relatively primitive B-1 B-cell subset, displaying CD5 antigen and responsible for the production of natural antibodies against bacterial and viral pathogens [34–37]. However, several studies have now clarified that clonal and normal B cells from II-MC do not significantly differ in their intensity of CD5 expression [7, 38]. This is consistent with our data from three patients with RF-IgM+ clonal B-cells that were CD5– and CD20+ [33]. Moreover, other studies describe that in the peripheral circulation of most HCV+MC+ patients there is a substantial population of clonally expanded IgM+Igk+IgDlow/-CD21lowCD27+ B cells that express RF-like Ig. The clonal restricted B-cells are CD5– [13] and CD27+, compatible with a memory B-cell phenotype.

261

In apparent contrast, circulating CD27+ B-cells were found to be reduced in persistently HCV-infected patients without II-MC, with a correlation between CD27+ B-cell percentage and plasma HCV load in persistently infected patients [39]. This discrepancy seems to reflect the unconventional and altered functional responsiveness of CD27+ B cells in persistently HCVinfected individuals. In fact, in these patients, in the absence of specific BCR engagement, memory B cells do not expand but instead overcome terminal differentiation as antibody-secreting cells and undergo apoptosis [39]. Consequently, we proposed that this self-attenuation process is incomplete in HCV II-MC but still sufficient to mitigate B-cell proliferation pathways, thus maintaining the overall clonal B-cell number at a relatively stable— albeit elevated in II-MC—steady state level.

33.2.6 B-Cell Immune Stimulation Mediated by HCV Proteins and Autoantigens An increased percentage of polyclonal proliferating B cells also results from the interaction of HCV-E2 envelope protein with the CD81 receptor, a tetraspanin widely expressed on the surface of different cell types, including B cells [40]. Surface contact between HCV and CD81 was also demonstrated to activate B cells in the absence of cell infection. In the case of E2-CD81 engagement, however, mainly naïve CD27– B cells proliferate, without any difference in the frequency between HCV+ patients and II-MC subset. Consequently, it may be that the polyclonal activation of naïve B-cells induced by HCV engagement of CD81 is the first step toward the polyclonal expansion of autoreactive clones of naïve B cells that, by becoming memory cells (CD27+), are more readily activated via the BCR to produce autoantibodies and to persist. In addition, anti-CD81 was recently demonstrated to have no effect on B-cell to hepatoma-cell transinfection, suggesting that CD81 is not implicated in HCV B-cell infection [12]. By contrast and similar to what occurs with HIV and Dengue-virus-specific antibodies [41, 42], HCVIgG immune complexes enhance the infection of B cells by Fc receptor uptake [12]. Consistent with this finding, we demonstrated that BCRs of clonal B cells cross-react with both HCV-NS3 and Fc-IgG [43, 44]. Thus, clonal self-reactive B cells could evolve into frank lymphoma, most likely in the presence of still unidentified genetic/environmental factors.

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V. De Re and M.P. Simula

Fig. 33.1 A model explaining the different behaviors of CD27+ B-cells in HCV+ patients. (a) Memory B-cells (CD27+) from persistently HCV-infected patients, overcome terminal differentiation as antibody-secreting cells (ASC) and rapidly undergo apoptosis. This mechanism would stop the potentially disastrous increase in B-cell proliferation triggered by an endless supply of stimulatory signals. (b) The above-described self-attenuation process may be incomplete in HCV II-MC but still sufficient to mitigate B-cell proliferation pathways. In that case, CD27+ cells can be more readily activated to produce autoantibodies and to persist. The regulatory elements involved in HCV-related B-cell

clonal expansions may include the chronic and specific B-cell receptor stimulation exerted by HCV (NS3) or self (IgG-Fc) antigens, the BLys receptor (BR3)/BLyS signaling mechanism, and the presence of specific HLA combinations. IgG-HCV immune complexes might also be involved, thus allowing B-cell infection, even if HCV does not more efficiently replicate in these cells. Chronically activated and rapidly proliferating B cells are at risk of sustaining polyploidy, as evidenced by the frequent observation of chromosomal alterations in which there is 3q gain. These events can determine the evolution of oligo/ monoclonal IgM+K+B-cells to indolent B-cell NHL

33.2.7 Possible Mechanisms Underlying the Process of Lymphoma Development

B cells are not active sites for viral replication [44]. Nonetheless, in both cases, class II histocompatibility molecules could play a role since different HLA combinations are associated with different clinical manifestations in patients with persistent HCV infection [51, 52]. The regression of established HCV-associated B-cell lymphoma in II-MC patients after antiviral treatment likely reflects the mechanistic role for HCV in lymphomagenesis. However, long-term antiviral therapy in NHL patients showed, by molecular IG-VDJ analysis, a persistence of the monoclonal B-cell population in the blood, albeit of only a few cells [18, 53]. Accordingly, lymphomas with an indolent clinical course, mainly splenic marginal zone lymphomas showing 3q gain, were shown to regress after HCV eradication, while 2q loss was found in lymphomas with an aggressive large-B-cell component [49]. Such findings indicate that some B-cell clones persist even after an effective clearance of HCV infection. Therefore, HCV infection corresponds to the initiation step that in some cases is followed by a perpetuation step in which, along with autoantigens and/or cell deregulation, in different mixtures, antigen stimulation induces B-cell lymphoproliferation.

Among the additional regulatory elements significantly affected by HCV-related B-cell clonal expansion are the Fas and BLyS signaling mechanisms [16, 45, 46]. Elevated BLys serum concentrations are reportedly associated with B-NHL activity progression and prognosis [47, 48] and with a decrease of the most abundant BLys receptor, BR3 [46]. Of note, BR3 expression gradually decreases to its lowest values in B-NHL and in the VH1-69+ B-cell population that is CD27+ IgD– CD38low or CD27+IgD+CD38low, suggesting a role in the pathogenesis of these disorders [46]. Nonetheless, the link between the HCV- and BLys-producing cells and the mechanism correlating BLys with HCVinduced B-cell NHL remain to be elucidated. Possible mechanisms underlying the process of lymphoma development also include the high rate of B-cell proliferation, which favors stochastic mutations reinforcing B-cell proliferation and furthering cell survival. In this regard, two distinct genetic events occur more frequently in HCV-driven lymphomagenesis: the gain of 3q, typically seen in indolent lymphomas, and the loss of 2q, associated with large B cells [49]. It was also demonstrated that HCV-infected B cells show chromosomal polyploidy [50]. Since polyploidy and translocations are caused by different mechanisms, the consequences of this difference deserve further study. However, while translocation would provide a model in which HCV causes DNA deregulation, HCV-associated NHLs only rarely contain HCV genome [9], and efficient HCV RNA replication in lymphocytes has not been fully verified [12]. Based on these findings, we proposed that HCV coupled with specific BCRs induces signals for cellular proliferation, or that BCR bound to anti-HCV IgG mediates HCV entry into the cell, even if

33.3

Conclusions

The evidence presented above together with the results of longitudinal studies seem to delineate a process that begins with polyclonal HCV-antigen mediated B-cell activation and evolves into an under-regulated oligoclonal/monoclonal expansion of the mature (CD27+) B-cell population through BCR-mediated proliferative signals (Fig. 33.1). These cells are responsible for the production of RF+ autoantibodies with cryoglobulinemic activity and evolve in some instances to frank

33

a

Molecular Features of Lymphoproliferation in Mixed Cryoglobulinemia

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Persistent HCV infection

C D8 1

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C D2 7

Fas R

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Type II MC and indolent B-NHL NS3

HCV

Fc lgG

R

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M

Fc

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BR3

Soluble Blys

Clonal B-cell expansion

malignant clones, which, at least in some cases, persist despite HCV eradication. Further studies are necessary to better define the pathogenetic mechanisms associated with HCV-related B-cell deregulation and overt lymphoma development.

References 1. De Re V, De Vita S, Marzotto A et al (2000) Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-Hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 96:3578–3584 2. Sansonno D, Carbone A, De Re V, Dammacco F (2007) Hepatitis C virus infection, cryoglobulinaemia, and beyond. Rheumatology (Oxford) 46:572–578 3. Sansonno D, Lauletta G, De Re V et al (2004) Intrahepatic B-cell clonal expansions and extrahepatic manifestations of chronic HCV infection. Eur J Immunol 34:126–136

Indolent B-NHL

4. Sansonno D, Tucci FA, De Re V et al (2005) HCV-associated B-cell clonalities in the liver do not carry the t(14;18) chromosomal translocation. Hepatology 42:1019–1027 5. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B-cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B-cell lymphoma. Arthritis Rheum 43:94–102 6. De Re V, De Vita S, Marzotto A et al (2000) Pre-malignant and malignant lymphoproliferations in an HCV-infected type II mixed cryoglobulinemic patient are sequential phases of an antigen-driven pathological process. Int J Cancer 87: 211–216 7. Ohtsubo K, Sata M, Kawaguchi T et al (2009) Characterization of the light chain-restricted clonal B-cells in peripheral blood of HCV-positive patients. Int J Hematol 89:452–459 8. Roulland S, Suarez F, Hermine O, Nadel B (2008) Pathophysiological aspects of memory B-cell development. Trends Immunol 29:25–33 9. De Vita S, De Re V, Sansonno D et al (2002) Lack of HCV infection in malignant cells refutes the hypothesis of a direct transforming action of the virus in the pathogenesis of HCVassociated B-cell NHLs. Tumori 88:400–406

264 10. Sansonno D, Tucci FA, Lauletta G et al (2007) Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia. Clin Exp Immunol 147:241–248 11. Inokuchi M, Ito T, Uchikoshi M et al (2009) Infection of B-cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C. J Med Virol 81:619–627 12. Stamataki Z, Shannon-Lowe C, Shaw J et al (2009) Hepatitis C virus association with peripheral blood B lymphocytes potentiates viral infection of liver-derived hepatoma cells. Blood 113:585–593 13. Charles ED, Green RM, Marukian S et al (2008) Clonal expansion of immunoglobulin M+CD27+ B-cells in HCVassociated mixed cryoglobulinemia. Blood 111:1344–1356 14. Duty JA, Szodoray P, Zheng NY et al (2009) Functional anergy in a subpopulation of naive B-cells from healthy humans that express autoreactive immunoglobulin receptors. J Exp Med 206:139–151 15. De Re V, Caggiari L, Simula MP et al (2007) B-cell lymphomas associated with HCV infection. Gastroenterology 132: 1205–1207 16. De Re V, De Vita S, Sansonno D, Toffoli G (2008) Mixed cryoglobulinemia syndrome as an additional autoimmune disorder associated with risk for lymphoma development. Blood 111:5760 17. Hermine O, Lefrere F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med 347: 89–94 18. Mazzaro C, De Re V, Spina M et al (2009) Pegylatedinterferon plus ribavirin for HCV-positive indolent nonHodgkin lymphomas. Br J Haematol 145:255–257 19. De Vita S, De Re V, Sansonno D et al (2000) Gastric mucosa as an additional extrahepatic localization of hepatitis C virus: viral detection in gastric low-grade lymphoma associated with autoimmune disease and in chronic gastritis. Hepatology 31:182–189 20. Sorrentino D, Ferraccioli GF, De Vita S et al (1997) Hepatitis C virus infection and gastric lymphoproliferation in patients with Sjogren’s syndrome. Blood 90:2116–2117 21. Cammarota G, Cianci R, Grillo RL et al (2002) Relationship between gastric localization of hepatitis C virus and mucosaassociated lymphoid tissue in Helicobacter pylori infection. Scand J Gastroenterol 37:1126–1132 22. Takeshita M, Sakai H, Okamura S et al (2006) Prevalence of hepatitis C virus infection in cases of B-cell lymphoma in Japan. Histopathology 48:189–198 23. Visco C, Arcaini L, Brusamolino E et al (2006) Distinctive natural history in hepatitis C virus positive diffuse large B-cell lymphoma: analysis of 156 patients from northern Italy. Ann Oncol 17:1434–1440 24. De Vita S, Sacco C, Sansonno D et al (1997) Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection. Blood 90:776–782 25. Tursi A, Brandimarte G, Torello M (2004) Disappearance of gastric mucosa-associated lymphoid tissue in hepatitis C virus-positive patients after anti-hepatitis C virus therapy. J Clin Gastroenterol 38:360–363 26. De Re V, Simula MP, Cannizzaro R et al (2008) HCV inhibits antigen processing and presentation and induces

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42. Halstead SB (2003) Neutralization and antibody-dependent enhancement of dengue viruses. Adv Virus Res 60:421–467 43. De Re V, Sansonno D, Simula MP et al (2006) HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 20:1145–1154 44. De Re V, Pavan A, Sansonno S et al (2009) Clonal CD27+ CD19+ B-cell expansion through inhibition of FC gammaIIR in HCV(+) cryoglobulinemic patients. Ann N Y Acad Sci 1173:326–333 45. Fabris M, Quartuccio L, Sacco S et al (2007) B-lymphocyte stimulator (BLyS) up-regulation in mixed cryoglobulinaemia syndrome and hepatitis-C virus infection. Rheumatology (Oxford) 46:37–43 46. Landau DA, Saadoun D, Calabrese LH, Cacoub P (2007) The pathophysiology of HCV induced B-cell clonal disorders. Autoimmun Rev 6:581–587 47. Briones J, Timmerman JM, Hilbert DM, Levy R (2002) BLyS and BLyS receptor expression in non-Hodgkin’s lymphoma. Exp Hematol 30:135–141 48. Novak AJ, Grote DM, Stenson M et al (2004) Expression of BLyS and its receptors in B-cell non-Hodgkin lymphoma:

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correlation with disease activity and patient outcome. Blood 104:2247–2253 Matteucci C, Bracci M, Barba G et al (2008) Different genomic imbalances in low- and high-grade HCV-related lymphomas. Leukemia 22:219–222 Machida K, Liu JC, McNamara G et al (2009) Hepatitis C virus causes uncoupling of mitotic checkpoint and chromosomal polyploidy through the Rb pathway. J Virol 83:12590–12600 De Re V, Caggiari L, Simula MP et al (2007) Role of the HLA class II: HCV-related disorders. Ann N Y Acad Sci 1107:308–318 De Re V, Caggiari L, Monti G et al (2010) HLA DR-DQ combination associated with the increased risk of developing HCV+ non-Hodgkin’s lymphoma is related to the type-II mixed cryoglobulinema syndrome. Tissue Antigens 75:127–135 Vallisa D, Bernuzzi P, Arcaini L et al (2005) Role of antihepatitis C virus (HCV) treatment in HCV-related, lowgrade, B-cell, non-Hodgkin’s lymphoma: a multicenter Italian experience. J Clin Oncol 23:468–473

The Higher Prevalence of B-Cell Non-Hodgkin’s Lymphoma in HCV-Positive Patients with and Without Cryoglobulinemia

34

Franco Dammacco and Domenico Sansonno

34.1

Introduction

It has been estimated that approximately 15% of all human tumors have a viral origin. The percentage of virus-related cancers is nearly three-fold higher in developing than in developed countries, reflecting the higher prevalence of infection by the causative viruses and possibly the exposure to enhancing co-factors [1]. Viruses are associated with a variety of human malignancies. Both hepatitis B virus (HBV) and hepatitis C virus (HCV) can cause hepatocellular carcinoma (HCC) [2]. Epstein-Barr virus (EBV) is etiologically linked to Burkitt’s lymphoma, nasopharyngeal carcinoma, post-transplant lymphoma, Hodgkin’s disease, and possibly other tumors [3]. The human papillomavirus may cause cervical cancer, skin cancers, and, likely, head and neck cancers as well as other ano-genital cancers [4]. Human T-cell leukemia virus type 1 (HTLV-1) induces adult T-cell leukemia [5]. Human herpes virus type 8 (HHV-8/KSHV) may be causally related to Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castelman’s disease [6]. Simian virus 40 (SV40) has been associated with brain tumors, osteosarcoma, and mesothelioma [7]. Some viruses are associated a single tumor type (i.e., HBV), whereas others are linked to multiple tumor types (i.e., EBV), presumably reflecting the tissue tropism of a given virus. A virus associated with human tumors may also induce non-neoplastic diseases in some hosts. For example, HTLV-1 is the cause of myelopathy or F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

tropical spastic paraparesis; EBV causes infectious mononucleosis; HPV causes a variety of benign hyperplasias, and both HBV and HCV cause acute and chronic hepatitis. The frequency of disease development varies widely, reflecting the basic characteristics of the particular virus and the virus-host relationships [8]. However, confirming an etiologic role for a virus in human cancer is not an easy task and almost always requires laborious and sustained efforts to prove a causal association. For example, it must be explained why viruses are ubiquitous, whereas cancer is relatively rare. A major role of host-related determinants in the susceptibility to the malignant process must be considered as well. In addition, there is usually a long lag time between virus infection and tumor appearance. Virus-related tumorigenesis requires the participation of environmental, genetic, and other viral and host-related cofactors. Indeed, it is evident that the incidence of cancer varies in different geographic areas and in different age groups. It also can be envisaged that virus strains differ in their biologic properties and oncogenic potential. Finally, an exact definition of the mechanisms of viral tumorigenesis has so far been hampered due to the difficulties in obtaining animal models of human cancers. The identification of a virus in a tumor does not prove its etiological role: the virus might have infected the tumor following tumor outgrowth, or the virus may be present in the lymphocytes that carry the infection from one anatomic site to another [9, 10]. Nonetheless, by using a combination of assays, a strong association was determined between HCV infection and the risk of HCC. This subsequently confirmed relationship is particularly noteworthy because HCV is a member of the

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_34, © Springer-Verlag Italia 2012

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F. Dammacco and D. Sansonno

Flaviviridae family, comprising RNA viruses not thought to be capable of tumorigenic activity [11]. Due to the genetic instability generated during virogenesis, several major genotypes of HCV, with different global distributions, are recognized [12]. A causative association between HCV and nonHodgkin’s lymphoma (NHL) has also been postulated [13]. This relationship is the subject of intense investigation and debate. On the strength of epidemiologic data, biological investigations, and clinical observations, HCV appears to be etiologically related to a subset of NHLs.

34.2

Epidemiology

The evidence linking HCV to NHL derives from a large number of epidemiological studies that have provided variable data, reflecting differences in study

design, patient populations, geographic areas, virus prevalence, and laboratory methods (Table 34.1). Despite the many inconsistencies, including a strong regional connotation, several reports strongly support a relationship between chronic HCV infection and an elevated risk of developing NHL. HCV-positive individuals from the Mediterranean basin, Japan, Brazil and Eastern Europe show an increased risk association, with an odds ratio (OR) of 2–4. By contrast, in Northern Europe, the UK, and many areas of Canada, no convincing epidemiological evidence has been provided to support a link between HCV and B-cell NHL. Two recent large studies carried out in the USA, involving the NCI-SEER registry and US Veterans Affairs health system, respectively, as well as an analysis by the European Multicenter EPYLYMPH Consortium have consistently shown a higher risk of B-NHL in HCVpositive patients than in HCV-negative controls (relative risk: 2–3) [29–31]. The International Lymphoma

Table 34.1 Prevalence of hepatitis C virus infection in patients with Non-Hodgkin’s lymphoma (NHL)

Country/reference Australia [14] Egypt [15] France [16] Italy [17] [18] [19] [20] [21] Japan [22] [23] South Korea [24] Spain [25] USA [26] [27] [28] a

NHLa Number of cases

Mean age (range)

Controlsb Number of cases HCV-positive

HCV-positive

Prevalence (%)

Prevalence (%)

597

3.0

0.5

55 (20–74)

522

2.0

0.4

220

94

42.7

48

222

52

23.4

212

6.0

2.8

60 (21–90)

974

20

2.1

67 (18–101)

175 300 111 530 225

65 48 28 83 44

37.1 16 25.2 15.7 19.6

350 847 226 396 504

32 66 17 22 45

9.1 7.8 7.5 5.6 8.9

55 (20–97)

134 187

17 23

12.7 12.3

63 (24–87) 63 (46–79)

516

34

6.6

214

7.0

3.3

52 (14–85)

865

19

2.2

376

25

6.6

55

599

22

3.7

120 813 464

26 32 8

21.7 3.9 1.7

52 (23–84) 57 (20–74) 64 (21–84)

114 684 534

6 14 5

5.3 2.0 0.9

63 (17–92) 59 (13–85) 59 (18–84)

Mean age (range)

63 (18–84)

Selected papers include studies of HCV prevalence carried out in over 100 patients and over 100 controls Controls included lymphoid tumors other than NHL, non-lymphoid cancers, hospital-based population, blood donors, and/or general population

b

34

B-Cell Non-Hodgkin’s Lymphoma in HCV-Positive Patients

Epidemiology Consortium (InterLymph), which encompasses centers in Europe, North America, and Australia, performed a pooled case–control study (4,784 B-NHLs and 6,269 controls). HCV infection was detected in 172 B-NHLs (3.6%) and in 169 controls (2.7%). Interestingly, however, in a subtype-specific analysis, HCV was associated with marginal zone lymphoma (OR: 2.47), diffuse large B-cell lymphoma (DLBC) (OR: 2.24) and lymphoplasmacytic lymphoma (OR: 2.57) [32]. From the pooled epidemiological data, the following conclusions were drawn: (a) there is a greater propensity to develop B-NHL in the setting of HCV; (b) the risk is dramatically higher in populations with high HCV prevalence; (c) geographic variability worldwide indicates additional environmental factors influencing the strength of the association. Some other aspects of the association remain to be elucidated, including the role of specific HCV genotypes [33]. Lymphoma subtypes, including Burkitt lymphoma, T-cell lymphoma, and Hodgkin’s lymphoma, have not been consistently linked to HCV infection [27]. The lack of association with these pathological entities is in line with the notion that proliferation of specific B-cell clones following chronic antigenic stimulation is the mechanism that drives subtypes of NHL. Notably, HIV/HCV co-infected individuals provide further evidence for the causal role of HCV in sustaining B-cell malignant lymphoproliferation. HIV-1 infection is a well-recognized risk factor for NHL [34]. In a large European study involving 5,832 HIV-1 positive patients, HCV was not associated with systemic HIV-positive NHL [35], suggesting that HCV does not increase the risk of NHL among HIV-positive patients. Following the hypothesized mechanism invoking chronic antigenic stimulation as a trigger of lymphomagenesis, an effect of HCV infection in immuno-compromised patients, such as HIV-positive individuals, is indeed not expected.

34.3

Clinical Features of NHLs

A growing number of clinical and biologic observations have strongly emphasized the potential role of HCV in causing a variety of extrahepatic disorders, whether dermatologic, hematologic, endocrinologic, rheumatic, or autoimmune. In this context, as already emphasized in several chapters of this book, a strong association between HCV and mixed cryoglobulinemia

269

(MC) has been established [36]. MC is considered an indolent B-cell lymphoproliferative disorder capable of evolving to frank malignancy. When HCV-infected patients were analyzed over the course of a long-term observation, progression to NHL was demonstrated in 5–10% of them. Symptoms indicative of progression are usually mild and include an expanding spectrum of autoimmune phenomena, namely, hemolytic anemia, thrombocytopenia, and granulocytopenia [37]. Indeed, two subsets of B-cell NHLs associated with HCV infection, with distinct clinical and pathologic features, have been described: (a) NHLs that complicate the course of MC: are usually low-grade, involve the bone marrow, and may evolve into an aggressive phenotype; (b) NHLs unrelated to MC, which frequently show an aggressive phenotype ab initio and often lack bone marrow involvement [13, 38]. The studies published so far are inadequate to assess the long-term impact of HCV infection on patients with NHL not associated with MC. These data should be interpreted with particular caution, in that it is difficult to know whether HCV infection occurred before NHL was diagnosed or was acquired in the course of the disease. It is likely that immunosuppression, which results from the lymphoma process and conditioning therapies, make patients more susceptible to viral infections. Analysis of clinical features shows that these patients present at onset with either extranodal B-cell NHL (especially liver and salivary glands), a diffuse large-cell histotype without any prior history of low-grade B-cell malignancy or bone marrow involvement, and a weak association with autoimmune disorders, as summarized in Fig. 34.1.

34.4

Pathogenesis of HCV-Associated NHLs

Although epidemiological data suggest a link between HCV infection and NHL, the pathobiological processes leading to clonal B-cell expansion and subsequent malignant transformation are still unclear. Viruses can persist in the host indefinitely, through latency or continuous low-level replication. In chronic HCV infection, it has been shown that peri-hepatic lymph node enlargement is directly related to viral replication rather than to the severity or activity of liver disease [39], supporting the concept that the actual

270

F. Dammacco and D. Sansonno

Fig. 34.1 Frequency of HCV chronic carriage in nonHodgkin’s B-cell lymphoma with and without mixed cryoglobulinemia

HCV-INFECTED PATIENTS ~ 15% MAJOR CLINICAL FEATURES • FREQUENTLY EXTRA-NODAL • HIGH-GRADE HISTOTYPE • ABSENCE OF BONE MARROW INVOLVEMENT

?%

~ 100% ~ 12% MIXED CRYOGLOBULINEMIA

B-CELL NONHODGKIN’S LYMPHOMA

~ 7% MAJOR CLINICAL FEATURES • FREQUENTLY NODAL • LOW-GRADE HISTOTYPE • BONE MARROW INVOLVEMENT

presence of replicating virus within immunocytes may be responsible for the virus-driven B-cell expansion. Indeed, the demonstration of active HCV replication in extra-hepatic sites remains a crucial issue due to many technical and biological confounding factors, including different proportions of negative and positive HCV strands during virogenesis. HCV, as a member of flaviviruses, favors the production of positive- over negative-stranded RNA [40]. This generates mispriming events during genomic amplification techniques, leading to false-positive products of HCV replication intermediates [41]. An additional biological confounding factor is the multiprotein cell surface receptor complex comprising CD81, human scavenger receptor SR-BI, claudin, occludin, heparan sulfate and low-density lipoprotein receptors [42], which are expressed on the surface of different cells, including hematological cells [43]. Appropriate methodological approaches capable of demonstrating incontrovertibly the occurrence of HCV replication in hematopoietic cells are biologically and clinically relevant. HCV may directly affect the function of immunocytes by interfering with their ability to eliminate the virus from infected cells and/or modifying their properties such that they are activated either in response to a suitable stimulant or to exert adequate effector functions. We have been able to demonstrate viral genomic sequences in the absence of viral protein production in malignant B-cells from low-grade NHLs, suggesting a

latency status of HCV in these cells. Analysis of lymph nodes with high-grade NHL showed persistently negative results, providing evidence of a relationship between the expression of viral gene products and certain stages of cell differentiation and emphasizing that high-grade malignant phenotypes are not permissive to HCV replication [44]. The virtual absence of HCVpositive lymphoid cells in neoplastic lymph nodes and the large number of HCV-carrying B-cells detected in hyperplastic reactive lymphadenopathy indicate that HCV infection contributes to the early steps in the development of malignancy. The potential transforming activity of HCV has been suggested by in vitro studies, in which the role of core protein in the transcriptional regulation of cellular proto-oncogenes affecting normal cell growth has been emphasized [45]. At this stage of lymphomagenesis, other factors are likely to contribute to the malignant process, including stimulation of cell proliferation and aberrant protein expression through cytogenetic abnormalities [46].

34.5

Conclusions

It is conceivable that a better understanding of the mode of HCV persistence and of the role played by HCV in the malignant process will permit the development of new approaches, aimed at the prevention and

34

B-Cell Non-Hodgkin’s Lymphoma in HCV-Positive Patients

treatment of HCV-associated NHLs. Virus-induced lymphomagenesis is strongly supported by the evidence that HCV-related NHL disappears after successful treatment of HCV infection. The largest reported series refers to a group of nine patients with splenic lymphoma and villous lymphocytes, the majority of whom had MC. Almost 80% of these patients responded completely to antiviral treatment, achieving sustained hematological and clinical responses [47]. These observations indicate that HCV-associated NHLs share some of the pathogenetic features of other recognized and potentially reversible antigen-driven NHLs, such as Helicobacter-pylori-related gastric marginal zone NHL [48]. Given that the immune system modulates the host response to infection, an important area for further epidemiologic investigation is a systematic evaluation of the interactions of polymorphisms in immune-related genes with specific infectious agents in causing NHL.

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272 29. Engels EA, Chatterjee N, Cerhan JR et al (2004) Hepatitis C virus infection and non-Hodgkin lymphoma: results of the NCI-SEER multi-center case-control study. Int J Cancer 111:76–80 30. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297:2010–2017 31. Nieters A, Kallinowski B, Brennan P et al (2006) Hepatitis C and risk of lymphoma: results of the European multicenter case-control study EPILYMPH. Gastroenterology 131: 1879–1886 32. de Sanjose S, Benavente Y, Vajdic CM et al (2008) Hepatitis C and non-Hodgkin lymphoma among 4784 cases and 6269 controls from the International Lymphoma Epidemiology Consortium. Clin Gastroenterol Hepatol 6:451–458 33. Besson C, Canioni D, Lepage E et al (2006) Characteristics and outcome of diffuse large B-cell lymphoma in hepatitis C virus-positive patients in LNH 93 and LNH 98 Groupe d’Etude des Lymphomes de l’Adulte programs. J Clin Oncol 24:953–960 34. Waters L, Stebbing J, Mandalia S et al (2005) Hepatitis C infection is not associated with systemic HIV-associated non-Hodgkin’s lymphoma: a cohort study. Int J Cancer 116: 161–163 35. Franceschi S, Polesel J, Rickenbach M et al (2006) Hepatitis C virus and non-Hodgkin’s lymphoma: findings from the Swiss HIV Cohort Study. Br J Cancer 95:1598–1602 36. Sansonno D, Dammacco F (2005) Hepatitis C virus, cryoglobulinaemia, and vasculitis: immune complex relations. Lancet Infect Dis 5:227–236 37. Ryan J, Wallace S, Jones P et al (1994) Primary hepatic lymphoma in a patient with chronic hepatitis C. J Gastroenterol Hepatol 9:308–310 38. De Vita S, Sacco C, Sansonno D et al (1997) Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection. Blood 90:776–782

F. Dammacco and D. Sansonno 39. Pal S, Sullivan DG, Kim S et al (2006) Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo: implications of HCV lymphotropism. Gastroenterology 130:1107–1116 40. Lescar J, Canard B (2009) RNA-dependent RNA polymerases from flaviviruses and Picornaviridae. Curr Opin Struct Biol 19:759–767 41. Sansonno D, Tucci FA, Lauletta G et al (2007) Hepatitis C virus productive infection in mononuclear cells from patients with cryoglobulinaemia. Clin Exp Immunol 147:241–248 42. Budkowska A (2009) Mechanism of cell infection with hepatitis C virus (HCV)–a new paradigm in virus-cell interaction. Pol J Microbiol 58:93–98 43. Sansonno D, Lotesoriere C, Cornacchiulo V et al (1998) Hepatitis C virus infection involves CD34(+) hematopoietic progenitor cells in hepatitis C virus chronic carriers. Blood 92:3328–3337 44. Sansonno D, De Vita S, Cornacchiulo V et al (1996) Detection and distribution of hepatitis C virus-related proteins in lymph nodes of patients with type II mixed cryoglobulinemia and neoplastic or non-neoplastic lymphoproliferation. Blood 88: 4638–4645 45. Irshad M, Dhar I (2006) Hepatitis C virus core protein: an update on its molecular biology, cellular functions and clinical implications. Med Princ Pract 15:405–4016 46. Koike K, Tsutsumi T, Fujie H et al (2002) Molecular mechanism of viral hepatocarcinogenesis. Oncology 62(Suppl 1):29–37 47. Hermine O, Lefrere F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med 347: 89–94 48. Psyrri A, Papageorgiou S, Economopoulos T (2008) Primary extranodal lymphomas of stomach: clinical presentation, diagnostic pitfalls and management. Ann Oncol 19(12): 1992–1999

Incidence and Characteristics of Non-Hodgkin’s Lymphomas in HCV-Positive Patients with Mixed Cryoglobulinemia

35

Pietro Enrico Pioltelli, Giuseppe Monti, Maurizio Pietrogrande, and Massimo Galli

The link between mixed cryoglobulinemia and aggressive lymphoproliferative disorders has a relatively long history. It begins in 1975 when, in the same issue of the New England Journal of Medicine in which Lukes described immunoblastic lymphadenopathy, Schultz reported a case of this disorder in a patient positive for cryoproteins, but with low complement levels, after repeated treatment with a liver extract [1]. Subsequently, the usual finding of nodular lymphoid infiltrates, clonally restricted, in the liver and bone marrow of cryoglobulinemic patients prompted some authors to classify this disorder as very low grade lymphoproliferative disease [2–4]. This perspective was supported by the frequent case reports correlating HCV positivity with the cryoglobulinemic syndrome and the nonHodgkin lymphomas [5–12]. Nevertheless, this theoretical approach conflicted with several features of the syndrome, as these patients suffer mainly from vasculitis, with organ damage resulting from immune complex deposition via phlogistic pathways, and only a minor proportion developed and eventually died from malignancy [13]. Hepatitis C virus (HCV) proteins bind to selected receptors on naïve lymphocytes thereby inducing antiapoptotic and clonogenic effects, such as the enhanced production of interleukin-6 and BLyS (B lymphocyte stimulator) [14–18], but this proliferative response is clonal only in a subset of patients [19] and is more often oligoclonal [20]. Furthermore, when overt lymphoma is found, the neoplastic cells do not permit

P.E. Pioltelli (*) Hematology Unit, Ospedale San Gerardo, Monza, Italy e-mail: [email protected]

viral replication, which is instead limited to neighboring cells [20, 21]. Thus, while the initial response to HCV in HCV-related cryoglobulinemia seems to involve immune pathways, it is the presence of metabolic abnormalities that ultimately fosters a neoplastic shift. Specifically, stimulatory and anti-apoptotic cytokines are over-secreted or their genes are overexpressed [16, 18, 22, 23] and the cognate B-cell receptor (BCR) for the HCV proteins is expressed. Furthermore, patients with certain MHC restriction patterns and HCV-related cryoglobulins are more likely to develop a lymphoma [24]. These features suggest the presence of a genetic background favoring an abnormal response to a persistent antigenic trigger, at the level of an immunological response as well as with respect to proliferative pattern. Moreover, while patients with mixed cryoglobulinemia may be able to overcome HCV infection, there is still an enhanced risk of NHL that is no longer associated with HCV infection [25–27]. While HCV infection seems to be linked to a specific subset of lymphoproliferative disorders, data definitively proving this association are lacking. However, there is general agreement that the more differentiated histotypes are more frequently observed, such as villous splenic lymphoma. Some of these associations have also been reported in cryoglobulinemic patients, albeit anecdotally. Indeed, this could not be confirmed in an evaluation of a larger series [28]. Instead, the histological representation in cryoglobulinemic patients with clinically relevant diseases did not differ from that of the general population of lymphoma patients. However, the conclusions of that study are biased by inadequate statistics as the considered population had been collected retrospectively from

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many centers and included patients observed over a wide time interval and submitted to a broad range of diagnostic and therapeutic tools. Furthermore, the characteristics of the study population itself limited a sound evaluation of treatment outcome, as there were frequent and heterogeneous co-morbidities in addition to the advanced age of many of the included patients. Nevertheless, the evidence supported an increased incidence of lymphoma, compared to either a general age-matched population or to HCV-infected patients without cryoglobulinemia. These data provide epidemiological support to the laboratory findings regarding the specific behavior of  the immune system, in which there is a unique response  to chronic antigenic challenge, with evolution first to an immune-complex-mediated illness and then to a monoclonal neoplastic disease. The serendipitous presence of a well-defined triggering antigen and easily detectable immune complexes has provided insight into lymphomagenesis in the context of a peculiar genetic background of the patient. By following this complicated path of clinical and biological observations, we will be able to confirm the hypothesis proposed by Shultz in his seminal paper published more than a quarter of a century ago.

References 1. Schultz DR, Yunis AA (1975) Immunoblastic lymphadenopathy with mixed cryoglobulinemia. A detailed case study. N Engl J Med 292(1):8–12 2. Monteverde A, Rivano MT, Allegra GC et al (1988) Essential mixed cryoglobulinemia, type II: a manifestation of a lowgrade malignant lymphoma? Clinical-morphological study of 12 cases with special reference to immunohistochemical findings in liver frozen sections. Acta Haematol 79(1):20–25 3. Mussini C, Mascia MT, Zanni G et al (1991) A cytomorphological and immunohistochemical study of bone marrow in the diagnosis of essential mixed type II cryoglobulinemia. Haematologica 76(5):389–391 4. Monteverde A, Sabattini E, Poggi S et al (1995) Bone marrow findings further support the hypothesis that essential mixed cryoglobulinemia type II is characterized by a ­monoclonal B-cell proliferation. Leuk Lymphoma 20(1–2): 119–124 5. Pozzato G, Mazzaro C, Crovatto M et al (1994) Low-grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia. Blood 84(9):3047–3053 6. Rasul I, Shepherd FA, Kamel-Reid S et al (1999) Detection of occult low-grade b-cell non-Hodgkin’s lymphoma in patients with chronic hepatitis C infection and mixed cryoglobulinemia. Hepatology 29(2):543–547

P.E. Pioltelli et al. 7. Rubbia-Brandt L, Bründler MA, Kerl K et al (1999) Primary hepatic diffuse large B-cell lymphoma in a patient with chronic hepatitis C. Am J Surg Pathol 23(9):1124–1130 8. Chowla A, Malhi-Chowla N, Chidambaram A et al (1999) Primary hepatic lymphoma in hepatitis C: case report and review of the literature. Am Surg 65(9):881–883 9. Licata A, Pietrosi G, Rizzo A et  al (2003) Disseminated non-Hodgkin’s lymphoma and chronic hepatitis C: a case report. Ann Ital Med Int 18(4):246–249 10. Lizardi-Cervera J, Poo JL, Romero-Mora K et  al (2006) Hepatitis C virus infection and non-Hodgkin’s lymphoma: a review and case report of nine patient. Ann Hepatol 5(4): 257–262 11. Vincent D, Gombert B, Vital A et  al (2007) A case of mononeuropathy multiplex with type II cryoglobulinemia, necrotizing vasculitis and low grade B cell lymphoma. Clin Neuropathol 26(1):28–31 12. Krishnan C, Cupp JS, Arber DA et al (2007) Lymphoplasmacytic lymphoma arising in the setting of hepatitis C and mixed cryoglobulinemia. J Clin Oncol 25(27):4312–4314 13. Ferri C, Sebastiani M, Giuggioli D et al (2004) Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 33(6):355–374 14. Machida K, Cheng KT, Pavio N et  al (2005) Hepatitis C virus E2-CD81 interaction induces hypermutation of the immunoglobulin gene in B cells. J Virol 79(13):8079–8089 15. Rosa D, Saletti G, De Gregorio E et al (2005) Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci USA 102(51):18544–18549 16. Feldmann G, Nischalke HD, Nattermann J et  al (2006) Induction of interleukin-6 by hepatitis C virus core protein  in hepatitis C-associated mixed cryoglobulinemia and B-cell non-Hodgkin’s lymphoma. Clin Cancer Res 12(15): 4491–4498 17. Landau DA, Saadoun D, Calabrese LH et  al (2007) The pathophysiology of HCV induced B-cell clonal disorders. Autoimmun Rev 6(8):581–587 18. Landau DA, Rosenzwajg M, Saadoun D et al (2009) The B lymphocyte stimulator receptor-ligand system in hepatitis C virus-induced B cell clonal disorders. Ann Rheum Dis 68(3):337–344 19. Charles ED, Green RM, Marukian S et al (2008) Clonal expansion of immunoglobulin M + CD27+ B cells in HCV-associated mixed cryoglobulinemia. Blood 111(3):1344–1356 20. De Vita S, De Re V, Gasparotto D et al (2000) Oligoclonal non-neoplastic B cell expansion is the key feature of type II mixed cryoglobulinemia: clinical and molecular findings do not support a bone marrow pathologic diagnosis of indolent B cell lymphoma. Arthritis Rheum 3(1):94–102 21. De Vita S, Sansonno D, Dolcetti R et al (1995) Hepatitis C virus within a malignant lymphoma lesion in the course of type II mixed cryoglobulinemia. Blood 86(5):1887–1892 22. Alisi A, Giannini C, Spaziani A et  al (2007) Hepatitis C virus core protein enhances B lymphocyte proliferation. Dig Liver Dis 39(Suppl 1):S72–S75 23. Fabris M, Quartuccio L, De Re V et al (2008) Fibronectin gene polymorphisms and clinical manifestations of mixed cryoglobulinemic syndrome: increased risk of lymphoma

35  Incidence and Characteristics of Non-Hodgkin’s Lymphomas in HCV-Positive Patients with Mixed Cryoglobulinemia 275 associated to MspI DD and HaeIII AA genotypes [in Italian]. Reumatismo 60(1):28–34 24. De Re V, Caggiari L, Monti G et  al (2010) HLA DR-DQ combination associated with the increased risk of developing human HCV positive non-Hodgkin’s lymphoma is related to the type II mixed cryoglobulinemia. Tissue Antigens 75(2):127–135, Epub 2009 Dec 9 25. Rosas SE, Tomaszewski JE, Feldman HI et  al (1999) Membranoproliferative glomerulonephritis type I, mixed cryoglobulinemia and lymphoma in the absence of hepatitis C infection. Am J Nephrol 19(5):599–604 26. Díaz-Peromingo JA, García-Suárez F, Saborido-Froján J et al (2001) Mixed cryoglobulinaemia and B-cell ­lymphoma

in the absence of hepatitis C virus infection. Haematologia (Budap) 31(3):225–230 27. Saadoun D, Sellam J, Ghillani-Dalbin P et  al (2006) Increased risks of lymphoma and death among patients with non-hepatitis C virus-related mixed cryoglobulinemia. Arch Intern Med 166(19):2101–2108 28. Monti G, Pioltelli P, Saccardo F et al (2005) Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed Cryoglobulinemias. Arch Intern Med 165(1): 101–105

Waldenström’s Macroglobulinemia Associated with Cryoglobulinemia: Pathogenetic, Clinical, and Therapeutic Aspects

36

Meletios A. Dimopoulos and Efstathios Kastritis

36.1

Introduction

Waldenström’s macroglobulinemia (WM) is a distinct B-cell lymphoproliferative disorder that is characterized by the infiltration of bone marrow with lymphoplasmacytic cells and by an IgM monoclonal gammopathy. WM is considered to be a lymphoplasmacytic lymphoma as defined by the REAL and WHO classification systems [1]. To establish the diagnosis of WM, a monoclonal IgM must be demonstrated by immunofixation [1]. The bone marrow is infiltrated by a lymphoplasmacytic cell population consisting of small lymphocytes with evidence of plasmacytoid/ plasma cell differentiation. Immunophenotypic studies (flow cytometry and/or immunohistochemistry) typically show sIgM+CD19+CD20+CD22+CD79+ [1]. In up to 20% of cases the expression of CD5, CD10, or CD23 is detected. In these patients, care should be taken to satisfactorily exclude chronic lymphocytic leukemia and mantle cell lymphoma. An increased number of mast cells, usually in association with the lymphoid aggregates, is commonly found in WM and their presence may help in differentiating WM from other B-cell lymphomas [2, 3]. In the USA, and throughout the world, WM is an infrequent disease, affecting approximately 1,500 Americans each year; it is approximately 10–20% as common as multiple myeloma and there is a slight male preponderance. The median age of WM patients is more than 65 years, and the disease is significantly M.A. Dimopoulos (*) Department of Clinical Therapeutics, University of Athens School of Medicine, Athens, Greece e-mail: [email protected]

more common among whites than blacks [4]. The etiology of WM is unknown, but a genetic predisposition has been suggested by the identification of family clusters and by the detection of the disease in monozygotic twins [5–8]. A role of HCV infection has also been proposed by some investigators but not supported by others [6, 9–12]. The clinical manifestations of WM are the result of: (a) direct infiltration of the bone marrow by the clonal cells, which may cause cytopenias, most commonly anemia and thrombocytopenia; (b) infiltration of lymphoid organs, such as lymph nodes or spleen, resulting in lymphadenopathy or splenomegaly; and (c) the amount as well as the physicochemical and immunologic properties of the monoclonal IgM immunoglobulin [13]. The properties of IgM give rise to a variety of symptoms and signs that characterize WM, such as hyperviscosity syndrome, in which there is abundant “heavy” IgM, immune reactions and complement activation, deposition of immunoglobulin fragments, or, under certain physicochemical conditions, precipitation of intact immunoglobulins [14, 15]. The term cryoglobulins refers to immunoglobulin molecules that precipitate at temperatures below 37°C. Based on the combinations of immunoglobulin types and their properties, Brouet established a classification which, although it dates back to 1974, is still in use [16, 17]. Thus, type I cryoglobulins are composed of a single monoclonal immunoglobulin, usually an IgM paraprotein, less frequently IgG and occasionally light chains only. Rarely, they belong to the IgA isotype and behave as pyroglobulins, precipitating irreversibly at 50°C. Type II cryoglobulins, the most common type, are characterized by a polyclonal fraction, mainly IgG, and a monoclonal IgM fraction that has rheumatoid

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factor activity, i.e., against polyclonal IgG. The monoclonal fraction is rarely represented by IgA or IgG. Most IgM reacts with both intact IgG and the F(ab)2 fragment as well as with the Fc fragment of autologous IgGs. Type III cryoglobulins are characterized by polyclonal IgG and IgM, although with high-resolution methods a small monoclonal component is frequently detected [18–20]. Cryoglobulinemia was described first in a myeloma patient by Wintrobe and Buell in 1933. Jan Waldenström was the first to describe WM, in 1944, in a report describing two patients with oronasal bleeding, severe anemia, lymphadenopathy, hypofibrinogenemia, elevated erythrocyte sedimentation rates, and large amounts of a high-molecular-weight gamma globulin in the serum; one of these two patients had cryoglobulins. Both type I and type II cryoglobulins may be associated with WM. About 10–20% of patients with the diagnosis of WM have cryoglobulins, but clinically evident type I cryoglobulinemia causing Raynaud’s phenomenon, palpable purpura, or glomerulonephritis occurs in less than 5% of patients [21–27].

36.2

Pathogenesis of Cryoglobulinemia in WM

Although most WM patients with circulating cryoglobulins are asymptomatic, cryoglobulins are often important in the pathogenesis of disease findings [4]. In type I cryoglobulinemia, the large molecule of monoclonal IgM typically precipitates at temperatures well below 37°C; however, in some cases, the cryoglobulin may precipitate or form a gel even a few degrees or just below 37°C. In type I cryoglobulinemia, the levels of monoclonal IgM are usually high and there may be an easily visible turbidity or precipitate [4, 20, 28]. Precipitation may take place quite rapidly during the blood-drawing procedure or it may be incidentally detected, during other laboratory manipulations of the blood sample or due to interference with other laboratory tests. A usually large peak in the gamma region is detectable on serum electrophoresis [18–20, 24, 29]. Electrophoresis of the precipitate similarly reveals a large gamma peak but the albumin and other peaks are much shorter than those obtained by the electrophoresis of whole serum. Immunofixation will reveal the monoclonal protein type, which in WM is of the IgM class. Type II cryoglobulinemia can also be associated with WM. In this case, monoclonal IgM acts

as rheumatoid factor and binds to the Fc portion of polyclonal IgG. Hepatitis C virus (HCV) infection is the most common factor associated with type II cryoglobulinemia [30]. Infection in patients with type II disease results in a spectrum of lymphoproliferative responses that are usually limited but may become overtly malignant, usually low grade non-Hodgkins’s lymphomas, in about 10% of patients [9, 11, 24, 31, 32]. WM is a frequent, but not the only, B-cell nonHodgkin lymphoma associated with HCV and mixed cryoglobulinemia. In typical non-WM-related type II cryoglobulinemia, serum electrophoresis does not usually reveal a clear monoclonal peak, which is detected only following electrophoresis of the precipitate. However, in the case of WM-related type II cryoglobulinemia, a monoclonal peak may also be detectable by serum electrophoresis and it will be more prominent following electrophoresis of the cryoprecipitate. In general, the IgM monoclonal peak is larger than in non-WM type II cryoglobulinemia. A bone marrow biopsy is fundamental in establishing the diagnosis of WM. Coexistence of HCV infection has been reported [10, 33–35]. Whether HCV infection with type II cryoglobulinemia precedes WM development or whether type II cryoglobulinemia develops in an HCV-infected patient whose condition eventually is complicated by the development of WM may not always be clear. Although the symptoms and signs are not able to differentiate among the two types, the pathogenesis of the manifestations of type I and type II cryoglobulinemia may differ significantly. In type I cryoglobulinemia, the signs and symptoms are related to the physicochemical properties of the monoclonal IgM. In this case, immune phenomena do not develop as IgM does not form immune complexes and does not activate complement [25, 36]. Rather the symptoms are related to the occlusion of small vessels due to the precipitation of the large IgM molecules at temperatures below 37°C. Accordingly, the severity of the symptoms is related to the amount of the IgM and to the “cold sensitivity” of the protein. Thus, in some cases in which IgM precipitates at temperatures just below 37°C, digital Raynaud’s phenomenon in the hands, feet, nose, and ear may be prominent, leading even to peripheral gangrene [4]. The kidney is also another common site of IgM cryglobulin precipitation, a consequence not only of the lower temperatures but also of the specific condition of the renal microenvironment, with its extreme osmotic conditions, pH, urea content, and salt concentration [37]. The kidney’s ultrafiltration process

36

Waldenström’s Macroglobulinemia Associated with Cryoglobulinemia

results in high concentrations of IgM in the capillary lumen thereby facilitating local deposition of the immunoglobulin. IgM can precipitate on the endothelial side of the glomerular basement membrane, occlude the capillary lumen, and cause nonselective proteinuria [38]. These lesions usually do not trigger any glomerular proliferation, may be asymptomatic, and are usually reversible. However, mesangial hypercellularity along with endocapillary proliferation is seen in some cases. Glomerular capillaries may have intraluminal PASpositive deposits; interstitial fibrosis is not extensive and intratubular casts are absent. Monoclonal cryoglobulinemia is characterized by dense intracapillary, subendothelial, and mesangial deposits organized as fibrils or microtubules, similar to AL amyloidosis or immunotactoid glomerulopathy. However these deposits are Congo-red negative [39]. A few patients have been described in whom IgM behaved as an antibody against the glomerular basement membrane, resulting in an immune-mediated glomerulonephritis, manifested as nephritic or nephrotic syndrome [40, 41]. Symptoms of peripheral neuropathy are also associated with type I cryoglobulinemia, although less often than in patients with type II cryoglobulinemia. The pathogenesis of cryoglobulinemic neuropathy in type I cryoglobulinemia is due to perineural small-vessel occlusion rather that vasculitis, as in type II cryoglobulinemia [42, 43]. Nerve biopsy will usually show perivascular inflammatory cuffing with axonal degeneration, which, if focal, may suggest ischemia. The pathogenesis of symptoms in type II cryoglobulinemia is related both to the immune phenomena that develop in response to the precipitation of IgM-IgG immune complexes in the small vessels and to complement activation. Vasculitis of small and medium-sized vessels is the most common histologic finding. The severity of the vasculitic manifestations in type II cryoglobulinemia is rather associated with the “thermal amplitude,” i.e., the temperature at which the immune complex precipitates, rather than the serum level of cryoglobulins (cryocrit) or complement. The skin is most commonly involved by palpable purpura, and leukocytoclastic vasculitis is a characteristic histologic finding. The kidneys are often affected and kidney damage may be severe and irreversible. Renal damage results from the activation of complement by the immune precipitates within the glomeruli and is characterized by proliferative glomerular damage. Membranoproliferative glomerulonephritis with subendothelial deposits is almost always observed in

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kidney biopsies from patients with type II non-HCVrelated mixed cryglobulinemia. Peripheral neuropathy is also common and is the result of vasculitis of the perineural vessels. Fatigue and weakness as well as arthralgias are frequently seen and are due to the widespread vasculitis. Liver involvement outside the context of HCV infection may also be detected.

36.3

Clinical Aspects

While the clinical features of types I and II cryoglobulinemia may overlap, in type I cryoglobulinemia the clinical picture may be dominated by symptoms and signs related to the overt malignancy (such as anemia, thrombocytopenia, organomegaly). Type I cryoglobulins do not easily activate complement and patients may be asymptomatic until the level of cryoglobulinemia is sufficiently high to cause hyperviscosity syndrome, resulting in a non-inflammatory vasculopathy. Thus, at this stage type I cryoglobulins are rather associated with signs and symptoms related to peripheral vessel occlusion. Typical manifestations of hyperviscosity syndrome include fatigue, bleeding from the gums and nose, and ocular, neurologic, and cardiovascular complications (oral mucosal bleeding, headaches, etc.). Symptoms usually appear when the relative serum or plasma viscosity is above 5 cp (normal values: 1.4–1.8 cp); in such cases, the corresponding serum IgM is usually >3 g/dL However, depending on the properties of the monoclonal type I IgM cryoglobulin, the symptomatic threshold may be significantly lower [4, 28]. Manifestations indicating the presence of cryoglobulins may include purpuric lesions, acrocyanosis, Raynaud phenomena, dystrophic manifestations, and the formation of torpid ulcers and gangrene. Cryoglobulinemic neuropathy of type I cryoglobulinemia is characterized by a distal, sensory, symmetric polyneuropathy or with mononeuropathy multiplex, which is often axonal. Symptoms of peripheral neuropathy may be the presenting symptom and indicate the need for treatment initiation even when other symptoms (such as cytopenia or symptomatic organomegaly) are lacking [44–47]. Central nervous system involvement is rare, although hemorrhagic vasculopathic encephalopathy associated with a type I cryoglobulinemia has been reported [48]. Renal involvement in type I cryoglobulinemia is manifested mainly in the form of albuminuria. Since there is no inflammatory reaction within the glomeruli,

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there should be no active urine sediment. However, in the presence of proliferative glomerular damage, a nephritic picture with hematuria and red cell casts may be seen. In patients with significant albuminuria, the possibility of renal amyloidosis should be considered. The presence of rapidly progressive glomerulonephritis should raise suspicion of cryoglobulinemia with immune activation and proliferative glomerular damage [10, 37, 40, 41, 49–51]. There are also a variety of cutaneous manifestations that may occur in the context of type I cryoglobulinemia, including palpable purpura, ischemic necrosis, cutaneous ulcerations, livedo reticularis infarctions, cold urticaria, inflammatory macules and papules, Raynaud phenomenon, scarring of the nose, pinnae, fingertips, and toes, and hyperkeratotic spicules on acral surfaces [4, 24, 28]. The clinical syndrome of type II cryglobulinemia (mixed cryoglobulinemia syndrome) is characterized by a typical clinical triad of purpura, weakness, and arthralgias. However, multi-system organ involvement, including chronic hepatitis, membranoproliferative glomerulonephritis, and peripheral neuropathy due to leukocytoclastic vasculitis of small and medium-sized vessels is common. Different clinical patterns may be present at the initial diagnosis. In asymptomatic patients, isolated serum mixed cryoglobulins may be the only feature and probably expresses early-stage disease while other patients may present with a complete cryoglobulinemic syndrome characterized by the combination of serological findings and clinicopathological features. Mixed cryglobulinemia syndrome may initially appear as an incomplete form, gradually evolving during long-term follow-up into an overt syndrome. In some patients, the symptoms of WM may dominate, with mixed cryglobulinemia becoming overt during the course of the disease, while in others clinically overt type II cryoglobulinemia may not be associated with the additional features of WM, except the clinicopathological findings in the bone marrow. All WM patients with cold-sensitive symptoms should be tested for the presence of cryoglobulins.

36.4

Treatment Options

Plasmapheresis/plasma exchange is very effective for the immediate reduction of the amount of circulating cryoglobulins [4, 24, 36, 52]. Circulating

levels of mixed cryoglobulins and monoclonal IgM cryoglobulins are more easily reduced than those of IgG cryoglobulins [53]. A dramatic response may be seen even after just one plasmapheresis procedure, but the effects may be temporary if it is not repeated at scheduled intervals. For some patients with type I cryoglobulinemia and symptomatic hyperviscosity syndrome, normal saline should be used instead of albumin as replacement fluid. It is important that the albumin or saline is pre warmed to 37°C in order to avoid exacerbation of the symptoms due to a decrease in core temperature, thus leading to cryoprecipitation. Blood transfusions should be avoided if possible; commonly, the hematocrit is factitiously low due to the high IgM levels, which lead to blood volume expansion. In any case, any blood product should be pre-warmed to 37°C. Cascade filtration is as effective as plasma exchange, and both can remove 50% of plasma IgM and reduce plasma viscosity by 60% [54]. Although cascade filtration selectively removes macroglobulins, it is not more effective than plasma exchange [55]. On-line cryopheresis, using special filters with an average pore size of 0.2 mm, was previously used but the technique is hindered by quick plugging of the filter, thus requiring frequent backwashing or filter changes, and its use therefore cumbersome [56]. Cryofiltration apheresis using a high-capacity cryofilter is the most selective procedure to remove cryoglobulins and is specific for the treatment of cryoglobulinemia [57]. In some patients with WM and cryoglobulinemia, the tumor load at presentation is low, anemia and organomegaly are absent, and the main discomfort is caused by the cryoglobulins. In such cases, an intensive series of plasma exchange may rapidly reduce the monoclonal protein and provide immediate symptomatic improvement. However, plasma exchange alone does not usually lead to the prolonged remission of symptoms, and systemic treatment is needed in almost all of these patients. Recently, treatment recommendations for patients with symptomatic WM were formulated and published by a panel of experts. They provide specific recommendations for initial and salvage treatment of patients with symptomatic WM [52]. The number of patients who are diagnosed with asymptomatic WM is increasing. These patients have the typical features of WM on bone marrow biopsy and a monoclonal IgM that may be quite large; however,

36

Waldenström’s Macroglobulinemia Associated with Cryoglobulinemia

they do not have symptoms reflecting WM. Specifically, these patients do not have anemia or thrombocytopenia but may have lymphadenopathy or some splenomegaly, although neither is symptomatic. According to the consensus criteria, these patients should be followed without treatment; rather, initiation of treatment should only be considered for patients with symptoms due to WM and not on the basis of the amount of IgM per se [58]. However, in patients presenting with symptoms due to the cryoglobulinemic properties of the monoclonal IgM, treatment should be considered even when other features of the disease, such as cytopenia or lymphadenopathy, are absent or when the levels of monoclonal IgM are low. Treatments targeting the IgM-producing clone of WM may take weeks to effectively reduce the amount of circulating immunoglobulin. After the initiation of plasma exchange, anti-lymphoma treatment should be started as soon as possible. A highly effective regimen should be used preferably; single agent agents such as chlorambucil are not indicated. The single agent rituximab is also not recommended for patients suffering symptomatic type I cryoglobulinemia, due to the slow response, and an increase in IgM levels may follow its administration. This surge of IgM does not indicate treatment failure, but in patients with cryoglobulinemia and hyperviscosity it may exacerbate symptoms. Indeed, after initiation of rituximab, exacerbations of symptoms or complications related to cryoglobulinemia in patients without previous symptoms of the disease have been reported [59]. Thus, close follow-up is needed, at least during the first months after initiation of rituximab. Bortezomib has shown activity in WM patients with relapsed/refractory disease and in newly diagnosed patients. It has the advantage that it rapidly reduces the levels of IgM and thus may be a useful option for some patients with symptoms of hyperviscosity. However, bortezomib is neurotoxic and in patients with cryoglobulinemic neuropathy its use may be contraindicated. Nucleoside analogues are very effective and may improve symptomatic cryoglobulinemic neuropathy but these drugs are associated with immunosuppression; in younger patients who may be candidates for autologous stem cell transplantation, they should probably be deferred. According to recent recommendations, a combination of chemotherapy and immunotherapy based on the monoclonal antibody rituximab is the preferable regimen for newly diagnosed previously untreated

281

patients. CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) combined with rituximab or the combination of DRC (dexamethasone, rituximab, cyclophosphamide) may be used. Time to response varies between 1 and 4 months, and repeated plasmapheresis may be needed. Bortezomib, as a single agent, may be used in some cases due to its rapid reduction of IgM levels. Supportive measures are also very important for patients suffering from symptoms of cryoglobulinemia. Thus, both the avoidance of exposure to cold temperatures and the use of gloves, hats, etc., should be encouraged. A warm room, or warming blankets in some cases, could be useful in reducing symptoms and complications [4, 50]. In WM patients with type II cryoglobulinemia, manifestations of the disease are triggered by the immunologic properties of the IgM-IgG complex and the amounts of IgM are not as high as in type I cryoglobulinemia. Plasmapheresis may not be as effective since even small amounts of the IgM-IgG complex can trigger vasculitis. Rituximab has been used successfully to target the B-cell clone and to reduce the immunologic phenomena of type II cryoglobulinemia. Steroids with or without cyclophosphamide have also been used as an immunosuppressive adjunct. Patients with WM and symptomatic type II cryoglobulinemia with an underlying HCV infection may need special management. Rituximab targets the WM clone and has shown activity in refractory HCV-related type II cryoglobulinemia, although it is mostly effective in the treatment of skin features and less so in renal complications of the syndrome. A flare of HCV RNA load has been reported in some patients, without deterioration of liver biology [60–71]. However, there are limited data on the use of chemotherapy combinations with rituximab in patients with HCV and symptomatic WM with or without cryoglobulinemia [72].

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41. Meyrier A, Simon P, Mignon F et al (1984) Rapidly progressive (‘crescentic’) glomerulonephritis and monoclonal gammapathies. Nephron 38(3):156–162 42. Ropper AH, Gorson KC (1998) Neuropathies associated with paraproteinemia. N Engl J Med 338(22):1601–1607 43. Garcia-Bragado F, Fernandez JM, Navarro C et al (1988) Peripheral neuropathy in essential mixed cryoglobulinemia. Arch Neurol 45(11):1210–1214 44. Meier C (1985) Polyneuropathy in paraproteinaemia. J Neurol 232(4):204–214 45. Vital C, Vallat JM, Deminiere C et al (1982) Peripheral nerve damage during multiple myeloma and Waldenstrom’s macroglobulinemia: an ultrastructural and immunopathologic study. Cancer 50(8):1491–1497 46. Nobile-Orazio E (2004) IgM paraproteinaemic neuropathies. Curr Opin Neurol 17(5):599–605 47. Vital A, Vital C, Ragnaud JM et al (1991) IgM cryoglobulin deposits in the peripheral nerve. Virchows Arch 418(1): 83–85 48. Mazzola L, Antoine JC, Camdessanche JP et al (2003) Brain hemorrhage as a complication of type I cryoglobulinemia vasculopathy. J Neurol 250(11):1376–1378 49. Zlotnick A, Rosenmann E (1975) Renal pathologic findings associated with monoclonal gammopathies. Arch Intern Med 135(1):40–45 50. Shaikh A, Habermann TM, Fidler ME et al (2008) Acute renal failure secondary to severe type I cryoglobulinemia following rituximab therapy for Waldenstrom’s macroglobulinemia. Clin Exp Nephrol 12(4):292–295 51. Yonemura K, Suzuki T, Sano K et al (2000) A case with acute renal failure complicated by Waldenstrom’s macroglobulinemia and cryoglobulinemia. Ren Fail 22(4): 511–515 52. Dimopoulos MA, Gertz MA, Kastritis E et al (2009) Update on treatment recommendations from the Fourth International Workshop on Waldenstrom’s Macroglobulinemia. J Clin Oncol 27(1):120–126 53. Berkman EM, Orlin JB (1980) Use of plasmapheresis and partial plasma exchange in the management of patients with cryoglobulinemia. Transfusion 20(2):171–178 54. Valbonesi M, Montani F, Guzzini F et al (1985) Efficacy of discontinuous flow centrifugation compared with cascade filtration in Waldenstrom’s macroglobulinemia: a pilot study. Int J Artif Organs 8(3):165–168 55. Hoffkes HG, Heemann UW, Teschendorf C et al (1995) Hyperviscosity syndrome: efficacy and comparison of plasma exchange by plasma separation and cascade filtration in patients with immunocytoma of Waldenstrom’s type. Clin Nephrol 43(5):335–338 56. Yamashita M, Malchesky PS, Omokawa S et al (1990) Limitation of plasmapheresis in cryoglobulinemia with high levels of cryoglobulins. Prog Clin Biol Res 337:491–494 57. Siami GA, Siami FS (2001) Current topics on cryofiltration technologies. Ther Apher 5(4):283–286 58. Kyle RA, Treon SP, Alexanian R et al (2003) Prognostic markers and criteria to initiate therapy in Waldenstrom’s

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Part VII Therapy of Cryoglobulinemia

Should HCV-Positive Asymptomatic Patients with Mixed Cryoglobulinemia Be Treated with Combined Antiviral Therapy?

37

José Luis Calleja Panero, Juan de la Revilla Negro, and Fernando Pons Renedo

37.1

Introduction

The term “cryoglobulinemia” refers to the presence of cryoglobulins in the serum. While cryoglobulinemia is frequently an asymptomatic condition, the deposition of circulating immune complexes, mainly cryoglobulins, and complement in the vascular lumen can cause a leukocytoclastic type of vasculitis called cryoglobulinemic vasculitis. Mixed cryoglobulinemia (MC) syndrome follows a benign clinical course in over 50% of patients, but a moderate-severe clinical course is observed in one-third of patients, with their prognosis severely affected by renal and/or liver failure. Before the discovery of HCV, MC was considered an essential disease. In 1990, three cases of HCVassociated MC were described [1]. This was followed by an increasing number of studies that ultimately established a crucial role for HCV in the pathogenesis of MC. Indeed, between 50% and 92% of patients with MC are HCV-positive. However, the incidence of detectable cryoglobulins in these patients is only 40–50% [2] and, within this group, only 5% will develop overt MC syndrome [3]. Some studies claim that the characteristics of patients with HCV-associated MC differ from those of HCV patients without cryoglobulinemia. The prevalence of female sex is higher, the average age at diagnosis is older, the duration of liver disease is longer,

J.L.C. Panero (*) Gastroenterology and Hepatology Department, Hospital Universitario Puerta de Hierro Majadahonda, Majadahonda, Madrid, Spain e-mail: [email protected]

the serum IgM concentration is higher and rheumatoid factor activity is noted, in addition to a higher prevalence of cirrhosis [2, 4]. In patients with symptomatic HCV-associated MC, the most frequent extrahepatic manifestation is palpable purpura. Anywhere from 10% to 91% of cryoglobulinemic patients suffer from arthralgias. The incidence of renal involvement varies from 8% to 58%, and polyneuropathy from 40% to 70% [5–7].

37.2

Treatment of HCV-Associated Mixed Cryoglobulinemia

The sequelae of symptomatic MC cause significant morbidity and mortality; therefore, this complication of HCV may require treatment independent of the severity of the liver disease. Spontaneous resolution is seen only in rare cases. Before the recognition of viral hepatitis as an etiologic factor, corticosteroids with or without additional immunosuppressives, mainly cyclophosphamide, and sometimes with the addition of intravenous immune globulin or plasmapheresis, were considered the standard therapy [8]. However, in most patients these efforts were only marginally effective and long-term remission was not achieved. Following the discovery of HCV as the etiologic agent of MC, new therapeutic opportunities but also problems emerged. One of the persisting issues is the potentially adverse effects of immunosuppressive therapy consisting of glucocorticoids and cytotoxic drugs on the underlying chronic viral infection. Another concern involves the cornerstone of HCV antiviral therapy, interferon (IFN), which has the potential to exacerbate

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_37, © Springer-Verlag Italia 2012

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autoimmune disease states [9]. In addition, IFN may newly induce vasculitis and worsen certain complications, such as neuropathy. Following the main steps of the etiopathogenetic process, from HCV infection to B-lymphocyte proliferation and to cryoglobulinemic vasculitis, MC can be managed at different levels [3, 10]: • Etiologic treatment: eradication of the causative agent by means of antiviral therapy; this is recommended in all patients with HCV-associated MC. • Pathogenetic treatment: reversal of the immunologic derangement producing circulating immune complexes; this is achieved with immunosuppressive therapy. • Symptomatic therapies: amelioration of the clinical manifestations of the disease, with aid of immunomodulators/anti-inflammatory agents. • In this chapter we analyze the scientific evidence regarding antiviral therapy in HCV-associated MC. Therapeutic effectiveness can be classified in terms of: (a) virological response, based on the concept of sustained virological response (SVR), (b) clinical response, in which there is symptom resolution, and (c) immunologic response, with the reduction or even sustained disappearance of cryoglobulins.

37.2.1 Treatment of MC with IFN Monotherapy The first report of the use of IFN in the treatment of MC was published in 1987, i.e., before the recognition of HCV [11]. Seven patients with refractory essential MC were treated with recombinant IFN-a2a for 4–12 months. A conspicuous reduction of circulating cryoglobulins and a remarkable improvement of the clinical pattern were noted, which seemed to be consistent and prolonged in some patients. The good results obtained with IFN were attributed to its immune regulatory and antiproliferative effects. Another prospective study [12], conducted in 21 patients with severe essential MC unresponsive to immunosuppresive regimens, showed a 77% rate of clinical and immunological response after long-term treatment (from 8 to 30 months) with recombinant IFN-a or natural IFN-b. In that study, the disease in five patients (24%) remained in prolonged remission (18–40 months) after withdrawal of the drug. These studies confirmed that complete remission of symptoms could be achieved in

J.L.C. Panero et al.

some patients with the use of IFN, in contrast to conventional immunomodulatory regimens, which offered only temporary responses. Following evidence of an etiopathogenetic link of HCV with MC, Misiani et al. [13] published a prospective randomized controlled trial in HCV-associated MC patients treated with IFN-a. The authors concluded that the efficacy of the drug in this group of patients was closely related to its antiviral activity, by means of the virological response of HCV-RNA. Quickly, IFN became the therapy of choice for MC. Several studies have addressed IFN monotherapy in MC, but they are not easy to compare due to different IFN doses and types as well as length of treatment. Moreover, they employed less sophisticated virological laboratory procedures and quantitative methods in the determination of viral titers. Additionally, universal criteria for the end of treatment and SVR were not widely applied, which leads to uncertainties in interpretation of the data. The most frequently used IFN is recombinant IFN-a, although in some reports natural interferon or lymphoblastoid IFN-a were preferred. The usual dose of IFN is 3 MU, administered subcutaneously three times a week for 6–12 months. However, induction regimens with daily doses in the first 1–3 months of therapy have been explored. The main studies conducted in patients with HCVassociated MC treated with conventional IFN-a monotherapy are reported in Table 37.1. One of the first questions that the initial trials sought to answer was whether IFN was effective in the control of MC symptoms. In two open trials [16, 17], 44 patients were treated with IFN-a at a dose of 2–5 MU/3 times a week (ttw) for 6–12 months. At the end of treatment, 65–72% of the patients had clinical and immunological responses and in 50% HCV was negative. The clinical benefits used to define response were limited to cutaneous and articular manifestations; renal and neurologic involvement was less sensitive. However, these global beneficial effects were often transient, except for the small proportion of patients achieving a sustained clearance of HCV. In a randomized, crossover-controlled trial [20], 20 patients were assigned to one of two groups and after the first 6 months with or without IFN therapy the patients were crossed over to the other half of the study for an additional 6 months. While all the patients who completed treatment experienced a significant improvement of purpura,

37

Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy?

289

Table 37.1 Published studies in which HCV-associated MC treated with interferon monotherapy Number of Author (year) patients Dammacco (1994) [14] A: 15 B: 17

Casato (1997) [15]

C: 18 D: 15 31

Migliaresi (1995) [16] 24 Akriviadis (1997) [17] 18 Pellicano (1999) [18] 32 HCV MC+ 30 HCV MC− A: 18 Mazzaro (1995) [19] B: 18 23 HCV MC+ Polzien (1997) [4] 36 HCV MC− 20 Ferri (1993) [20]

Cresta (1999) [21] Misiani (1994) [13]

Calleja (1999) [22]

43 HCV MC+ 44 HCV MC− 27

18 HCV MC+ 195 HCV MC−

IFN regimen A: IFN 3 MU/3tw B: IFN 3 MU/3tw + PRED 16 mg/4tw C: PRED 16 mg/day D: No therapy IFN 3 MU/day 3 months + IFN 3 MU/3tw ³9 months IFN 3 MU/3tw IFN 3–5 MU/3tw IFN lymphoblastoid 3 MU/3tw IFN 3 MU/3tw IFN 3 MU/3tw IFN 2 MU/d 1 month + INF 2 MU/3tw 5 months IFN 3 MU/3tw IFN 1.5 MU/3tw 1 week + IFN 3MU/3tw 23 weeks IFN 3 MU/3tw

Duration of therapy 12 months

³12 months

SVR (%) A: 28 B: 28

CR to therapy (%)a A: 53.3 B: 52.9

Sustained CR (%)b A: 25 B: 33

C: 0 D: 0 16

C: 26.7 D: 6.7 62

C: 0 D: 0 48

46 100 –

12.5 11 –

12 months 6–12 months 6–12 months

8 5 HCV MC+: 22 HCV MC−: 20 A: 6 months A: 11 B: 12 months B: 22 6–14 months HCV MC+: 5 HCV MC−:22 6 months 10

A: 28 B: 39 –

–

100

0

6 months

43

21

60

0

55

27

6 months

12 months

HCV MC+: 14 HCV MC−: 18 0

HCV MC+: 27 HCV MC−: 19

–

SVR sustained virological response, IFN interferon, tw times a week, CR (complete response): improvement/reduction or disappearance of symptoms and/or cryoglobulins a CR to therapy: at the end of therapy b Sustained CR: after withdrawal of therapy (on follow-up)

transaminases, and cryoglobulins, a rebound phenomenon involving clinical and serological parameters was observed after IFN discontinuation. Some studies have asked whether the presence of circulating cryoglobulins affects antiviral therapy and whether treatment efficacy is similar in patients with symptomatic and asymptomatic MC [4, 18]. The data collected in these trials unanimously demonstrated that the antiviral effects of IFN were not altered by the presence of cryoglobulins, with SVR rates of 14–35% in the MC group vs. 18–22% in the control HCV group. Although there was a trend for a higher SVR in cryoglobulin-positive patients, this did not reach statistical significance. The influence of symptoms on the virological response was evaluated in only one trial [21]. No differences were detected in virological response but the disappearance of cryoglobulins occurred less frequently in symptomatic patients. Viral and host

predictive factors associated with response to therapy did not differ between the groups; in some studies, a low probability of response has been associated with high cryocrit levels [15, 19]. There have been several attempts to establish an optimal therapeutic regimen in HCV-associated MC. Two studies evaluated the duration of therapy, showing that a prolonged regimen (³1 year of therapy) seems to offer better efficacy, with higher long-term response off therapy, although associated with higher toxicity [15, 23]. For patients who relapse off drug and have a high risk of progressive MC vasculitic disease, longterm IFN maintenance therapy may be an option [24]. In summary, early studies demonstrated that treatment with IFN monotherapy in HCV-associated MC patients is as effective as in HCV patients without MC, with improvement of MC complications linked to viral clearance, normalization of alanine aminotransferase,

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Table 37.2 Published studies in which HCV-associated MC was treated with IFN plus ribavirin combination therapy

Author (year) Calleja (1999)c [22]

Number of patients 13

Zuckerman (2000)c [26] 9 Donada (1998)c [27] Parise (2007) [25] Cacoub (2002) [28]

Schmidt (2001) [29]

17 HCV MC+ 19 HCV MC− 31 HCV MC+ 71 HCV MC− 14

25 HCV MC+ 64 HCV MC−

IFN and ribavirin regimen IFN 3 MU/3tw + Riba 1,200 mg/day IFN 3 MU/3tw + Riba 1,000–1,200 mg/day IFN 6 MU/3tw 3 months + IFN 3

Duration of therapy 12 months

SVR (%) 54

CR to therapy Sustained CR (%)b (%)a 77 54

6 months

22

77

55

6 months

HCV MC+: 38.5d HCV MC−: 35.7 HCV MC+: 29 HCV MC−: 31 64

–

–

–

–

–

71

–

–

IFN 3 MU/3tw + Riba 1,000–1,250 mg/day

6–12 months

IFN 648 MU + Riba 895 ± 250 mge

IFN: 20 ± 14 months Riba: 14 ± 12 monthsf 6–12 months

IFN 3 MU/3tw + Riba 1,000–1,200 mg/day

HCV MC+: 52 HCV MC−: 18

SVR sustained virological response, IFN interferon, Riba ribavirin, tw times a week, CR (complete response): improvement/reduction or disappearance of symptoms and/or cryoglobulins a CR to therapy: at the end of therapy b Sustained CR: after withdrawal of therapy (on follow-up) c Includes only HCV non-responders to and patients with disease relapse after IFN monotherapy d Limited to patients with disease relapse e Median cumulative IFN dose and mean daily Riba dose f Mean duration of IFN and Riba therapy

and reduction of circulating cryoglobulins. The side effects and tolerance profile of IFN in patients with HCV-associated MC are similar to what is seen in the HCV general population, with a treatment discontinuation rate of 10–20% of patients.

37.2.2 MC Treatment with IFN Plus Ribavirin The discovery of the anti-inflammatory effect of ribavirin in HCV-infected patients led to a pilot study in which five patients with symptomatic MC who were HCVpositive but non-responders to a previous IFN course received oral ribavirin alone [25]. Ribavirin therapy resulted in clinical improvement in all patients and a decrease in alanine aminotransferase concentrations but without clearance of the virus. Clinical relapses occurred after drug withdrawal in all patients. Between 1998 and 2007, several studies on combination therapy with IFN and ribavirin in MC patients were published (Table 37.2). Almost all of them used the same doses of IFN-a (3 MU, 3 days weekly) and

weight-adjusted ribavirin doses (15 mg/kg or 1,000– 1,200 mg/day).All trials except one administered the drugs for 6–12 months. Four studies used combination antiviral therapy in naïve HCV-associated MC. Parise et al. [29] compared the virological response of 31 HCV MC patients (8 symptomatic) with that of 102 HCV patients without MC. SVR rates were similar (30%) in the two groups. No differences were found between symptomatic and asymptomatic patients with respect to virological response. In the majority of patients with SVR, cryoglobulins were reduced or eliminated. Another study of similar design obtained different results [28], reporting SVR in HCV-associated MC patients of 52% compared to only 28% in HCVinfected cryoglobulin-negative patients, with no significant differences in the distribution of viral genotypes. Of particular interest was the low virological response to IFN and ribavirin in the control group compared to the response reported in pivotal trials. A cohort of severely symptomatic patients with HCV and systemic vasculitis were treated with longterm IFN and ribavirin (average of 20 months) [30]. SVR was obtained in 66% of patients, with a significant

37

Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy?

improvement in difficult to treat symptoms, such as neurologic and renal involvement. This clinical response could have been related to the longer duration of therapy used in this trial. The immunological response of cryoglobulins to combination therapy was evaluated in another retrospective study of 25 asymptomatic HCV-associated MC patients who reached SVR [26]. Only one of the 25 patients had persistent cryoglobulins at the end of follow up. The results of patients unresponsive to previous courses of IFN therapy are different. As expected, patients without circulating cryoglobulins had a lower virological response, especially null non-responders. The virological response in previous non-responders ranged from 0% to 39%; however, the duration of therapy varied from 6 to 12 months. Two trials investigated IFN-a (3–6 MU/ttw) pus ribavirin (15 mg/kg/day) for 6 months. The first recruited nine severely symptomatic HCV MC patients refractory to previous therapies, including immunesuppressives and IFN [27]. Only 22% of the patients achieved virological, clinical, and immunological sustained responses. The second study compared 17 HCV MC asymptomatic patients with 19 HCV patients without circulating cryoglobulins [22]. Both groups received 3 months of induction therapy with 6 MU of IFN. None of the non-responders and 39% of the relapsers maintained the virological response off therapy. Response rates were very similar in patients with or without serum cryoglobulins. The results of trials employing a longer duration of combination therapy have been better. Calleja [31] treated 13 HCV-associated MC patients, either IFN non-responders or with disease relapse following IFN monotherapy, with 12 months of combination therapy. Seven achieved SVR (54%), demonstrating the superiority of the combination regimen over monotherapy (28%) in the same patient cohort. The response was higher in the relapse group than in the non-responders (80% vs. 38%). The conclusions drawn from this study can be extended to the HCV-associated MC combination therapy experience in general. The presence of cryoglobulins does not affect the response to antiviral treatment since the rate of SVR is similar in patients with or without cryoglobulins. These cold-precipitable proteins can therefore be considered as an epiphenomenon that does not modify the response of HCV to treatment. IFN, either alone or in combination with

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ribavirin, is a safe and effective treatment for HCVassociated MC. Eradication of the virus is linked to an improvement or the disappearance of MC-associated clinical manifestations.

37.2.3 MC Treatment with Pegylated IFN-a Plus Ribavirin The evolution of HCV treatment modalities for HCV has been broadened to include HCV cryoglobulin-positive patients. However, data on the effectiveness of the recently introduced pegylated IFN combination therapy in this subgroup of patients are limited to small pilot studies. Mazzaro [32] treated 18 HCV-symptomatic MC-naïve patients with pegylated IFN-a2b (1 mg/kg/ week) plus ribavirin (1,000–1,200 mg/day) for 48 weeks, regardless of the viral genotype. At the end of therapy, HCV RNA was undetectable in 83% of patients. At the end of follow-up, SVR was determined in 44%. The same results were obtained for clinical (disappearance of purpura and arthralgias) and biochemical (aminotransferase normalization) responses. Only 33% of the patients obtained a complete immunological response (disappearance of circulating cryoglobulins). When genotypes were analyzed separately, higher rates of SVR were obtained with genotype 2 or 3 than with genotype 1 infections (71% vs. 27%). One of the major weaknesses of this study was a pegylated IFN dosage lower than that usually recommended in HCV therapeutic guidelines. A French group published the results of the largest study of combination therapy performed thus far [33, 34]. Their series consisted of 72 unselected HCV MC patients treated with either standard IFN-a2b (n = 32, 3 MU/ttw) or pegylated IFN-a2b (n = 40, 1.5 mg/kg/ week) combined with ribavirin (600–1,200 mg/day) for at least 6 months (mean 16.63 ± 7.8 months). Twenty patients had received prior first-line therapy with standard IFN-a2b, either alone or in combination with ribavirin. After a long-term follow up of 40 months (± 24 months) after discontinuation of antiviral therapy, SVR was determined in 58.3%. There was a higher rate of virological responses in patients receiving pegylated IFN plus ribavirin than in those treated with IFN plus ribavirin (62.5% vs. 53.1%), regardless of viral load or genotype. In addition, with pegylated IFN combination therapy there was a trend toward a

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shorter duration of anti-HCV therapy (13.25 vs. 18.35 months), less frequent concomitant use of corticosteroids (35% vs. 47%), and a lower rate of death (5% vs. 18.8%). Clinical and virological responses were closely correlated, with a rate of complete clinical response of 62.5%. Although a significant decrease in proteinuria was observed in sustained virologic responders, serum creatinine levels did not significantly differ. In fact, renal insufficiency was a negative predictive factor of complete clinical response. In spite of the paucity of treatment data regarding pegylated IFN combination therapy in patients with MC, we can conclude that this regimen is safe and more effective than standard IFN plus ribavirin, with higher virological and clinical sustained responses. This evidence strongly suggests the combination of pegylated IFN and ribavirin as the therapy of choice in patients with HCV-associated MC.

37.2.4 Other Combinations with IFN-Based Therapies 37.2.4.1 MC Treatment with Standard IFN Plus Prednisone The largest source of information available on this topic comes from early publications, when monotherapy with IFN was the only antiviral therapy for HCV infection. Only two studies evaluated the effectiveness of IFN and prednisone combination therapy in HCVassociated MC [14, 35]. Although combination therapy with prednisolone resulted in a more prompt response and delayed relapse, the increasing viremia found with prednisolone and the marginal effects of combined therapy did not warrant the use of this protocol. 37.2.4.2 MC Treatment with Standard IFN Plus Plasmapheresis Plasma exchange has been used to treat life-threatening MC manifestations, such as progressive renal or central nervous system involvement or multivisceral vasculitis. This technique is usually performed three times weekly for several weeks, removing cryoglobulins from the circulation. Plasma exchange should be used in association with others therapies to avoid a rebound in cryoglobulins after the discontinuation of apheresis. It may be a way to buy time while awaiting HCV therapy efficacy. An investigation of the pharmacokinetics of IFN-a during plasma exchange sessions [36] showed increased IFN clearance between the initiation and

J.L.C. Panero et al.

termination of apheresis, which may decrease the pharmacological activity of the treatment. However, large amounts of newly produced virions were introduced into the vascular compartment just after the plasmapheresis-mediated drop in HCV viremia. Taken together, if plasma exchange is used in combination with anti-HCV treatment, IFN should be given after each apheresis session in order to maintain the virological effect of the drug.

37.2.4.3 MC Treatment with Pegylated IFN-Ribavirin Plus Rituximab Rituximab is a monoclonal antibody directed against CD20, a transmembrane protein expressed on pre-B lymphocytes and mature lymphocytes. As such, it interferes with monoclonal IgM production, cryoglobulin synthesis, and the deposition of immunocomplexes. Recently, Ahmed and Wong [37] reviewed the literature of rituximab in HCV MC patients. Most publications consisted of case series and case reports [38, 39], with no randomized controlled trials. Rituximab was used as rescue therapy in almost all patients despite previous treatments with IFN-a and/ or immunosuppressive therapies. In the majority of studies, four weekly doses of rituximab 375 mg/m2 were administered. The majority of patients achieved a complete clinical response with rituximab (73%), and some of the relapsing patients who were rechallenged with the antibody had a good response. A relatively small number of side effects were reported. One potential concern regarding this therapy is rituximab’s ability to increase HCV viremia. However, neither significant variation of serum transaminases nor deterioration of liver disease has been noted in most series [40]. In this setting, rituximab seems to be a suitable rescue therapy in recalcitrant HCVassociated MC but it cannot be considered a curative treatment as long as the viral antigen triggering the vasculitis remains. Based on the limitations of each therapy (antiviral and rituximab), with 20–30% of MC patients remaining symptomatic despite anti-CD20 monoclonal antibody or combined antiviral therapy, the association of rituximab with pegylated IFN-a plus ribavirin appears to be logical. Thus far, one single case and a small case series have been reported. The first publication was in 2003 [41]. A 45-year-old woman diagnosed with HCV MC and presenting with general symptoms, polyneuropathy and a low-grade lymphocytic NHL, was treated initially with standard IFN-a

37

Should HCV+ Asymptomatic Patients with MC Be Treated with Antiviral Therapy?

plus ribavirin, without clinical or virological response. Afterward, she received plasmapheresis combined with cyclophosphamide pulses, but with no control of vasculitis activity. Finally, she was treated with six weekly sessions of rituximab, which yielded an excellent clinical response (including NHL). HCV was eliminated with pegylated IFN-a2b combined with ribavirin for nearly 12 months. The second study [42], published in 2008, assessed 16 patients with severe HCV-MC who were resistant to or had disease relapse after combination treatment with standard or pegylated IFN-a2b plus ribavirin. Treatment consisted of four weekly doses of rituximab followed by pegylated IFN-a2b (1.5 mg/week) plus ribavirin (600–1,200 mg/day) for 12 consecutive months. SVR was achieved in 68.7%, with a complete clinical response in all patients. In 62.5% of the patients, serum cryoglobulins disappeared. After a mean follow-up of 19.4 months, only two patients (12.5%) experienced clinical relapse, associated with the simultaneous reappearance of HCV RNA and cryoglobulin. Peripheral neuropathy and nephropathy, the most challenging complications to treat in MC, achieved a complete response in 38.4% and 57.2% of cases, respectively. Treatment was well tolerated, with only two patients experiencing side effects linked to peg-IFN treatment that required discontinuation of antiviral therapy.

37.3

Conclusions

The eradication of HCV with antiviral therapy should be attempted in all cases of symptomatic HCVassociated MC, irrespective of the severity of liver disease, since even in the absence of SVR there may be marked symptomatic improvement. Although the response rate of the general and skin manifestations is high, the majority of reports describe only a partial improvement in neuropathy and glomerulonephritis. However, a recently published review of therapy in HCV cryoglobulinemic glomerulonephritis recommended antiviral therapy as the initial treatment except in the presence of nephritic-range proteinuria and/or rapidly progressive kidney failure [43]. The clinical response of HCV-associated MC is linked to the disappearance of HCV RNA, while a relapse of symptoms usually follows the reappearance of HCV viremia. However there are two reports in which cryoglobulinemic symptoms recurred despite

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successful antiviral therapy. In some of those patients, recurrence was associated with the development of lymphoproliferative disorders [44, 45]. In HCV-infected patients with circulating cryoglobulins but without clinical involvement, the indications for antiviral therapy are the same as in HCV patients without these immune complexes. Virological responses and tolerance of antiviral therapy in HCVassociated MC patients and in HCV matched controls are similar. Although only scarce evidence is available regarding the use of pegylated IFN plus ribavirin in HCVassociated MC patients, the higher clinical and virological response rates reported thus far make this combination the therapy of choice. Immunosuppression should only be used in MC patients whose symptoms severely impact the quality of life or persist despite SVR [10]. The therapeutic strategies suggested [9] for HCVassociated MC are based on the severity of MC symptoms (Fig. 37.1). Patients with mild to moderate disease severity, i.e., the Meltzer triad, isolated urine analysis alterations, or polyneuropathy, should be treated with pegylated IFN-a plus ribavirin. The duration of therapy has not been rigorously determined, but at least 12 months seems to be mandatory irrespective of HCV genotype. Patients presenting with severe disease, i.e., extensive skin manifestations with deep ulcers or distal necrosis, worsening of renal function, or monopolyneuritis, require an induction phase of immunosuppression while awaiting the initial slow response of antiviral treatments. Traditionally, a combination of high-dose steroids (1.0–1.5 mg/kg/day) plus cyclophosphamide or azathioprine has been used. With the recent availability of biologic B-cell-directed therapy, combination therapy with rituximab followed by pegylated IFN plus ribavirin is a logical strategy, as these drugs target both the viral trigger and the downstream B-cell arm of autoimmunity. For patients with the most fulminant life-threatening presentations, including necrosis involving the extremities, rapidly progressive glomerulonephritis, or central nervous system/systemic organ vasculitis, plasma exchange can bring initial rapid control of the disease but it needs to be combined with immunosuppression (cytotoxic agents, steroids) to avoid a postapheresis rebound of MC. Combination treatment with pegylated IFN and ribavirin should be initiated after the critical phase.

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HCV associated MC

Asymptomatic

Symptomatic

Is there any indication for antiviral therapy based on hepatic disease?

Mild-moderate disease

Life-threatening symptoms

Severe disease

PLASMA EXCHANGE YES

NO

INDUCTION Steroids ± Immunosuppressants ± Rituximab

RITUXIMAB FOLLOW UP PEGYLATED INTERFERON + RIBAVIRIN

2

nd

PHASE

PEGYLATED INTERFERON + RIBAVIRIN

Fig. 37.1 Proposed algorithm for management of patients with hepatitis C virus (HCV)-associated mixed cryoglobulinemia

References 1. Pascual M, Perrin L, Giostra E, Schifferli JA (1990) Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 162:569–570 2. Kayali Z, Buckwold VE, Zimmerman B, Schmidt WN (2002) Hepatitis C, cryoglobulinemia, and cirrhosis: a meta-analysis. Hepatology 36:978–985 3. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 3:25–42 4. Polzien F, Schott P, Mihm S et al (1997) Interferon-a treatment of hepatitis C virus-associated mixed cryoglobulinemia. J Hepatol 27:63–71 5. Schott P, Hartmann H, Ramadori G (2001) Hepatitis C virusassociated mixed cryoglobulinemia. Clinical manifestations, histopathological changes, mechanisms of cryoprecipitation and options of treatment. Histol Histopathol 16:1275–1285 6. Braun GS, Horster S, Wagner KS et al (2007) Cryoglobulinemic vasculitis: classification and clinical and therapeutic aspects. Postgrad Med J 83:87–94 7. Sterling RK, Bralow SP (2006) Extrahepatic manifestations of hepatitis C virus. Curr Gastroenterol Rep 8:53–59 8. Tavoni A, Mosca M, Feri C et al (1995) Guidelines for the management of essential mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl):191S–195S 9. Saadoun D, Delluc A, Piette JC, Cacoub P (2008) Treatment of hepatitis C-associated mixed cryoglobulinemia vasculitis. Curr Opin Rheumatol 20:23–28

10. Kayali Z, LaBrecque DR, Schmidt WN (2006) Treatment of hepatitis C cryoglobulinemia: mission and challenges. Curr Treat Options Gastroenterol 9:497–507 11. Bonomo L, Casato M, Afeltra A, Caccavo D (1987) Treatment of idiopathic mixed cryoglobulinemia with alpha interferon. Am J Med 83:726–730 12. Casato M, Lagana B, Antonelli G et al (1991) Long-term results of therapy with interferon-alpha for type II essential mixed cryoglobulinemia. Blood 78:3142–3147 13. Misiani R, Bellavita P, Fenili D et al (1994) Interferon alfa2a therapy in cryoglubulinemia associated with hepatitis C virus. N Engl J Med 330:751–756 14. Lauta VM, DeSangro MA (1995) Long-term results regarding the use of recombinant interferon alpha-2b in the treatment of II type mixed essential cryoglobulinemia. Med Oncol 12:223–230 15. Casato M, Agnello V, Pucillo LP et al (1997) Predictors of long-term response to high-dose interferon therapy in type II cryoglobulinemia associated with hepatitis C virus infection. Blood 90:3865–3873 16. Migliaresi S, Tirri G (1995) Interferon in the treatment of mixed cryoglobulinemia. Clin Exp Rheumatol 13(Suppl):175S–180S 17. Akriviadis E, Xanthakis I, Navrozidou C, Papadopoulos A (1997) Prevalence of cryoglubulinemia in chronic hepatitis C virus infection and response to treatment with interferonalpha. J Clin Gastroenterol 25:612–618 18. Pellicano R, Marietti G, Leone N et al (1999) Mixed crioglobulinemia associated with hepatitis C virus infection: a

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32. Mazzaro C, Zorat F, Caizzi M et al (2005) Treatment with peg-interferon alpha-2b and ribavirin of hepatitis C virus associated mixed cryoglobulinemia: a pilot study. J Hepatol 42:632–638 33. Cacoub P, Saadoun D, Limal N et al (2005) PEGylated interferon alfa-2b and ribavirin treatment in patients with hepatitis c virus-related systemic vasculitis. Arthritis Rheum 52:911–915 34. Saadoun D, Resche-Rigon M, Thibault V et al (2006) Antiviral therapy for hepatitis C virus-associated mixed cryoglobulinemia vasculitis. Arthritis Rheum 54:3696–3706 35. Dammacco F, Sansonno D, Han JH et al (1994) Natural interferon-alpha versus its combination with 6-methyl-prednisolone in the therapy of type II mixed cryoglobulinemia: a long-term, randomized, controlled study. Blood 84:3336–3343 36. Hausfater P, Cacoub P, Assogba U et al (2002) Plasma exchange and interferon-alpha pharmacokinetics in patients with hepatitis C virus-associated systemic vasculitis. Nephron 91:627–630 37. Ahmed MS, Wong CF (2007) Should rituximab be the rescue therapy for refractory mixed cryoglobulinemia associated with hepatitis C? J Nephrol 20:350–356 38. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101: 3827–3834 39. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon a with an anti-CD20. Blood 101:3818–3826 40. Sansonno D, Tucci FA, Montrone M et al (2007) B-cell depletion in the treatment of mixed cryoglobulinemia. Dig Liver Dis 39(Suppl):116S–121S 41. Lamprecht P, Lerin-Lozano C, Merz H et al (2003) Rituximab induces remission in refractory HCV associated cryoglobulinemic vasculitis. Ann Rheum Dis 62:1230–1233 42. Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinemia vasculitis. Ann Rheum Dis 67:1431–1436 43. Fabrizi F, Lunghi G, Messa P, Martin P (2008) Therapy of hepatitis C virus-associated glomerulonephritis: current approaches. J Nephrol 21:813–825 44. Levine JW, Gota C, Fessler BJ, Calabrese LH, Cooper SM et al (2005) Persistent cryoglobulinemic vasculitis following successful treatment of hepatitis C virus. J Rheumatol 32: 1164–1167 45. Landau DA, Saadoun D, Halfon P et al (2008) Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response. Arthritis Rheum 58:604–661

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

38

Francesco Zaja, Stefano Volpetti, Stefano De Luca, and Renato Fanin

38.1

Introduction

Type II mixed cryoglobulinemia (MC) is a systemic vasculitis largely mediated by immune complexes, usually associated with hepatitis C virus (HCV) infection, and characterized by the expansion of rheumatoid factor (RF)-positive B cell clones, leading to cryoglobulin production. It typically affects adult patients with a median age of 56 years [1] and a female prevalence. The link between HCV and lymphoproliferative disorders was clearly established based on epidemiological data as well as biological and pathological evidence. HCV is a lymphotropic virus [2–4] that is able to infect B cells and other components of the immune system, initiating an antigen-driven multistep lymphomagenic process. Chronic HCV infection is associated with a higher incidence of non-Hodgkin lymphoma [3, 4]. Probably the strongest association between lymphoproliferative disorders and HCV is the one linking viral hepatitis with MC: in 90–95% of patients with MC [1, 5] a concurrent or previous HCV infection is evidenced by serological tests; 10–70% of HCVpositive patients will develop MC [6]; and antiviral therapy may contrast lymphoid expansion with improved cryoglobulinemic manifestations. B-cell proliferation serves as the biological substrate of MC. It is generally represented by non-neoplastic oligoclonal B cell expansion, with pathological and phenotypic similarity to chronic lymphocytic leukemia F. Zaja (*) Clinica Ematologica, DISM, Azienda Ospedaliero Universitaria S. Maria Misericordia, Udine, Italy e-mail: [email protected]

(CLL) and lymphoplasmocytic lymphoma. These B-lymphoid infiltrates are CD20-positive, present in the bone marrow, liver, and lymph nodes, and tend to remain unmodified for years or even decades. Based on these characteristics, the term “monotypic lymphoproliferative disorder of undetermined significance (MLDUS)” has been suggested. In a minority of patients, a typical picture of overt B-cell lymphoma is already documentable at diagnosis; in nearly 10% of patients, lymphoma will develop during the clinical history of MC. The occurrence of B-cell lymphoma represents the final event of a multistep and multifactorial process that usually requires several years to develop. Diffuse large B-cell lymphoma, observed in 40–50% of cases, is the most frequent subtype of lymphoma; in the remaining cases, marginal-zone lymphoma (extranodal, nodal or splenic) or, rarely, CLL and lymphoplasmacytic lymphoma are documented. Beyond the characteristic features secondary to HCV infections and lymphoid expansion, patients with MC may exhibit variable autoimmune manifestations, with impairment of single or multiple organs, such as skin, kidney, nerves, lungs, and joints. More frequently, these features are the expression of a small-vessel vasculitis secondary to cryoprecipitation. Other autoimmune hematological phenomena, such as hemolytic anemia, immune neutropenia, and thrombocytopenia, may also occur. Recent studies evidenced an increase in serum B cellactivating factor (BAFF) in patients affected by MC. BAFF is usually described in systemic lupus erythematosus, rheumatoid arthritis, and Sjögren syndrome. It seems to be required for B-cell survival and may prevent autoreactive B cells from undergoing apoptosis [7].

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_38, © Springer-Verlag Italia 2012

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The chimeric monoclonal antibody (IgG1/k) rituximab (RTX) is directed against the CD20 antigen, expressed on the surface of normal (from pre-B to mature B lymphocytes) and malignant B lymphocytes [8]. The action of this drug, while not yet fully understood, seems to be related to complement-dependent cytotoxicity (CDC) [9], antibody dependent cell mediated cytotoxicity (ADCC) [8, 9], and the induction of apoptosis [10, 11]. RTX proved to be highly effective in the therapy of lowgrade and high-grade CD20-positive B-cell lymphomas and its use has now been registered in combination with chemotherapy or as maintenance therapy. Initial studies in animals and, subsequently, in patients treated with RTX, showed the development of a marked (even transient) B-cell depletion from peripheral blood, bone marrow, and lymph nodes, but only mild changes in immunoglobulin and complement serum levels [12]. These results, along with the good tolerance and handling of the drug, have recently prompted its use in several autoimmune diseases. Encouraging results were obtained in immune thrombocytopenia, hemolytic anemia, thrombotic thrombocytopenic purpura, different rheumatological disorders, anti-MAG polyneuropathies, pemphigus vulgaris, and several other autoimmune disorders. The therapeutic effect of B-cell depletion in autoimmune phenomena is not yet clearly defined but it likely involves the inhibition of auto-antibody production and the prevention of immune complex formation, and thus stimulation of an inflammatory response. B cells may also act by producing specific cytokines and have antigen-processing activity. Moreover, a regulatory effect of B cells on T cells has been documented, suggesting a primary role of the former in the pathophysiology of some autoimmune disorders. There is a strong rationale for using RTX in the treatment of MC, since this disorder is sustained by a low-grade B lymphoproliferation exhibiting autoimmune features. Biological evidence of effective targeting of RF-positive B cell clones by RTX has been reported, both in preliminary clinical studies and in animal models [13]. In addition, RTX was demonstrated to be effective and safe in B-cell lymphoma as well as in autoimmune diseases. The first use of RTX in MC dates to 1999 [14], when a 58-year old man, unresponsive to interferon (IFN), cyclophosphamide, and steroids, achieved a clinically significant response after two doses of RTX, with a progressive improvement in all signs of disease, including the nearly complete disappearance of purpura and arthralgia and

F. Zaja et al.

a progressive reduction in RF level. The response lasted for 3 months, when the patient again complained of purpura and arthralgia. On the basis of this report and the preliminary results obtained in autoimmune diseases characterized by pathogenic RF [14–16], RTX was tested in a larger cohort of patients. The antibody was administered i.v. generally at the standard dose of 375 mg/m2 weekly for 4 weeks, according to the previous experience in B-cell lymphoma. More recently, a schedule consisting of two bi-weekly 1,000 mg doses was proposed. Here we review data from the literature regarding the role of RTX in MC. Our literature sources were drawn from the electronic databases of MEDLINE (from 1998), which were searched using the explore function for the Medical Subject Heading (MeSH) terms “rituximab,” “rituxan,” “mabthera,” “anti-CD20,” and “cryoglobulinemia.” Only those papers reporting data on at least one patient with MC who was treated with RTX and only those papers in which the response to RTX was reported according to specific organ involvement were taken into account.

38.2

Rituximab and Cutaneous Lesions in MC

The skin is a very frequent target of MC, involved in >90% of patients [1]. Cutaneous manifestations are characterized by the development of purpura, skin ulcers, and urticarial lesions. Two major studies [13, 17] confirmed the therapeutic activity of RTX on MC cutaneous manifestations. In our experience, 11 out of 12 patients achieved complete disappearance of purpura while one patient had a partial response. Response duration was 6 months or more in nine patients. Similarly, we documented complete ulcer healing in five out of five patients, with response duration of 6 months or more in all of them. One of these patients has maintained a response after 10 years. One single case of urticaria had a complete and long-lasting response upon RTX therapy. Similar results were documented by Sansonno et al. [17]; their study showed improvement of purpura in 12 out of 14, skin ulcers in three out of five, and urticaria in three out of three patients. Other favorable experiences, even if involving a small number of cases, have been reported by other authors [18, 19] (Table 38.1). Notably, Visentini et al. [26] reported a complete disappearance of purpura in

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The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

299

Table 38.1 Results with rituximab (RTX) therapy for cutaneous lesions in mixed cryoglobulinemia References Zaja et al. [13]

Cutaneous lesions Purpura

Patients 12

Response OR: 12 CR: 11

Ulcers

5

OR: 5 CR: 5

Saadoun et al. [27]

Urticaria Purpura Ulcers Urticaria Ulcers Purpura Purpura Ulcers Purpura Purpura Purpura Ulcers Purpura

1 14 5 3 1 1 1 1 1 1 3 3 13

OR: 1 CR: 1 OR: 12 OR: 3 OR: 3 OR = 1 OR: 1 CR: 1 OR: 1 CR: 1 OR: 1 CR: 1 OR: 1 CR: 1 Brief improvement, then flare OR: 3 CR: 3 OR: 2 CR: 2 OR: 11

Roccatello et al. [18] Tallarita et al. [28]

Ulcers Ulcers Ulcers

2 3 1

OR: 2 OR: 3 OR: 1 CR: 1

Ulcers Purpura Ulcers Urticaria

1 46 22 4

OR: 1 CR: 1 OR: 41 (89%) OR: 19 (86%) OR: 4 (100%)

Sansonno et al. [17]

Arzoo et al. [20] Lamprecht et al. [21] Koukoulaki et al. [22] Ghobrial et al. [23] Ghijsels et al. [24] Cohen et al. [25] Visentini et al. [26]

Da Silva et al. [19] Total

Response duration < 6 months in 3 Response duration > 6 months in 9 Response duration < 6 months in 1 Response duration > 6 months in 4 Response duration > 6 months in 1

Low-dose RTX RTX in combination with PEG-IFN and ribavirin

RTX in combination with plasma exchange

OR overall response, CR complete response, IFN interferon

three out of three patients and complete ulcer healing in two out of three patients using a lower-dose RTX schedule (250 mg/m2 × 2). Taken together, these results highlight the effective therapeutic activity of RTX for the treatment of cutaneous lesions secondary to MC. Overall, the response rate to purpura, ulcer, and urticaria manifestations were 90%, 89% and 100%, respectively. In many cases, the response was complete and durable.

38.3

Rituximab and Renal Involvement in MC

Renal involvement occurs in nearly one-third of cases and represents the worst prognostic factor for patients with MC. In the study of Ferri et al. [1], renal involvement was clinically evident at diagnosis and during follow-up in 20% and 30% of patients, respectively, and was associated with significantly lower survival

rates than in the remaining patient population (10-year survival: 33.1% vs. 62.1% ). Cryoglobulinemic nephropathy is generally an expression of immune complex membranoproliferative glomerulonephritis and is associated with proteinuria and nephritis. The activity of RTX in MC nephritis has been described in nearly 40 patients, in most cases with clinical improvement (approximately 80% response rate; Table 38.2). The response duration was variable, with some patients showing medium to long periods of remission. Re-treatment was generally effective and in some patients a maintenance policy was favorably adopted [29] (Table 38.2). Remarkably, the group of Saadoun et al. [27] administered RTX in association with pegylated IFN plus ribavirin in seven patients with MC nephritis in order to simultaneously treat both the B-cell disorder and HCV. Treatment was generally well tolerated and was associated with renal improvement in four out of the seven patients. Basse et al. [30] treated seven patients with cryoglobulinemic nephritis that

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Table 38.2 Results with rituximab (RTX) therapy for renal involvement in mixed cryoglobulinemia (MC) References Zaja et al. [13] Sansonno et al. [17] Arzoo et al. [20] Ghijsels et al. [24] Koukoulaki et al. [22] Quartuccio et al. [29]

Patients 2 1 1 1 1 5

Response OR: 1 CR: 1 OR : 0 OR: 1 OR: 1 OR: 1 OR: 5

Basse et al. [30]

7

OR: 7

Visentini et al. [26] Saadoun et al. [27] Roccatello et al. [18]

1 7 7

OR: 1 CR: 1 OR : 4 OR: 7

Total

33

OR: 28 (87.5%)

Response duration > 6 months Response duration > 8 months Response duration > 24 months Two patients maintained response status after 15 and 21 months. Three patients relapsed after 5, 7, and 12 months, respectively. Retreatment with RTX was efficacious in 2 patients who subsequently underwent maintenance treatment. MC after renal transplant. Severe infectious complications in two patients

Good safety profile No increase in HCV load Response duration: 12–18 months

OR overall response, CR complete response

developed after renal transplant, documenting a sustained remission or an improvement of nephrotic syndrome in five patients and the disappearance of nephritic syndrome in one patient, with a sustained clearance of cryoglobulins in six. However, in two patients the clinical course was complicated by severe infections.

38.4

Rituximab and Neuropathy in MC

Peripheral neuropathy is a common manifestation of MC and is often refractory to standard treatment. Neuropathic inflammation is secondary to vasculitis of the vasa nervorum. It more frequently affects the nerve structures of the inferior limbs, impairing various levels of sensitivity and/or motility. In our study [13], seven out of seven patients unresponsive to conventional treatments improved after salvage therapy with RTX. A decrease of 50% or more in neuropathic pain was recorded in six out of the seven patients, with complete disappearance in one of them after 6 months of treatment. Similarly, a decrease in paresthesias in the lower limbs was noted in seven of seven patients, with complete disappearance in two of them after 6 months. Symptom improvement usually occurred during the second and third months, and it was still present at the end of the sixth month in all patients. Electromyography was consistent with axonal and myelinic damage and detected sensory and mild motor

involvement in all patients. In four out of four patients who agreed to repeat the test after 6 months, it remained unchanged with respect to baseline. In a similar study by Sansonno et al. [17], an improvement of neuropathic symptoms was observed in six out of 14 patients who had impaired nerve conduction velocity, in four out of 11 patients with paresthesias, and in two out of three patients with dysesthesia. In a more recent study, Cavallo et al. [31] treated 13 patients presenting with paresthesias (11 patients), burning feet (six patients), restless legs syndrome (one patient), and asthenia (12 patients). After six doses of RTX 375 mg/m2, a clinical improvement was evident in five out of 11 patients (45%) with paresthesias, in four out of six (67%) with burning feet, and in 10 out of 12 (83%) with asthenia. One patient who was unable to walk began to walk again following the third infusion. The mean clinical neuropathy disability score (CNDS) was 46.08 ± 7.62 before treatment and 50.23 ± 7.17 after RTX (p < 0.001). The electrophysiological data were also consistent with a beneficial effect of anti-CD20 therapy, showing an improvement in motor response and conduction. Moreover, two patients regained sensory response after therapy. In conclusion, RTX therapy seems to be effective in the majority of patients with neuropathy, even if the long-term efficacy of the drug is difficult to assess due to the lack of longer studies.

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The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

38.5

Rituximab and Arthritis in MC

Arthralgia is present in most patients and is generally caused by non-erosive lesions of the hands, ankles, knees, and elbows. Administration of RTX has been shown to improve MC arthritis. Seven different studies, collecting data from 59 patients, analyzed the effect of RTX on arthralgia: overall, 62% of patients experienced the disappearance or a significant reduction of arthralgias. In particular, in our series [13], all four patients rapidly responded after treatment and in two of them the response was maintained for 6 months or more. Our observations were confirmed by Sansonno et al. [17], in a study of 16 patients, eight of whom responded to treatment. Other studies, with fewer patients, reported higher overall response rates. Roccatello et al. [18] reported a clinical response in all patients treated (6/6, 100%) in terms of a >50% decrease in the Visual Analogue Scale. In the study of Saadoun et al. [27], which investigated the activity of combination therapy with RTX, pegylated IFN, and ribavirin, five out of six patients had clinical improvement upon RTX treatment. In conclusion, RTX seems to be highly active for arthralgia, either alone or together with antiviral therapy, although prospective trials are needed to confirm the safety profile and overall response of combination treatment.

38.6

Rituximab and Hyperviscosity Syndrome in MC

Hyperviscosity syndrome occurs in patients with systemic rheumatic diseases and other conditions associated with high titers of RF and large amounts of circulating immune complexes [32, 33]. In MC patients, hyperviscosity syndrome is relatively rare and present only in those with higher cryoglobulin levels. These patients are treated with plasma exchange, which is generally successful, even if only in the short term, in normalizing hemo-rheological parameters and controlling symptoms. At present, only a few cases of MC-related hyperviscosity syndrome treated with RTX have been described; one particularly impressive report was the case of a patient who had an extremely high cryocrit (78%) that became negative after treatment, as did the IgM monoclonal component (from 30.5 g/L to undetectable levels after therapy) [26].

301

These results matched the experience reported in patients with Waldenström macroglobulinemia (WM), suggesting that RTX is useful and safe in the treatment of hyperviscosity syndrome. Similar to what has been described in WM, in MC patients with higher cryoglobulins there may be a flare effect after RTX therapy. In particular, Sene et al. [34] reported four cases of life-threatening flare vasculitis that developed 1–2 days after the administration of RTX, particularly if given at high dose (1,000 mg). This phenomenon may be secondary to the possibility that RTX, almost immediately and especially at higher doses, enhances cryoprecipitation, by forming complexes with IgM/k that have rheumatoid activity. Sene and colleagues analyzed this hypothesis in in vitro experiments and found that the addition of RTX to serum containing an RF-positive IgM/k type MC was associated with visibly accelerated cryoprecipitation. For these reasons, in patients with high baseline levels of cryoglobulins RTX should be very cautiously prescribed, initially administering standard (375 mg/m2) or even lower doses after the removal of IgM/k by plasma exchange and accompanied by strict clinical and laboratory monitoring.

38.7

Modification of Specific Laboratory Parameters After Rituximab in MC

The specific laboratory findings of MC include cryoglobulins, IgM/k monoclonal gammopathy, RF positivity, and a decreased level of the complement component C4. The effect of RTX on these parameters has been investigated in different studies aimed at determining a correlation with clinical response. In our experience [13], a significant reduction in serum RF levels, cryoglobulins, and IgM was noted after treatment, while serum C4 levels increased significantly. Roccatello et al. [35] observed that in six patients with MC and nephritis, proteinuria, erythrocyte sedimentation rate, and cryocrit were significantly decreased at 2, 6, and 12 months. RF and IgM significantly decreased at 6 months whereas C4 significantly increased at 2 and 6 months and IgG remained stable. These findings were confirmed in three of the patients even after 18 months, although in one of them RF levels increased and C4 decreased at 19 months following the initiation of therapy. Another study [26] confirmed a reduction in serum cryoglobulins in four out of five evaluable patients, with an impressive negativization of the

302

F. Zaja et al.

cryocrit in a woman who had a 78% cryocrit before treatment. Correlating blood tests and response, Sansonno et al. [36] demonstrated a decrease in the mean cryocrit value of patients who responded (16/20, 80%); in three patients (18.7%), cryoglobulins stably disappeared throughout the observation period. In terms of duration of response, 12 months after the discontinuation of RTX, the response maintenance rate was 75%. In conclusion, the results of these studies seem to demonstrate a significant improvement in several MC-specific laboratory parameters after treatment with RTX, in particular a decrease in serum cryoglobulin levels, RF, and IgM/k and an increase in C4 levels.

38.8

Effect of Rituximab on B-Cell Expansion in MC

A key pathological and molecular feature in MC is the B-cell oligo/monoclonal expansion in both peripheral blood and the bone marrow. These B-cell lymphoid infiltrates, which are CD20-positive, usually remain unmodified for years or even decades, but in a small percentage of patients (nearly 10%) they can evolve to lymphoma. Sansonno et al. [36] analyzed the dynamics of peripheral CD20+ B cells in 20 patients with cryoglobulinemia (13 patients with mixed type II and 7 with type III) who were treated with RTX. As expected, RTX reduced the number of peripheral blood CD20+ cells to <1% after the first infusion, both in patients with clinical response and in non-responders. The immunophenotype of the lymphocyte subsets was monitored during 12 months of follow-up: the number of B cells decreased distinctly compared to baseline values and remained low for 6–7 months, while T-cell counts were not modified. B-cell recovery started after 6 months, both in responders and in non-responders. To determine B-cell clonal restriction and its persistence or clearance, PCR directed at IgH VDJ genes on DNA was performed on peripheral blood samples from 11 responders and four non-responders before and after each infusion. Sequential analyses demonstrated the disappearance of individual B-cell clones in patients who responded to RTX and the appearance of new and distinct clonotypes at varying time points, while the persistence and stability of initially expanded clonotypes were found in non-responders, suggesting that their B cell clones were less sensitive to treatment. Quartuccio et al. [37] analyzed B-cell clonality in the bone marrow of patients with MC and renal

involvement. A monoclonal expansion at baseline was determined in two patients and an oligoclonal pattern in another patient. Six months after RTX therapy, the molecular pattern of B-cell expansion had changed in all three cases, from clonal to polyclonal, suggesting that the efficacy of RTX in MC glomerulonephritis is due to the selective clearance of pathogenic autoreactive B-cell clones, even in the absence of a complete B-cell depletion. In MC, the chronic presentation of specific antigens may stimulate lymphocytes, with subsequent progression to lymphoproliferative disorders and lymphomas. Support for this hypothesis is described in several publications [38–40].

38.9

HCV Viral Load After Rituximab Therapy

As a lymphotropic virus, HCV infects different cellular agents of the immune system, in particular B cells [2–4]; thus, B-cell depletion secondary to RTX therapy may impair humoral immune functions. A great deal of interest has therefore emerged in monitoring HCV viral load and hepatitis re-activation upon RTX treatment, with several studies analyzing HCV-RNA levels before and during treatment with RTX and throughout follow-up. In our experience [13], the HCV viral load in serum remained above the upper limit of detection by quantitative PCR in three out of eight patients (37.5%), increased to above this limit in two of eight patients (25%), showed minimal fluctuations in two patients (25%), and decreased in one patient (12.5%). Similar results were described by Visentini et al. [26], who measured viral load at baseline and at weeks +20 and +40. HCV viremia remained unchanged in two patients (40%), decreased in two (40%), and transiently increased in one (20%). In a third study, Sansonno et al. [17] reported higher levels of HCV RNA in patients who responded to treatment. In particular, a progressive increment of viral load was documented after RTX, reaching approximately twice the baseline after 12 months. In non-responders, HCV RNA levels remained unchanged over sequential time points, despite B-cell depletion. Interestingly, Lake-Bakaar et al. [41] observed that in MC patients treated with antiviral therapy and RTX there was an important increase in viral load within 2 weeks of initiating RTX that continued for up to 23 weeks, corresponding to

38

The Role of Rituximab in the Therapy of Mixed Cryoglobulinemia

B-cell recovery. After B-cell reconstitution, the viral load progressively decreased, only to increase again after the second cycle of RTX. A more recent publication by Roccatello et al. [35] described a substantial stability or even a reduction of HCV RNA in 11 patients treated with RTX and evaluated after 6 and 12 months for viremia. The authors hypothesized that the HCV RNA increment reflected virus shedding through RTXinduced B-cell cytotoxicity. However, this explanation is contradicted by the data of Sansonno et al. in nonresponder patients in whom viral load remained unchanged [17]. A more convincing explanation is found in the B-cell depletion induced by RTX and the consequent reduction of IgG anti-HCV antibody levels, which suggests that humoral immunity exerts immune control on HCV replication. It must be underlined that the increased viremia did not correlate with a relevant worsening of liver function in the cited series, in terms of transaminase levels and clinical symptoms or signs. Nevertheless, the potential risk of hepatic and systemic complications due to a viral flare always should be considered and the monitoring of HCV RNA and transaminases is strongly recommended.

303

syndrome 7 and 9 days after the first RTX administration (1,000 mg). Patients with drug reactions had higher mixed cryoglobulin levels and lower C4 levels than patients without reactions. In vitro assays showed that RTX formed a complex with IgM/k with RF activity, leading to accelerated cryoprecipitation. Accordingly, the authors suggest that RTX be administered with caution in MC vasculitis, recommending plasma exchanges prior to infusion in patients with high baseline levels of mixed cryoglobulins. Moreover, Ruch et al. [45] reported the case of a patient who developed an anti-Pr cold agglutinin that manifested with hemolysis and microvascular occlusion, causing mesenteric ischemia and cerebral infarction. In conclusion, RTX can be considered as a safe therapeutic option, burdened by minimal toxicity. Nonetheless, caution is required in patients with high baseline levels of mixed cryoglobulins, to prevent drug reactions and cryoprecipitation. Moreover, careful monitoring for infectious events is needed, and early symptoms of complications should not be underestimated.

38.11 Conclusions 38.10 Safety and Toxicity of Rituximab in MC The safety of RTX in the treatment of patients with non Hodgkin lymphomas and autoimmune disorders is generally good. In MC, two studies [13, 17] reported only two major adverse events, one case of retinal artery thrombosis and one of sepsis. In subsequent publications, a relatively small number of side effects was reported [26, 30, 42], including bradycardia (3 cases), hypotension (2 cases), infection (3 renal transplant patients), mild transaminase elevation (3 cases), panniculitis of the elbows and knees (1 case), and serum sickness syndrome (1 case). There were three deaths: one occurred in an HCV-infected patient with renal insufficiency, one in a patient with meningoencephalitis caused by Acanthamoeba spp. [43], and one in a renaltransplant HCV-negative patient who developed Cryptococcus neoformans meningoencephalitis [44]. During long-term follow-up (>12 months), two cases of lymphoma and one case of breast cancer were noted. Recently, Sene et al. [34] reported six cases of systemic drug reactions after RTX: four patients developed a severe flare of MC vasculitis within 2 days after infusion while two patients developed serum sickness

RTX appears to be an active agent for the treatment of patients with MC. Based on its unique biologic activity, it acts specifically against B-cell lymphoproliferative disorders characterized by sustained cryoglobulin production and organ involvement. Almost all clinical manifestations may benefit from RTX treatment. Overall, RTX is well tolerated and safe; the possible increase of HCV viremia rarely causes hepatitis re-activation. However, caution should be exerted in patients with high cryoglobulin levels or hyperviscosity at baseline, because of possible flare syndrome. Prospective comparative trials are warranted in order to better evaluate the therapeutic impact of RTX and to define the best treatment approach, taking into consideration the other available therapies and in particular antiviral agents.

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Rituximab in Cryoglobulinemic Vasculitis: First- or Second-Line Therapy?

39

Peter Lamprecht and Paul Klenerman

39.1 Introduction: Cryoglobulinemic Vasculitis Cryoglobulinemic vasculitis is a potentially life-threatening systemic vasculitis resulting from the deposition of circulating immune complexes and complement, predominantly in small vessels (capillaries, venules, and/or arterioles) and, less frequently, in medium-sized vessels [1]. The major cause of cryoglobulinemic vasculitis is chronic hepatitis C virus (HCV) infection, with HCV-negative “idiopathic” disease and secondary forms in rheumatoid arthritis, connective tissue diseases, other chronic infections, non-Hodgkin lymphomas, and other hematologic disorders accounting for the remainder. The clinical spectrum of cryoglobulinemic vasculitis encompasses mild to moderate disease manifestations (e.g., Meltzer’s triad with purpura, arthralgia, and asthenia), severe disease (e.g., worsening renal function, polyneuropathy, mononeuritis ­multiplex, and skin ulcers, acral necrosis), and lifethreatening disease (e.g., rapidly deteriorating renal function, gastrointestinal vasculitis, central nervous system involvement, hyperviscosity syndrome). Purpura and arthralgia are the most frequent symptoms of cryoglobulinemic vasculitis, affecting nearly all patients (³95%), followed by polyneuropathy, renal involvement, and Raynaud’s phenomenon in ³50% of the patients [2].

P. Lamprecht (*) Department of Rheumatology, Vasculitis Center UKSH & Clinical Center Bad Bramstedt, University of Lübeck, Lübeck, Germany e-mail: [email protected]

39.2 Rationale for B-Cell-Depleting Therapy in Cryoglobulinemic Vasculitis Mixed cryoglobulinemia is present in 40–60% of the patients with chronic HCV infection, but symptomatic cryoglobulinemic vasculitis develops in <5%. The disease is characterized by an immunoglobulin (Ig)M antibody directed against the Fc segment of an IgG (rheumatoid factor), with polyclonal IgG as the antigen. In HCV-associated cryoglobulinemic vasculitis, the IgM component is either monoclonal (type II cryoglobulinemia) or, less frequently, polyclonal (type III cryoglobulinemia). Mixed cryoglobulinemia is a result of the clonal proliferation of B cells, which produce an IgM rheumatoid factor. While this lymphoproliferative disorder is benign, transition into malignant non-Hodgkin lymphoma (NHL) and primary co-presentation of cryoglobulinemic vasculitis and NHL have been reported in a number of cases [2]. Recently published studies have shown that chronic HCV infection confers a 20–30% increased risk of NHL and a three-fold increased risk of developing Waldenström’s macroglobulinemia compared to normal controls [3]. Moreover, the risk for certain B-cell NHL subtypes, such as diffuse large B-cell lymphoma, marginal-zone lymphoma, including splenic marginal-zone lymphoma with circulating villous lymphocytes, and lymphoplasmacytic lymphoma, is higher in patients with chronic HCV infection [4]. Those with HCV-negative cryoglobulinemic vasculitis also have a four-fold higher risk of NHL. Moreover, these patients have an increased risk of renal involvement and a poor outcome [5]. HCV is primarily a hepatotropic virus but may also be lymphotropic. The virus is thought to induce the

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above-described polyclonal B-cell expansion, which in turn gives rise to a benign monoclonal proliferation and in some patients a transition to malignant B-NHL. B-cell proliferation causes mixed cryoglobulinemia and the induction of various autoantibodies and autoimmune phenomena [2]. Clonal B-cell expansions are found in the blood, bone marrow, and liver of patients displaying HCV-associated mixed cryoglobulinemia. Peripheral blood, intrahepatic, and bone marrow clonal B-cell expansions are associated with extrahepatic manifestations, such as mixed cryoglobulinemia and glomerulonephritis [6, 7]. Chronic antigenic stimulation may drive clonal B-cell selection in chronic HCV infection [2]. Moreover, HCV induces B-cell proliferation via interaction with various host cellular molecules, e.g., binding of the HCV envelope glycoprotein E2 to human CD81, expressed on various cell types, including hepatocytes and B cells. CD81 associates with CD19 and CD21 to form the B-cell co-receptor complex, which reduces the threshold for B-cell ­activation [8]. Clonal proliferation of circulating memory phenotype marginal-zone-like B-cells with restricted expression of the Ig heavy chain variable (VH) 1–69 gene, encoding IgM rheumatoid factor of the WA idiotype, is  also detected in patients with HCV-associated ­cryoglobulinemic vasculitis [9, 10]. B-lymphocyte ­stimulator (BLyS)-mediated anti-apoptotic effects are maintained in clonally expanded VH 1-69+ B cells in spite of down-regulated BLyS receptor 3 (BR3) expression, suggestive of an increased sensitivity of the BLys intracellular signaling cascade in VH 1-69+ B-cells. BLyS is a physiological inducer of anti-apoptotic Bcl2, sustaining B-cell survival [11]. An increased frequency of the t(14;18) translocation in circulating B-cells resulting in activation of the bcl-2 proto-oncogene has also been reported in patients with HCVassociated cryoglobulinemic vasculitis [12]. In HCV-associated cryoglobulinemic vasculitis, clonal VH 1-69+ B-cell expansion shows reversion and clinical remission is induced by treatment with the monoclonal anti-CD20 antibody rituximab [13]. Similarly, increased BLyS-mediated activity is normalized by rituximab treatment [11]. Concomitant abnormalities within the T-cell compartment including a decreased frequency of circulating regulatory T cells (Treg) and an increased frequency of activated CD8T cells, are also normalized with successful rituximab treatment [13]. In patients unresponsive to rituximab

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treatment, however, clonal B-cell expansions and T-cell compartment abnormalities persist [13, 14]. CD81 is also expressed on T cells, but chronic HCV is not associated with highly activated or monoclonally expanded CD8+ or CD4+ T-cell populations in vivo. In contrast, T-cell responses against the virus in chronic HCV are typically weak or poorly functional in peripheral blood, compared to other chronic infections [15]. A  number of mechanisms have been suggested to explain this, including exhaustion through repetitive antigen exposure and up-regulation of inhibitory signaling molecules such as PD-1. Induction of CD4+ Treg subsets has also been demonstrated and a significant fraction of CD4+ T cells within the liver express the Treg marker FoxP3. It is hypothesized that induction of Treg populations protects the liver against excess T-cellmediated immunopathology and contributes to the control of B-cell mediated immunopathology [16, 17]. The impact of both HCV infection and impaired anti-viral and anti-proliferative interferon (IFN)-a activity on lymphoproliferation was recently demonstrated in a mouse model, in which HCV core protein expression and impaired IFN-a-mediated signaling synergize to promote lymphoproliferation and the development of lymphoma [18]. Of note, polymorphisms in two genes, IFN regulatory factor (IRF)-1 and signal transducer and activator of transcription (STAT)-1, are associated with chronic HCV infection [19]. The virus also uses a number of other mechanisms to impair IFN signaling, including induction of USP18 and cleavage of CARDIF by NS3/4a protease [15]. HCV replication is extraordinarily dynamic, necessitating high IFN-a doses for viral eradication and the prevention of viral resistance that results from the rapid development of quasi-species diversity [20].

39.3 Rituximab as Firstand Second-Line Treatment in Cryoglobulinemic Vasculitis As noted above, rituximab is a monoclonal chimeric anti-CD20 antibody selectively targeting B cells. CD20 is expressed on B cells from the pre-B stage to mature B cells and disappears during their differentiation to plasma cells. It acts through the generation of a calcium flux and by mediating signal transduction, thereby activating B cells. Antibody-dependent cell-mediated cytotoxicity, complement-dependent

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Table 39.1  Summary of clinical outcome in uncontrolled case series using rituximab as first- or second-line therapy for the ­induction of remission in HCV-associated and HCV-negative cryoglobulinemic vasculitis Author [reference] Sansonno et al. [14]

Number of patients 20

RTX as first- or second-line therapy Second-line

Zaja et al. [24]

15 (3/15 HCV-negative) Second-line

Roccatello et al. [25] Quartuccio et al. [26]

6 5

First line in 2, second-line in 4 First line in 3, second-line in 2, maintenance in 2

Clinical outcome CR 16/20 (80%) Re 4/20 (20%) CR/PR 14/15 (93%) Re 4/15 (26%) CR/PR 6/6 (100%) CR/PR 6/6 (100%) Re 2/5

RTX Rituximab, CR complete remission, PR partial remission, Re relapse during follow-up

cytotoxicity, inhibition of cell proliferation, and B-cell apoptosis have been implicated in the mechanisms of rituximab action. In vitro studies suggest a dose– response relationship up to a 10–25 mg/ml threshold, after which the curve levels off due to receptor saturation. The half-life of riuximab is about 1 week, but the median persistence of active levels is about 3 months [21, 22]. Rituximab completely depletes circulating B cells for about 6 months, whereas B-cell depletion from secondary lymphoid organs and central compartments (thymus and bone marrow) is subject to considerable inter-individual and inter-site variability [23]. In 2003, two seminal studies showed the successful induction of remission in HCV-associated cryoglobulinemic vasculitis [14, 24]. In one study, 20 patients with HCV-associated cryoglobulinemic vasculitis who were positive for Meltzer’s triad (purpura, arthralgia, asthenia) but resistant to IFN-a therapy were treated with four weekly doses of 375 mg/m2 rituximab intravenously. Sixteen patients (80%) achieved complete remission, with 10 (62.5%) in complete remission within 2 months and the remainder within 5 months. Circulating B cells were depleted after the first infusion and started recovering from 6  months onwards after rituximab treatment. Viremia increased approximately two-fold from baseline levels following rituximab infusions [14]. As discussed above, clonal B-cell expansion is reversed in successfully treated patients with HCV-associated cryoglobulinemic vasculitis, but not in non-responders [13, 14]. Four of the 16 patients (25%) relapsed within 1 year following the cessation of rituximab infusions, starting from 7 months onwards [14]. In the other study, 15 patients with ­HCV-associated cryoglobulinemic vasculitis who were ­positive for Meltzer’s triad and had skin ulcers, ­peripheral neuropathy, autoimmune hemolytic anemia, ­autoimmune neutropenia, and/or glomerulonephritis ­unresponsive

to conventional treatment, including IFN-a and various immunosuppressive therapies, were treated with four weekly doses of 375  mg/m2 rituximab intravenously. Treatment resulted in rapid improvement in vasculitic skin manifestations (ulcers, purpura, or ­urticaria) in all patients. Furthermore, arthralgias, hemolytic anemia, fever, and peripheral neuropathy improved. Among the 15 patients, two complete responses and one partial response were obtained in three patients with B-cell NHL. Of the two patients with glomerulonephritis, proteinuria and nephritic sediment disappeared in one patient with membranoproliferative glomerulonephritis, whereas nephritic activity and renal insufficiency persisted in another, in whom rituximab was interrupted after two infusions due to side effects. Serum rheumatoid factor and mixed cryoglobulinemia decreased and C4 complement ­levels increased, reflecting clinical efficacy. In addition, prednisolone doses could be tapered [24]. The successful induction of remission, including the reversal of bone marrow abnormalities and improvement of renal function, was also obtained in two case series reporting on six and five patients with HCV-associated cryoglobulinemic vasculitis, respectively. Of note, rituximab was successfully used as first-line therapy in two of the six and three of the five patients in the two studies [25, 26]. Relapse in two patients was successfully treated with another round of rituximab infusion. Maintenance therapy with rituximab was continued in two patients who showed sustained remission of nephritic activity [26]. Table 39.1 summarizes the data on clinical outcomes in the four discussed studies [14, 24–26]. Apart from the two large uncontrolled series mentioned above [14, 24] and the two smaller case series [25, 26], there have been a number of case reports on rituximab in HCV-associated cryoglobulinemic vasculitis, involving either one or

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two patients (reviewed in [22]). Induction of remission  is also achieved in patients with HCV-negative ­cryoglobulinemic vasculitis resistant to conventional immunosuppressive therapy [24, 27]. Among the few adverse effects reported so far are bradycardia, hypotension, infection (in 3 renal-transplant patients), mild transaminase elevation, retinal artery thrombosis, self-limiting panniculitis, and serum-sickness [22]. Furthermore, a modest increase in viral load following rituximab treatment has been detected, as discussed above [14].

39.4 Rituximab and Peg-IFN-a Plus Ribavirin in Cryoglobulinemic Vasculitis Treatment of HCV currently relies on combination therapy with pegylated (PEG)-IFN-a and ribavirin. This approach provides a sustained virological response (SVR) in 45–85% of patients, dependent on host and viral genotype. The action of IFN-a is both anti-viral and immunomodulatory, while the nature of the effect of ribavirin is unclear, although it is not anti-viral. Treatment of patients presenting with acute disease is highly effective (SVR in >90% of patients), although very few individuals present at this stage. It is likely that such treatment reverses the excess risk of lymphoproliferation-related disease, although the time course for the induction of autonomous B-cell activity is not known [28]. Given HCV’s highly dynamic virion production, its escape mechanisms from cellular immunity, and its resistance to IFN, coupled with its inherent propensity for the promotion of lymphoproliferative disease, it seems highly desirable to eliminate the virus early, at the same time as the prevention of HCV-induced lymphoproliferative disease [20–22]. Relapses of HCV-associated cryoglobulinemic vasculitis in patients with chronic HCV who exhibit a SVR to combination therapy have been reported, underscoring the need for the control of clonal B-cell proliferation in addition to virus elimination. In some patients, the withdrawal of IFN-a caused a short-lived relapse of cryoglobulinemic vasculitis; in others, the underlying lymphoproliferative disease evolved to malignant B-cell NHL [29]. Treatment of HCV-associated cryoglobulinemic vasculitis with PEG-IFN-a and ribavirin results in sustained clinical and virological responses in up to 60%

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of patients. Clearance of mixed cryoglobulinemia is observed in about half of the patients [22]. Successful anti-viral therapy with IFN-a in HCV-associated cryoglobulinemic vasculitis is accompanied by a reversal of monoclonal B-cell proliferation [30]. Thus, antiviral treatment combined with PEG-IFN-a plus ribavirin is an appropriate first-line therapeutic option for patients with mild to moderate disease severity and activity (i.e., Meltzer’s triad). However, about 30% of the patients continue to have active disease while receiving anti-viral therapy. Thus, B-cell depleting therapy is warranted in patients lacking early virological and clinical responses and in patients with severe disease activity, in whom rapid clinical improvement is the goal and a delayed clinical response, as seen with anti-viral therapy, is not tolerable. While rituximab induces a complete clinical response in 60–70% of the patients with HCV-associated cryoglobulinemic vasculitis, reverses clonal VH 1-69+ B-cell expansion and concomitant Treg abnormalities, and evokes a cryoglobulin clearance in about 30% of the patients, viremia persists or even increases, as discussed above. Moreover, 30% of the patients are subject to disease relapse during peripheral blood B-cell recovery [13, 14, 22, 24–26]. Thus, the combination of rituximab and PEG-IFN-a plus ribavirin may well be a logical approach to successfully combine the anti-proliferative impact and anti-viral potencies of both treatment approaches [22]. Our group was the first to report the successful induction of remission with a combination therapy of rituximab and PEG-IFN-a2b plus ribavirin, in a patient with refractory HCV-associated cryoglobulinemic vasculitis. The patient suffered from Meltzer’s triad, severe polyneuropathy, and intestinal involvement. Leukocytoklastic vasculitis of the skin and small-vessel vasculitis of the colon were diagnosed from biopsies, and a low-grade lymphocytic NHL discovered upon further investigation. Interestingly, the patient showed neither a clinical nor a virological response to an initial treatment approach consisting of standard IFN-a2b and ribavirin. She was subsequently treated with prednisolone, cyclophosphamide, and plasmapheresis, but did not respond clinically. Clinical remission of the cryoglobulinemic vasculitis and complete remission of the NHL were ultimately induced with six thrice-weekly infusions of 500 mg rituximab. Remission was maintained and HCV successfully eliminated with PEG-IFN-a (which had meanwhile

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Table 39.2  Summary of clinical outcome in a case report and uncontrolled case series using rituximab or combined rituximab and PEG-IFN-a2b plus ribavirin as first- or second-line therapy for the induction of remission in HCV-associated cryoglobulinemic vasculitis Author [reference] Lamprecht et al. [31] Saadoun et al. [32]

Number of patients 1 16

RTX as first- or second-line therapy Second line Second line

Terrier et al. [33]

32

First- and second-line (two treatment arms: RTX alone vs. combined RTX and PEG-IFN-a2b plus ribavirin)

Clinical outcome CR CR 10/16 (63%) Re 2/16 (13%) CR 7/12 (58%) in RTX alone CR 16/20 (80%) in combined RTX and PEG-IFN-a2b plus ribavirin Re 7/32 (22%)

PEG-IFN-a Peglyated interferon-a, RTX rituximab, CR complete remission, Re relapse during follow-up

become available) plus ribavirin. The patient has remained in remission for over 6 years and is assumed to be cured [31]. More recently, a pilot study in 16 patients demonstrated the efficacy of combined rituximab and PEGIFN-a2b plus ribavirin in patients with severe HCV-associated cryoglobulinemic vasculitis who were either resistant to or experienced a relapse on previous combination treatment with standard IFN-a or PEGIFN-a2b plus ribavirin. The treatment schedule consisted of four weekly doses of 375  mg/m2 rituximab intravenously followed 1 month later (to avoid cumulative side effects) by PEG-IFN-a2b (1.5  mg/kg per week subcutaneously) plus ribavirin (600–1,200  mg/ day orally) for 12  months. During treatment, 15 patients (94%) showed a clinical response (improvement of rheumatologic and other symptoms) and 10 patients (63%) achieved a complete clinical response. HCV-RNA and cryoglobulins both became undetectable in the group of complete responders. Early virological response (within 3 months) to PEG-IFN-a plus ribavirin and vasculitis of shorter duration than the previous episode were associated with a complete response. Whereas skin manifestations (purpura and leg ulcers) were successfully treated, peripheral neuropathy and renal involvement responded less favorably. Three patients had a NHL; in two, complete remission of the NHL could be induced whereas in the third partial remission was achieved. Of note, peripheral blood B-cell reconstitution was delayed until the end of anti-viral treatment. During follow-up, the two complete responders experienced a relapse, with the simultaneous reappearance of HCV-RNA and cryoglobulins. Adverse events in two patients (worsening of polyneuropathy and flare of psoriasis) were attributed to PEG-IFN-a2b [32].

The results of the pilot study were confirmed in a larger study of 32 patients with severe HCV-associated cryoglobulinemic vasculitis who were treated with either rituximab alone or rituximab and PEG-IFN-a2b plus ribavirin according to the above-mentioned treatment schedule (plus steroids and/or plasmapheresis in patients with life-threatening vasculitis). A complete clinical response was determined in 7/12 (58%) and 16/20 (80%) patients treated with rituximab alone and rituximab and PEG-IFN-a2b plus ribavirin, respectively. Thus, the response was better in patients treated with the combined protocol. After a 23 ± 12  months (mean ± SD), seven patients (22%) experienced a clinical relapse, which was associated with the failure of virological control (either non-responders to PEGIFN-a2b plus ribavirin or patients treated with rituximab without anti-viral therapy) and recovery of peripheral blood B-cells. Retreatment with rituxmab successfully induced remission in these patients. In contrast, none of the patients with SVR and B-cell recovery experienced a relapse. The main side effects were attributable to rituximab and consisted of serum sickness in 6/32 (19%) and neutropenia in 2/32 (6%) patients [33]. Systemic reactions may be associated with higher doses of rituximab (1,000  mg) [34]. Table 39.2 summarizes the data on clinical outcomes in the three discussed studies [31–33].

39.5 Conclusion: Rituximab as First- or Second-Line Therapy in Different Clinical Settings The current clinical evidence, based on uncontrolled non-randomized trials in different clinical settings, suggests the use of PEG-IFN-a plus ribavirin as

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­ rst-line therapy for patients with mild to moderate fi disease severity and activity (i.e., Meltzer’s triad) in HCV-associated cryoglobulinemic vasculitis. In patients with active disease resistant to anti-viral treatment and those with severe manifestations and activity of cryoglobulinemic vasculitis (e.g., worsening of renal function, polyneuropathy, mononeuritis multiplex, skin ulcers, acral necrosis), combined treatment with rituximab and PEG-IFN-a plus ribavirin should be considered. In fulminant and life-threatening cryoglobulinemic vasculitis (e.g., rapidly deteriorating renal function, gastrointestinal vasculitis, central nervous system involvement, hyperviscosity syndrome), the initial therapy should combine plasmapheresis and immunosuppressive therapy (e.g., cyclophosphamide and steroids). Immunosuppressive treatment is continued until the clinical response is sustained and steroids can be safely tapered. Thereafter, PEG-IFN-a plus ribavirin, either alone or in combination with rituximab, may help to eliminate the HCV and terminate B-cell clonal proliferation. Relapses of HCV-associated cryoglobulinemic vasculitis can be treated with a second round of the same rituximab regimen [21, 22]. Since rituximab as a first- or second-line treatment option, alone or in combination with PEG-IFN-a plus ribavirin, induces a sustained clinical and virological remission in only about 60% of the patients with HCVassociated cryoglobulinemic vasculitis, further treatment options are needed to both eliminate HCV and revert clonal B-cell proliferation. Accordingly, randomized controlled trials are necessary to verify and extend our current treatment options in HCV-associated cryoglobulinemic vasculitis [21, 22, 35]. Fortunately, a number of strategies to improve the control of HCV replication using specific anti-viral drugs are now in advanced clinical trials [28, 35]. The most promising of these to date is telaprevir, a targeted inhibitor of HCV protease. In clinical trials, telaprevir was given together with combination therapy and led to improvement in SVR rates from 45% to 65%, even using shortened treatment regimens (6 vs. 12  months) [36, 37]. However, the drug is associated with substantial excess side effects, mainly rash, which may necessitate its withdrawal, but the above figures are cited from intention-to-treat analyses [38]. Unfortunately, telepravir and other targeted therapies in early-phase trials show rapid selection of HCV escape mutants during 1–2 weeks of therapy. Thus, these drugs will need to be given in combination with the current standard of

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care, until, potentially, a range of two or three highly active drugs (nucleoside inhibitors, non-nucleoside inhibitors, and protease inhibitors) can be given in combination, analogous to the strategy currently used to treat HIV patients [28, 35, 38]. An alternative strategy to improve treatment outcome for HCV is the use of immunotherapy. Therapeutic vaccine trials for HCV have been attempted, although the tested vaccines were not highly immunogenic and had only a modest impact on viral load in a subset of patients. Alternative strategies include blockades of interleukin-10 and the inhibitory molecule PD-1. However, with immunomodulators such as these there is the risk of exacerbating the autoimmune disease. Consequently, they may not be appropriate for patients with mixed cryoglobulinemia, even if they prove to be successful in the treatment of uncomplicated HCV infection [28, 35]. In HCV-negative “idiopathic” cryoglobulinemic vasculitis, rituximab represents a new treatment option, also in disease resistant to conventional immunosuppressive therapy [24, 25]. Moreover, IFN-a was able to induce remission in two patients with severe HCVnegative cryoglobulinemic vasculitis, presumably based on its anti-proliferative effect [39].

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Franco Dammacco and Domenico Sansonno

40.1 HCV-Related Mixed Cryoglobulinemia and Therapeutic Implications For many years, the etiology of mixed cryoglobulinemia (MC) remained unknown, thus accounting for its designation as “essential” MC. Since the end of the 1980s, an overwhelming body of evidence has consistently shown that over 90% of cryoglobulinemic patients are infected with hepatitis C virus (HCV) [1– 5], suggesting that the virus plays a crucial role in the development of this intriguing immune-complexmediated vasculitis. With rare exceptions, patients are therefore more properly diagnosed as having HCVassociated, rather than essential MC. Interestingly, the use of an anti-viral drug such as interferon (IFN)-a in the therapy of MC was first proposed by Bonomo et al. [6] a few years before the association between MC and HCV infection was clearly established. These authors reasoned that if a chronic viral infection is involved in the etiopathogenesis of MC, then the IFN-mediated inhibition of viral replication could play an important role in its treatment. In addition, if one considers MC as a low-grade B-cell lymphoma-like disorder, IFN-a might prove effective because of its anti-proliferative action. And indeed, the use of recombinant IFN-a in seven patients diagnosed with type II idiopathic MC resulted in each case in a striking clinical improvement and a remarkable reduction of circulating cryoglobulins, with usually mild and transient side effects. F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

A number of subsequent reports [7–9] confirmed the efficacy and safety of this treatment strategy, although relapse occurred in a significant proportion of patients a few months after drug discontinuation. Since corticosteroids (CS) were widely prescribed for MC prior to the introduction of IFN-a, we asked whether a combination of CS plus IFN-a would induce a better response than obtained with the administration of IFN-a as a single drug [10]. Following a one-year treatment of 65 patients who were then monitored for 8–17  months after treatment discontinuation, a complete response was determined in approximately 53% of the patients receiving either IFN-a alone or the IFN-a plus 6-methyl-prednisolone (PDN) combination, with combined therapy resulting in a quicker (within 3  months) response. Partial responses were also largely comparable in the two groups. However, the probability of relapse within 3  months from the end of therapy was 75% in patients receiving IFN-a alone vs. none of the patients given IFN-a plus PDN. The pegylated (p) IFN-a/ribavirin (RBV) combination has now become the standard of care worldwide for HCV chronically infected patients [11–13]. This treatment induces a sustained virological response (SVR) rate of 45–50% in patients infected with HCV genotype 1 and 70–80% in those infected with HCV genotypes 2 and 3 [14, 15]. The same combined therapy has been administered to patients with HCV-positive MC, with the obvious aim of eradicating the underlying HCV infection. A remarkable improvement was observed [16] in up to 54% of patients unresponsive to IFN-a alone, as they achieved both clinical remission and an SVR. Even better results were reported in another study [17], in which >70% of the patients attained both an SVR and a complete clinical response.

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Although the achievement of an SVR in MC patients receiving pIFN-a/RBV combination therapy is usually associated with regression or remarkable improvement of vasculitis, little change, if any, occurs in terms of neuropathy and/or glomerulonephritis, strongly suggesting that factors other than viral infection are involved in their pathogenesis. In addition, more recent observations indicate that cryoglobulinemic vasculitis can persist in spite of the treatment-induced disappearance of viremia [18] or it may unexpectedly relapse in patients who had achieved an SVR [19]. A possible explanation of these phenomena stems from biomolecular studies, which have clearly shown that: (a) restriction of the humoral immune response occurs in MC patients, in whom B-cell clonal expansions may be detected in the liver, bone marrow, and circulation [20, 21], and (b) restriction of V-gene usage is invariably associated with extrahepatic manifestations of HCV infection [22]. At variance from these observations, B-cell clonal expansions are rarely detectable in HCV-infected patients without MC [21, 23].

40.2 Rituximab in Cryoglobulinemic Vasculitis Based on these observations, a therapeutic approach aimed at deleting expanded B-cell clones was attempted by two independent Italian groups [24, 25] using the chimeric monoclonal antibody, rituximab (RTX), which is directed against the transmembrane CD20 antigen expressed on pre-B and differentiated B lymphocytes. Treatment resulted in a striking improvement of cutaneous vasculitis, arthromyalgias, and weakness in approximately 80% of the 20 patients studied [24]. Parallel decreases of the cryocrit, serum IgM levels, and rheumatoid factor titers were also recorded, in step with improvements in C3 and C4 hypocomplementemia, sometimes including the restoration of normal values. A complete response was maintained in 75% of 16 responders throughout the follow-up. In addition, molecular monitoring of the B-cell response revealed disappearance of peripheral clones in the responders. RTX administration was found to be well tolerated and no remarkable adverse events were reported. However, a matter of concern was the demonstration that RTX increased HCV RNA to approximately twice the baseline levels in responders, whereas viremia remained mostly unchanged in non-responders.

F. Dammacco and D. Sansonno

The effectiveness and safety of RTX was confirmed in several studies thereafter, summarized by Cacoub et al. in a systematic review of the literature [26]. After excluding review papers, the authors selected 13 contributions reporting data on a total of 57 patients with MC, 75% of whom had HCV-related disease. Independent of the few variations in the treatment schedules among the different studies, RTX was found to positively affect the main signs of vasculitis and to induce a clinical response in 80–93% of the patients; however, a relapse was recorded in 39%. It should also be emphasized that the safety and efficacy of RTX were demonstrated in MC patients with advanced liver disease [27] and in those with non-viral cryoglobulinemic vasculitis [28].

40.3 PIRR Therapy as a New Therapeutic Approach to Mixed Cryoglobulinemia The occurrence of HCV infection in the large majority of patients with MC, chronic antigenic (viral) stimulation of the immune system, and expansion of B-cell clones have been established as crucial factors in the pathogenesis of cryoglobulinemia [29]. However, since enhanced viremia is a potentially harmful outcome of RTX administration and the ensuing B-cell depletion, it seems reasonable to combine this monoclonal antibody with antiviral drugs in order to achieve and stabilize the deletion of expanded B-cell clones, on the one hand, and induce an SVR or at least avoid an increase in the viral load, on the other. In line with these goals, a few trials have been carried out, which consistently indicated the effectiveness of the respective therapeutic measures.

40.3.1 Personal Experience We administered a combination of pIFN-a plus RBV plus RTX (PIRR) to 22 naïve HCV-positive patients with MC. Fifteen additional MC patients received pIFN-a plus RBV combined therapy without RTX [30]. All of the patients were included in a prospective, single-center, randomized study on the basis of the ­following criteria: (a) occurrence of anti-HCV antibodies and detectable HCV RNA in the serum; (b) cryocrit ³ 3% associated with the classical symptom triad of purpura, arthralgia, and asthenia; (c) recent (£ 3 months) liver biopsy showing chronic active hepatitis; (d) no

40  PIRR Therapy in HCV-Related Mixed Cryoglobulinemia Fig. 40.1  General design of pegylated interferon-a (pIFN-a) plus ribavirin plus rituximab (PIRR) therapy. RTX Rituximab, RBV Ribavirin, ST start of therapy, A-ETR assessment of end-of-treatment response, A-SR assessment of sustained response, A-LTR assessment of long-term response

317

THERAPY

ST

FOLLOW-UP

A-ETR

pIFN-α

A-LTR

RBV

RTX

0

A-SR

RTX

2

4

6

RTX

8

10

12

14

16

18

24

36

MONTHS

previous administration of IFNs or immunosuppressive factors; (e) lack of co-infection with HBV or HIV. Exclusion criteria were neuropsychiatric disorders, primary biliary cirrhosis, and cardiovascular, metabolic, neoplastic, and autoimmune diseases. Pregnant women and patients ingesting >40  g of alcohol per day were also excluded. The study design, which conformed to the Declaration of Helsinki and followed the Good Clinical Practice Guidelines, was approved by the Ethical Committee of the University of Bari. As shown in Fig.  40.1, patients who entered the PIRR arm received weekly subcutaneous injections of 180 mg pIFN-a2a or 1.5 mg/kg pIFN-a2b, in combination with 1,000 or 1,200  mg/day RBV according to their body weight (£75  kg or >75  kg, respectively). Both antiviral drugs were given for 48 weeks, independent of HCV genotype and viral load. In addition, RTX infusions (375 mg/m2) were administered to all PIRR patients once a week in the first month and then after 5 and 11 months. As already stated, patients assigned to the control arm received the same pIFN-a/RBV combination, without RTX. The patients were followed for up to 36 months from the end of treatment. Table  40.1 summarizes the main demographic, clinical, immunological, and virological features of the patients enrolled in the PIRR and control arms. All patients, regardless of the enrollment arm, completed the study and were examined for the entire duration of the follow-up. Definitions of clinical, immunological, virological, and molecular responses as well as the diagnostic criteria of complete response,

Table 40.1  Main clinical, immunological and virological features of 37 patients with HCV-related mixed cryoglobulinemia enrolled in two therapeutic arms Parameters Age/sex Purpura (%) Asthenia (%) Arthralgias (%) Chronic active hepatitis (%) Renal impairment (%) Peripheral neuropathy (%) Cryocrit (%; mean ± SD) Cryoglobulin type II/ type III (%) Rheumatoid factor (IU/mL) Complement fractions (mg/dL) C3 (mean ± SD) C4 (mean ± SD) Anti-HCV antibodies (%) HCV RNA (IU/mL × 106; mean ± SD) Genotype 1/genotype 2 (%)

PIRR therapy (22 patients) 63 (51–68)/ 15F-7M 100 95 74 91 22.7 27.2 6.4 ± 3.9 91/9

pIFN-a/RBV (15 patients) 59 (50–66)/ 10F-5M 100 87 73 93 26.6 20 5.5 ± 3.0 80/20

415.2 ± 138.55 482.5 ± 283.5 119.3 ± 40.7 116 ± 31 4.8 ± 3.0 3.7 ± 1.9

100 1.6 ± 0.5

100 1.9 ± 0.7

40.9/59

46.6/53.3

partial response, and no response are reported in Table 40.2. Assessment of the end-of-treatment response indicated that 54.5% of the patients enrolled in the PIRR arm achieved a complete response, compared to 33.3% of those included in the control arm (p < 0.05). The dif-

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Table 40.2  PIRR therapy: definition of responses

Type of response Complete response (a) Sustained (b) Long-term Partial response No response Relapse

Immunological Virologic response Molecular response Clinical response (CR) response (IR) (VR) (MR) Disappearance of B-cell Disappearance or remarkable clonalities from the Disappearance of Undetectability of improvement of clinical blood cryoglobulins HCV RNA features Achievement of CR + IR + VR + MR Persistence of all responses at month 6 after cessation of therapy Persistence of all responses at month 36 after cessation of therapy Achievement of any 3 of the 4 complete response criteria Absence of all response criteria Recurrence of clinical and/or immunological and/or virologic and/or molecular features at any time during the follow-up

Fig. 40.2  Probability of complete response (%) in patients receiving PIRR therapy (pIFN-a plus RBV plus RTX: study arm) and, by comparison, in patients treated with pIFN-a plus RBV (control arm)

(%) 60

54.5%

50

45.5%

40

pIFN-α+ RBV + RTX (PIRR)

36.4%

30

33.3% 27.5%

pIFN-α + RBV

22.7% 20

13.3%

10

3

6

9

12

MONTHS

ference in the probability of response between the two arms was already evident at month 5 of treatment and reached the overall response rate at month 10 for both arms (Fig. 40.2). An even more striking difference was observed in terms of response duration, defined as the time elapsed from end of treatment to the occurrence of relapse. When the maintenance rates were assessed at month 6 (sustained response) and at month 36 (longterm response) from the end of treatment, the corresponding percentages were 100% vs. 80% (p < 0.05) and 83% vs. 40% in the PIRR arm and control arm, respectively (p < 0.01). Careful monitoring of clinical, virological, immunological, and molecular parameters indicated that relapse was heralded by the reappearance of detectable HCV RNA levels, followed by a new clonal B-cell expansion, a progressive return of the complement C4 fraction to the low levels present

prior to starting therapy, and finally the reappearance of serum cryoglobulins. Recurrence of clinical signs and symptoms also followed a slowly progressive course, with the initial features usually being arthromyalgias and purpuric patches. Altogether, relapse was fully blown within a couple of months. A partial response was diagnosed in 23% and 33% of the patients in the PIRR arm and the control arm, respectively. It is worth emphasizing that, regardless of the type of treatment, patients achieving a partial response usually exhibited a good clinical response and cleared HCV RNA from the serum, but cryoglobulins and B-cell clonal expansion were mostly unchanged. Finally, the same percentages of patients (23% of those receiving PIRR therapy and 33% of those receiving pIFN-a plus RBV) were considered non-responsive, in that clinical, immunological, viro-

40  PIRR Therapy in HCV-Related Mixed Cryoglobulinemia

logical, and molecular parameters were poorly affected or remained unmodified during and after treatment. As expected, RTX administration had no influence on T-cell events throughout the study period, in contrast to the dramatic decrease of circulating CD20-positive cells. In fact, following the second infusion, CD20positive cells were as low as 1% and remained virtually undetectable in the following months before slowly reappearing in the circulation 7–10 months after the end of therapy. The extent of the impact of PIRR therapy in patients achieving a complete response was clearly shown by a number of effects, namely, undetectable levels of HCV RNA in the serum, liver, peripheral blood cells, and bone marrow; substitution of B-cell clonal expansions with polyclonal B-cell populations; and a remarkable improvement of portal and periportal inflammation, comparably shown in the seven patients who underwent a second biopsy at the end of follow-up. As already stated, although RTX is capable of deleting expanded clones, it may result in enhanced viremia; this has been demonstrated in some trials [24, 31], but not in others [32]. PIRR combination therapy seems to exert a synergistic effect. Indeed, compared with the standard of care (pIFN-a plus RBV), the rate of complete responses, including a remarkable clinical improvement, eradication of HCV RNA, disappearance of circulating cryoglobulins, and regression of clonally expanded B cells, was significantly increased following PIRR administration, occurring in over half of the patients. Obviously, a crucial point of PIRR therapy was the persistence of its therapeutic effects, especially regarding HCV RNA disappearance, during the follow-up period. Table 40.3 summarizes the results of HCV RNA monitoring in four different compartments (serum, peripheral blood cells, bone marrow, and liver) in seven responsive patients. At variance from patients showing a favorable response, the characterizing feature of patients who achieved a partial response, and especially of those totally unresponsive to PIRR therapy, was resistance of the dominant B-cell clones to RTX. Thus, analysis of the molecular profile of naïve patients may help to single out those who are more likely to respond. Conversely, whether patients with non-abatable B cell clonality are more prone to progressively develop nonHodgkin’s lymphoma remains to be established through a more detailed molecular analysis and/or a much more prolonged follow-up.

319 Table 40.3  Sequential analysis of HCV RNA levels in different biological compartments in seven MC patients who achieved a complete response 6 months after cessation Before starting of therapy therapy 1,160,441 ± 992,405 BDT

HCV RNA Serum levels (IU/mL, mean ± SD) Peripheral 196,800 ± 95,300 blood cells (IU/106 cells, mean ± SD) Liver 240,260 ± 180,600 (IU/mg tissue, mean ± SD) Bone marrow 166,440 ± 110,300 (IU/106 cells, mean ± SD)

36 months after cessation of therapy BDT

BDT

BDT

BDT

BDT

BDT

BDT

BDT below detection threshold

40.3.2 Other Experiences The effects of RTX administration in mixed (type II or III) or, more rarely, single (type I) cryoglobulinemia have been described in several papers, reviewed by Wink et al. [33]. The review was based on 142 patients with cryoglobulinemic vasculitis (mostly published as case reports) who received RTX after failure on other treatments. A much more limited experience may be retrieved in the literature regarding the standard pIFN-a plus RBV antiviral therapy in combination with RTX. Terrier et  al. [34] compared 20 MC patients treated with PIRR and 12 antiviral-intolerant patients treated with RTX alone. PIRR induced a complete clinical response in 80%, a complete immunological response in 67%, and a sustained virological response in 55% of the patients. Treatment with RTX alone induced appreciable, albeit significantly lower percentages for all responses. Using an approach roughly similar to our own [28], Saadoun et al. [35] studied 93 MC patients. Compared with those receiving the standard of care, patients treated with PIRR had a shorter time to clinical remission, better renal response rates, and a higher rate of cryoglobulin clearance. A more frequent suppression of clonal VH(1–69)-positive B cells was also observed. In addition, HCV RNA levels were not found to be enhanced in patients receiving RTX.

320

40.3.3 Adverse Events of PIRR Therapy The administration of pIFN-a plus RBV (standard of care) is frequently associated with a vast array of undesired effects. As these are very well known by physicians experienced in treating HCV-infected patients, for the sake of brevity they are not further discussed here. Instead, we briefly describe the side effects related to the use of RTX. Our own experience and that of many other groups indicate that in the large majority of patients RTX is well tolerated. However, since it is a chimeric monoclonal antibody, there may be acute mild to severe adverse events, such as fever, shivers, asthenia, headache, abdominal pain, backache, bronchospasm and chest pain, rash, and malaise. Reducing the speed of RTX infusion usually results in the mitigation or disappearance of these symptoms. Isolated literature reports of extremely rare complications include a catastrophic multi-organ ischemia due to anti-Pr cold agglutinins developing in a MC patient after treatment with RTX [36], and severe immune-mediated ulcerative gastrointestinal disease in a pediatric patient with refractory minimal-change nephrotic syndrome 42 days after RTX therapy [37]. In addition, medium- to long-term side-effects have been, although rarely, described in patients administered several infusions of RTX. These include infections, serum sickness syndrome, and severe flares of purpuric eruptions. In some patients, in  vitro immunochemical assays have shown that RTX forms a complex with the cryoprecipitating IgMk monoclonal component with rheumatoid factor activity, resulting in enhanced cryoprecipitation and severe systemic reactions [38]. Additional risks of long-term serious adverse events include progressive multifocal leukoencephalopathy, which has indeed been found to occur more frequently (rate difference 2.2 every 1,000 patient-years) in nonHodgkin’s lymphoma patients administered RTX than in those receiving standard chemotherapy regimens [39]; but, to the best of our knowledge, this has not been reported in MC patients. A “black-box” warning might nonetheless be justified.

40.4 Future Perspectives Discovery of the strong association between HCV infection and MC has deeply changed our approach to the therapy of both conditions. Compared with the

F. Dammacco and D. Sansonno

pIFN-a plus RBV standard of care, the PIRR protocol more frequently results in a successful clinical outcome and has been shown to be reasonably safe in patients with MC. However, further improvement may be expected as a number of still unclarified issues are eventually addressed. A usually neglected point is the pharmacokinetic behavior of RTX. In a study of different schedules of administration in patients with follicular lymphoma, autoimmune disorders, primary amyloidosis, and relapsed or refractory follicular and mantle cell lymphomas, the steady-state plasma concentration of RTX varied according to the different schedules of drug administration [40]. The dose of RTX that we employed in the PIRR protocol was largely drawn from its use in the oncological setting. Our own unpublished observations indicate that much lower doses of RTX may be equally effective in terms of complete response in a usually non-progressive lymphoproliferative disorder such as MC. The correct sequence and the best chronological order of the PIRR drugs also need to be established. Should pIFN-a, RBV, and RTX be given simultaneously, as we have done, or would it be better to try first to eradicate HCV infection by the pIFN-a plus RBV standard of care and then to administer RTX, with the aim of deleting the expanded B-cell clones? Or might it not be more convenient to abate the B-cell clonal expansion with RTX, despite the risk of enhanced viremia, knowing that the viral load would, in a second step, be targeted by the antiviral treatment? Should antiviral drugs be given for periods shorter than 48 weeks when MC patients are infected with HCV of genotype 2 or 3, as shown in non-cryoglobulinemic patients [41]? Can the search for interleukin (IL)-28B polymorphism in MC be used as a powerful predictor of a higher probability to achieve SVR and to tailor the duration and type of therapy to the individual patient, as is now emerging for non-cryoglobulinemic HCV-infected patients [42]? Should RTX [28] or natural IFN-a [7] be considered the standard of care for patients with type II non-viral (essential) cryoglobulinemia vasculitis? Obviously, multi-center, well-controlled clinical trials are required to answer these questions. Finally, the recent availability of new, specific drugs promises to open new avenues. Figure 40.3 is a schematic representation of the potential therapeutic approaches to HCV-positive MC, according to the severity of the clinical picture. The future treatment of HCV infection will probably be based on the addition

40  PIRR Therapy in HCV-Related Mixed Cryoglobulinemia CRYOGLOBULINEMIC HCV CARRIERS WITHOUT CRYOGLOBULINEMIC VASCULITIS

pIFN-α plus RBV (Tailored length)

321

CRYOGLOBULINEMIC VASCULITIS WITH NO OR MILD NEPHROPATHY AND/OR NEUROPATHY

CRYOGLOBULINEMIC VASCULITIS WITH OVERT NEPHROPATHY AND/OR NEUROPATHY

pIFN-α plus RBV (Tailored length)

RAPIDLY PROGRESSIVE CRYOGLOBULINEMIC VASCULITIS

CS, PE, CFM UNTIL MITIGATION; THEN:

NON NON RESPONDERS RESPONDERS RESPONDERS RESPONDERS (follow-up) (follow-up) /RELAPSERS /RELAPSERS

RESPONDERS (follow-up)

PIRR THERAPY

PARTIAL RESPONDERS

RETREATMENT

NON RESPONDERS /RELAPSERS

NEW TARGETED THERAPIES: • PROTEASE INHIBITORS: TELAPREVIR, BOCEPREVIR • MoAbs: OFATUMUMAB,90Y-IBRITUMOMAB

Fig. 40.3  Treatment algorithm of personalized (“tailored”) therapy according to the clinical severity of HCV-associated cryoglobulinemic vasculitis. pIFN-a Pegylated interferon-a,

of specifically targeted antiviral therapy for HCV, such as protease and/or polymerase inhibitors, to the standard of care. Promising results are indeed being reported in patients with HCV genotype 1 infection, by addition of one of two protease inhibitors, telaprevir or boceprevir, to pIFN-a plus RBV. This combination is able to increase the SVR rates from approximately 50% (p-IFN-a plus RBV) to 70% [43]. In addition, new anti-CD20 monoclonal antibodies have been developed, such as ofatumumab, which, compared with RTX, binds to a different epitope of CD20 and is able to induce a better complement- and antibodydependent cellular cytotoxicity. MC patients who have progressed to low-grade or follicular B-cell NHL and have relapsed or are refractory to RTX might also benefit from the administration of ibritumomab tiuxetan, a CD20-directed radiotherapeutic antibody. Again, the possible extension of these new approaches to patients with MC awaits further studies.

RBV ribavirin, RTX rituximab, CS corticosteroids, CFM cyclophosphamide, PE plasma exchange, PIRR pegylated IFN-a plus ribavirin plus rituximab combined therapy

Acknowledgements D. Sansonno is supported by the AIRC (Italian Association for Cancer Research), Milan, Italy.

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F. Dammacco and D. Sansonno 23. Vallat L, Benhamou Y, Gutierrez M et al (2004) Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection. Arthritis Rheum 50: 3668–3678 24. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 25. Zaja F, De Vita S, Mazzaro C et al (2003) Efficacy and safety of rituximab in type II mixed cryoglobulinemia. Blood 101:3827–3834 26. Cacoub P, Delluc A, Saadoun D et  al (2008) Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand? Ann Rheum Dis 67:283–287 27. Petrarca A, Rigacci L, Caini P et al (2010) Safety and efficacy of rituximab in patients with hepatitis C virus-related mixed cryoglobulinemia and severe liver disease. Blood 116:335–342 28. Terrier B, Launay D, Kaplanski G et al (2010) Safety and efficacy of rituximab in nonviral cryoglobulinemia vasculitis: data from the French autoimmunity and rituximab registry. Arthritis Care Res 62(12):1787–1795, Aug 25 [Epub ahead of print] 29. Dammacco F, Sansonno D, Piccoli C et al (2001) The cryoglobulins: an overview. Eur J Clin Invest 31:628–638 30. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a longterm study. Blood 116:343–353, Epub 2010 Mar 22 31. Lake-Bakaar G, Dustin L, McKeating J et al (2007) Hepatitis C virus and alanine aminotransferase kinetics following B lymphocyte depletion with rituximab: evidence for a significant role of humoral immunity in the control of viremia in chronic HCV liver disease. Blood 109:845–846 32. Terrier B, Saadoun D, Sene D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 60:2531–2540 33. Wink F, Houtman PM, Jansen TL (2011) Rituximab in cryoglobulinaemic vasculitis, evidence for its effectivity: a case report and review of literature. Clin Rheumatol 30(2):293– 300, Epub 2010 Oct 31 34. Terrier B, Saadoun D, Sène D et al (2009) Efficacy and tolerability of rituximab with or without PEGylated interferon alfa-2b plus ribavirin in severe hepatitis C virus-related vasculitis: a long-term followup study of thirty-two patients. Arthritis Rheum 60:2531–2540 35. Saadoun D, Resche Rigon M, Sene D et al (2010) Rituximab plus Peg-interferon-alpha/ribavirin compared with Peginterferon-alpha/ribavirin in hepatitis C-related mixed cryoglobulinemia. Blood 116:326–334 36. Ruch J, McMahon B, Ramsey G et al (2009) Catastrophic multiple organ ischemia due to an anti-Pr cold agglutinin developing in a patient with mixed cryoglobulinemia after treatment with rituximab. Am J Hematol 84:120–122 37. Ardelean DS, Gonska T, Wires S et al (2010) Severe ulcerative colitis after rituximab therapy. Pediatrics 126:e243–e246, Epub 2010 Jun 21 38. Sène D, Ghillani-Dalbin P, Amoura Z et al (2009) Rituximab may form a complex with IgMkappa mixed cryoglobulin and

40  PIRR Therapy in HCV-Related Mixed Cryoglobulinemia induce severe systemic reactions in patients with hepatitis C virus-induced vasculitis. Arthritis Rheum 60:3848–3855 39. Tuccori M, Focosi D, Blandizzi C et al (2010) Inclusion of rituximab in treatment protocols for non-Hodgkin’s lymphomas and risk for progressive multifocal leukoencephalopathy. Oncologist 15:214–1219, Epub 2010 Nov 1 40. Regazzi MB, Iacona I, Avanzini MA et  al (2005) Pharmacokinetic behavior of rituximab: a study of different schedules of administration for heterogeneous clinical settings. Ther Drug Monit 27:785–792

323 41. Mangia A (2011) Individualizing treatment duration in hepatitis C virus genotype 2/3-infected patients. Liver Int 31:36–41 42. Pearlman BL (2011) The IL-28 genotype: how it will affect the care of patients with hepatitis C virus infection. Curr Gastroenterol Rep 13(1):78–86, 2010 Nov 16 [Epub ahead of print] 43. Asselah T, Estrabaud E, Bieche I et al (2010) Hepatitis C: viral and host factors associated with non-response to pegylated interferon plus ribavirin. Liver Int 30:1259–1269

Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications

41

Franco Dammacco, Cinzia Conteduca, and Domenico Sansonno

41.1 Introduction In 1975, Schultz and Yunis [1] described a patient with immunoblastic lymphoma, mixed cryoglobulinermia (MC), and depressed serum complement levels who had received liver extract injections and orally over several years. A single cycle of the chemotherapeutic regimen cyclophosphamide, vincristine, and prednisone resulted in complete remission of the lymphoproliferative disorder, which, according to the authors, might have been due to chronic antigenic stimulation by the extracts. Thirteen years later, Monteverde et al. [2], studying multiple liver and bone marrow biopsies from 12 patients with type II “essential” MC, described cytological characteristics that closely resembled those of an immunocytoma. They advanced the hypothesis that type II MC is a manifestation of a low-grade malignant lymphoma. At that time, the close association of MC with HCV infection had not yet been detected, but it is reasonable to assume that most, if not all of the patients were indeed HCV-infected. In a conventional light microscopy study and based on immunohistochemistry, the same authors [3] examined 116 consecutive bone marrow biopsies from 76 HCVpositive patients with type II MC. They were able to provide further evidence to support the hypothesis that type II MC is characterized by an indolent monoclonal B-cell proliferation which, possibly through the action of environmental stimuli and the occurrence of mutagenic events, eventually results in overt lymphoma. F. Dammacco (*) Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy e-mail: [email protected]

Since the 1990s, a growing number of studies have been published that support an association between chronic HCV infection, with [4–6] or without [7, 8] MC, and a subset of B-cell non-Hodgkin’s lymphoma (B-NHL). In roughly 50% of the patients with chronic HCV infection and clinically active MC, low-grade B-NHL is clinically occult but can be detected by morphological and molecular analysis of bone marrow biopsy [9]. However, a remarkable geographical heterogeneity has been described in the prevalence of HCV-positive B-NHL (reviewed in [10]), suggesting that, along with persistent antigenic stimulation exerted by HCV and required for maintaining a clonal B-cell proliferation, additional factors, such as genetic and environmental features, play a role in the progression to frank lymphoid malignancy, an event that occurs in a small subgroup of patients [11, 12]. The increased risk of developing B-NHL following HCV infection was convincingly shown by Giordano et al. [13], who in a retrospective cohort study examined almost 150,000 HCV-infected patients and, for purposes of comparison, over 572,000 patients uninfected with HCV. Their results indicated that HCV infection confers a 20–30% increased risk of NHL, thus supporting an etiological role for HCV in lymphoproliferation and eventually NHL. Moreover, in patients with HCV-positive MC, the overall risk of B-NHL has been estimated to be approximately 35 times higher than in the general population [14]. In a review of 18 Italian studies including 2,736 NHL patients, the prevalence of anti-HCV antibodies was found to be 19.7% (range 8.3–37.1%). In addition, the distribution of B-NHLs by histological subtypes in case-control studies showed that the lymphoplasmacytoid subtype was apparently more frequent among

F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4_41, © Springer-Verlag Italia 2012

325

326

anti-HCV-positive cases [15]. The prevalence of HCV infection in patients with B-NHL, assessed by a metaanalysis, was approximately 15% [16], although with wide ethnic and geographical variability [16, 17]. In Italy, epidemiologic data indicate that at least about 5% of all B-NHLs are causally related to HCV [18]. Thus, if HCV chronic infection is truly a risk factor for the onset of B-NHL, even though this effect may be regional and related to the background prevalence of the virus in the community [19], then the risk of developing NHL should be lower in HCV-positive patients who achieve a treatment-induced sustained virological response (SVR) than in untreated patients. And indeed, the SVR induced by pegylated interferon-a (pIFN-a) therapy reportedly exerts a preventive effect on lymphomagenesis [20] in patients with chronic HCV infection. Since monoclonal B cells that use a restricted repertoire of immunoglobulin VH region genes are frequently detectable in HCV-positive patients with B-cell lymphoproliferative disorders, and given that this monoclonal expansion may become undetectable following a successful response to antiviral treatment, it seems reasonable to suggest that antigen persistence is crucial for B cell proliferation [21]. Hence, clearance of the virus should be able to block a process potentially capable of malignant transformation.

41.2 Therapeutic Implications Based on the evidence of a link between HCV infection and the onset of a small subset of B-NHLs, as discussed above, it was obvious to hypothesize that an effective antiviral therapy capable of inducing an SVR would result in the regression of an overt NHL or in the prevention of its occurrence. Hermine et  al. [22] studied 15 patients with splenic lymphoma characterized by villous lymphocytes: nine patients were HCVpositive and were given IFN-a2b either alone or in combination with ribavirin (RBV), whereas the remaining six patients tested negative for HCV infection but were similarly treated. Complete remission (CR) was achieved in eight patients and partial remission (PR) in one of the nine HCV-positive patients, consistent with the loss of detectable HCV RNA. Relapse of NHL was diagnosed in one patient and coincided with the reappearance of detectable serum HCV RNA. By contrast, no response was recorded among the six HCV-negative patients treated with

F. Dammacco et al.

IFN-a2b, indicating that the induction of remission in patients of the first group was ascribable to the antiviral rather than to the anti-proliferative effect of IFN. A number of subsequent therapeutic studies, summarized in a systematic review [23], have substantially confirmed these data, in that 75% of HCV-infected patients with lymphoproliferative disorders treated with pIFN-a (with or without RBV) achieved a CR, which was maintained as long as there was no detectable recurrence of HCV RNA. Moreover, no such response has been described in HCV-uninfected patients with similar disorders. A multicenter Italian study, carried out on 13 HCV-positive patients with low-grade B-NHL reported a 74% rate of complete plus partial responses. It also showed, as perhaps expected, that while hematological responses were significantly associated with clearance of the HCV viral load following antiviral treatment, they did not correlate with viral genotype and that an SVR was more frequently observed in patients infected with HCV genotype 2 [24].

41.3 Personal Experience We carried out a retrospective, single-center, open study with the aim of assessing the effect of antiviral therapy and/or chemotherapy on the infection and disease of 28 HCV-positive patients with NHL, with or without MC. All patients but one, whose diagnosis was T-cell NHL, had B-NHL and were either at first diagnosis or undergoing relapse. The REAL (Revised European-American Lymphoma) classification criteria proposed by the International Lymphoma Study Group [25] were adopted to establish the diagnosis and histotype of the NHLs. Tumor stage and possible extranodal involvement were assessed according to the Ann Arbor classification by thorough clinical examination, CT scan, and bone marrow biopsy. No patient had bulk disease. Six patients were therapeutically naïve, in that they had never been treated with antiviral therapy and/ or chemotherapy whereas the remaining seven patients had been previously treated with different chemotherapy regimens but had not received anti-neoplastic treatments for at least 6  months. All of the patients were HBV- and HIV-negative; concomitant overt neoplastic disease of any other kind was not detected. Since this was a retrospective analysis, the patients were heterogeneous and were thus divided into the

41  Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications

following subgroups: (a) three naïve patients who received first-line pIFN-a plus RBV antiviral therapy; (b) five patients who, regardless of occasional previous treatments, were given pIFN plus RBV plus rituximab (RTX), a combination referred to by the acronym PIRR and shown to be effective in patients with refractory or relapsing MC [26]; (c) five patients who had been treated (in our own or in other hospitals) with chemotherapy regimens, chosen according to the NHL histotype, and who, after an interval of at least 6 months, received pIFN-a alone (one patient) or pIFN-a plus RBV (four patients); (d) three patients who were treated with the simultaneous administration of chemotherapy plus IFN-a (two patients) or else chemotherapy plus IFN-a plus RBV (one patient); (e) the remaining 12 patients, who had been administered different chemotherapeutic regimens but not antiviral therapy. The doses of pIFN-a, RBV, and RTX are those specified elsewhere [26] and are not reported here, for the sake of brevity. The length of the antiviral treatment was 48 weeks or longer and was independent of the HCV genotype. All patients provided written informed consent and the study was approved by the Ethical Committee of our university. Response criteria, with reference to baseline values, were established by clinical evaluation, abdominal ultrasonographic examination and total body CT scan, determination of biochemical parameters, peripheral blood cell count and blood smears examination, bone marrow biopsy, and quantitative HCV RNA measurement. CR was defined as disease regression with no evidence of NHL, and PR as a 50% or greater decrease in lymph node size or a decrease in the size of the spleen by at least 50%, associated with a parallel improvement in hematological parameters over baseline values. No response and disease progression were defined as persistence or a further increase by at least 50% in lymph node and spleen size. Relapsing patients were those who, after achieving a CR or PR, showed a progressive increase in disease to lymph node involvement and/or splenomegaly. Table  41.1 summarizes the main features of 13 HCV-positive patients with B-NHL, including five naïve, one who had been treated with pIFN-a alone for short and irregular periods of time, and the remaining seven patients who had been previously given chemotherapy. Liver biopsy was performed in all but one patient and revealed chronic active hepatitis in nine

327

patients and cirrhosis in three. Six patients had cryoglobulinemic vasculitis, with a cryocrit ranging from 2% to 20% and with traces of cryoglobulins in a seventh patient. As can be inferred from Table 41.1, the interval between the diagnosis of HCV infection and the onset of B-NHL in eight patients was anywhere from 1 to 16 years. In four patients, viral infection and hematological tumor were diagnosed in the same year, whereas in one patient a gastric lymphoma was diagnosed 6 years prior to the detection of HCV. Antiviral therapy administered to this group of patients consisted of pIFN-a alone in one patient, pIFN-a plus RBV in seven patients, and the combination of pIFN-a plus RBV plus RTX in the remaining five patients. Strikingly positive effects of the antiviral treatment were recorded for both the HCV infection and the hematological tumor, in that normalization of transaminase levels (sustained biochemical response, SBR) and sustained undetectable HCV RNA (SVR) in serum for over 6  months occurred in 10 out of 13 patients (77%). Figure  41.1 depicts the clinical course of a patient (number 1 in Table 41.1) who achieved a CR following 1 year of pIFN-a plus RBV therapy. In terms of hematological response, two patients were considered unresponsive and HCV RNA was not cleared in either one. Three additional patients achieved PR but one, in whom viral clearance was not achieved, suffered disease relapse within 3  months. At the end of the antiviral therapy, two patients were judged to have a CR but this was short-lived, in that both had disease relapse after 6 and 10 months, respectively, in spite of a persistent SVR. In the remaining six patients, both an SVR and a hematological CR of variable length were determined and are still present at the time of this writing, thus ranging from 1 to 6 years. Although the limited number of patients does not allow definite conclusions to be drawn, there was no apparent correlation between hematological response and HCV genotype. Finally, consistent with the clearance of HCV RNA, a remarkable improvement was recorded in five of the six patients with MC, including regression of purpura and other signs of cryoglobulinemic vasculitis, disappearance of serum cold-precipitable proteins, normalization of complement C3 and C4 levels, and reduction of rheumatoid factor. In the sixth patient, however, antiviral therapy induced a greater than two logarithms reduction but not the disappearance of the HCV RNA load, and all the MC-related features remained mostly unchanged.

72, M

73, F

63, M

71, F

64, F

71, M

65, M

72, M

58, F

66, F

68, F

60, F

Patient 1

2

3

4

5

6

7

8

9

10

11

12

13

2004

2009

2008

2003

CAH

CAH

CAH

2003

2003

2008

2001 Biopsy not performed CAH 2005

CAH

Cirrhosis

Cirrhosis

CAH

Centroblastic centrocytic lymphoma/IVA

Centroblastic centrocytic lymphoma/IIIB Follicular lymphoma/ IIIA Diffuse large B-cell lymphoma, IIA

Main liver involvement/IVA Gastric maltoma/IVA

Gastric lymphoma, large B cells/IVA Marginal zone lymphoma/IVA Small B-lynphocytes lymphoma/IVA

Histology of liver Dx of biopsy B-NHL Histotype/stage Cirrhosis 2006 Follicular B-cell lymphoma/IIIA CAH 2007 Diffuse large B-cell lymphoma/IVA CAH 2008 Small B-lymphocytes lymphoma/IVA CAH 2006 Diffuse large B-cell lymphoma/IIA None

None

None

Prior therapy None

No/ –

No/ –

Yes/9%

Yes/5%

No/ –

Yes/2%

pIFN + RBV

pIFN + RBV + RTX

pIFN + RBV + RTX

pIFN + RBV + RTX

pIFN + RBV + RTX

pIFN + RBV + RTX

pIFN + RBV

pIFN + RBV

Antiviral therapy pIFN + RBV

R-CHOP, 6 cycles

pIFN + RBV

CBL + PDS, pIFN for 2 years R-CHOP, 6 pIFN + RBV cycles R-CHOP, 6 pIFN + RBV cycles

CEOP, 7 cycles R-CVP, 6 cycles

No/traces R-CVP, 6 cycles Yes/20% pIFN, irregularly No/ – None

No/ –

Yes/2%

Yes/3%

MC/ cryocrit No/ –

HCV RNA positive BR, VR

BR, VR

BR, VR

BR, VR

HCV RNA positive BR, VR

BR, VR

HCV RNA positive BR, VR

BR, VR

BR, VR

HCV EoT response BR, VR

SBR, SVR SBR, SVR HCV RNA positive SBR, SVR

HCV response after 1 year SBR, SVR SBR, SVR SBR, SVR HCV RNA positive SBR, SVR SBR, SVR HCV RNA positive SBR, SVR SBR, SVR

CR from 6 years

No response

PR from 2 years

CR from 5 years

CR from 6 years

CR from 1 year

No response

CR from 1 year

PR from 1 year

PR for 3 months, then relapse

NHL response and duration CR for 2 years, then relapse CR for 1 year, then relapse Still in CR

Dx diagnosis, MC mixed cryoglobulinemia, EoT end of treatment, NHL non-Hodgkin’s lymphoma, CAH chronic active hepatitis, pIFN pegylated interferon alfa, RBV ribavirin, BR biochemical response, VR virological response, SBR sustained (³6 months) biochemical response, SVR sustained (³6 months) virological response, CR complete remission, PR partial remission, RTX rituximab, R-CVP rituximab-cyclophosphamide, vincristine, prednisone, CEOP cyclophosphamide, epirubicine, vincristine, prednisone, CBL + PDS chlorambucil + prednisone, R-CHOP rituximab-cyclophosphamide, doxorubicin, vincristine, prednisone

2003, 2a/2c

2002, 2a/2c

2008, 2a/2c

1992, n.d.

2001, 1b

2004, 2a/2c

2008, 1b

2005, 1b

2009, 1b

2000, 2a/2c

1996, 2a/2c

1991, 1b

Dx of HCV Age (years)/ infection and genotype sex 74, F 2004, 1b

Table 41.1  Characteristics of 13 HCV-infected patients with B-NHL treated with antiviral therapy

328 F. Dammacco et al.

41  Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications 0

1

2

3

4

5

329 6

12 months

INTERFERON-α2a: 180 µg/week

10

a

HCV RNA ALT

7 5

2.5 2 1.5

3

ALT (x ULN)

HCV RNA (IU/mL (log )

Ribavirin: 800 mg/day

1

1

b

c

Fig. 41.1  Patient DCA, a 74-year-old housewife, was found to be HCV-positive, with genotype 1b, in 2004 but was probably infected (through undefined modalities) many years before, since she was aware that her serum transaminase levels were persistently, though mildly elevated. In the same year, liver biopsy showed macronodular cirrhosis. Serum cryoglobulins were consistently absent. In 2006, laterocervical, axillary and inguinal lymphadenopathy was detected and a lymph node biopsy led to the diagnosis of B-NHL (a). Bulky lymph node involvement was confirmed by total-body CT (b). She was

administered combined pegylated IFN-a plus RBV therapy for 1  year, which resulted in progressive normalization of serum ALT levels, undetectable serum HCV RNA, and regression of lymph node enlargement (c). Therefore, she was considered to have reached a hematologic CR. As reported in Table  41.1, 1 year after discontinuation of the antiviral treatment, sustained biochemical and virologic responses were unchanged. The patient maintained the CR of the NHL for 2  years but then relapsed. ULN upper limit of normal

The main characteristics of an additional 15 HCVinfected patients with NHL who were consistently treated with chemotherapy are summarized in Table  41.2. Liver biopsy, performed in all but two patients, resulted in the diagnosis of chronic active hepatitis in eight patients and liver cirrhosis in the remaining five. MC was detected in four patients, who had cryocrits ranging from 2% to 3%. In two other patients, cryoglobulins were initially absent but later became detectable in small amounts (cryocrit 2% in both cases). Three patients received chemotherapy in association with IFN-a, whereas in one patient chemotherapy was combined with IFN-a plus RBV. In contrast to the patients described in Table 41.1, those in Table 41.2 included five patients with high-grade NHL, two with splenic lymphoma with villous lymphocytes,

and one with T-cell NHL. Independently of histotype, seven patients had stage IVB disease. HCV infection was diagnosed 1–10  years prior to the detection of NHL in six patients, whereas serum HCV RNA and NHL were detected in the same year in eight patients. In one patient, the chronological relationship between HCV infection and NHL remained undefined. Only six patients were evaluable in terms of virological response after 1 year from the end of therapy. One patient achieved a SVR and a partial hematological response; the remaining five patients tested positive for HCV RNA. Sequential determination of their viral loads showed a progressive and remarkable increase compared to baseline values in four of these patients (data not shown). In terms of NHL response and duration, seven patients (46.6%), including the only one

71, F

78, F

58, F

70, F

67, F

78, M

85, F

70, F

78, M

82, F

Patient 2

3

4

5

6

7

8

9

10

11

12

2004, 1b

2000, 2a/2c

1994, n.d.

2005, 2a/2c

1995, n.d.

2010, 1b

1990, 1b

1993, n.d.

2005, 2a/2c

1990, 2a/2c

Dx of HCV Age (years)/ infection and genotype sex 72, F 1993, 2a/2c

Cirrhosis

CAH

CAH

Cirrhosis

CAH

CAH

Cirrhosis

CAH

CAH

2004

2006

1997

2005

1999

Nov. 2010

?

2008

2005

Nodular centrocytic lymphoma/IIIB

Diffuse T-cell lymphoma/IVA

Diffuse large B-cell lymphoma/IVB Diffuse large B-cell lymphoma/IIIA

Small B-lymphocytes lymphoma/IIIB Splenic lymphoma with villous lymphocytes Diffuse large B-cell lymphoma/IIIB Small B-lymphocytes lymphoma/IVB Small B-lymphocytes lymphoma/IVB Diffuse large B-cell lymphoma/IVB VNCOP-B 6 cycles, then ProMACEcytaBOM 6 cycles R-CHOP, 6 cycles

VNCOP-B 6 cycles, then ProMACEcytaBOM 6 cycles CHOP 6 cycles

No/ –

No/ –

No/ –

No, Yes/1%

No/ –

R-CHOP 6 cycles

R-CHOP 1 cycle

No/ –

No/ –

CHOP 6 cycles, then DHAP 3 cycles + IFN R-CHOP 1 cycle

Treatment R-CHOP 6 cycles, then R-CVP 6 cycles + IFN R-CVP 6 cycles, then CEOP 6 cycles + IFN + RBV R-CHOP 6 cycles

Yes/2%

No/ –

Yes/3%

Dx of MC/ B-NHL Histotype/stage cryocrit 2003 Follicular B-cell Yes/3% lymphoma/IVB

Biopsy not 1990 performed

Histology of liver biopsy CAH

HCV RNA-positive

n.d.

n.d.

HCV RNA-positive

HCV RNA-positive

HCV RNA-positive

HCV RNA-positive

n.d.

n.d.

HCV RNA-positive

Not evaluable

HCV RNA-positive

–

SVR

VR

HCV RNA-positive

HCV RNA-positive

HCV RNA-positive

HCV RNA-positive

HCV RNA-positive

HCV response after 1 year –

HCV EoT response HCV RNA-positive

Table 41.2  Characteristics of 15 HCV-infected patients with NHL treated with chemotherapy combined with or without antiviral therapy

No response, death after 10 months No response, death after 1 year

No response, death after 1 year

No response

CR from 9 years

So far, stable disease

Death after 3 months

PR for 6 months, then relapse

CR for 3 years, then relapse

PR for 1 year, then relapse

NHL response and duration No response

330 F. Dammacco et al.

83, F

80, M

14

15

2004, 1b

2002, 2a/2c

1999, 2a/2c

2002

2000

Biopsy not 2004 performed

Cirrhosis

CAH

Splenic lymphoma with villous lymphocytes Gastric maltoma/IVA No/ –

No, Yes/1%

Follicular B-cell No/ – lymphoma/IVB

R-CHOP 6 cycles

HCV RNA-positive

HCV RNA-positive F-ara + DHAD 6 cycles, then RTX 4 cycles Splenectomy, then n.d. undefined chemotherapy CR for 6 months, then relapse

CR for 2 years, then HCC nodules

n.d.

n.d.

CR for 3 years, then relapse

n.d.

VNCOP-B Cyclophosphamide, mitoxantrone, vincristine, etoposide, bleomycin, and prednisone, DHAP cytarabine, cisplatin, and dexamethasone, ProMACE-cytaBOM prednisone, adriamycin, cyclophosphamide, etoposide, ara-c, bleomycin, vincristine, and methotrexate, F-ara + DHAD fludarabine + mitoxantrone, n.d. not determined, HCC hepatocellular carcinoma. All other abbreviations are defined in Table 41.1

65, F

13

41  Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications 331

332

with T-NHL, were unresponsive to treatment and died within a few months to 1 year. Two additional patients achieved a PR and their disease relapsed shortly. One patient was not evaluable, and the remaining five patients were considered in CR (33.3%). Despite both the small number of patients examined and their heterogeneity, the data reported in Tables 41.1 and 41.2 and discussed above can be summarized as follows: Firstly, in 14 patients, HCV infection was detected before the diagnosis of NHL; in 12 additional patients, infection and lymphoma were diagnosed at the same time, during a thorough examination before the start of chemotherapy, but it is reasonable to assume that infection occurred long before the onset of NHL. The fact that in 92.8% of the patients HCV infection preceded, often by several years, the development of NHL again points to a role for HCV in its pathogenesis. Secondly, unless the patient has advanced-stage and life-threatening NHL requiring prompt chemotherapeutic intervention, a first-line antiviral approach consisting of pIFN-a plus RBV seems justified by the likelihood of achieving a double objective, namely an SVR to the infection and a CR of the hematological malignancy. Both objectives, along with the regression of cryoglobulinemic vasculitis, when present, are more likely attainable by the addition of RTX to pIFN-a plus RBV, as already shown in MC patients [25]. Thirdly, the relatively high number of unresponsive patients among those included in Table 41.2 suggests that chronic HCV infection adds to the severity of NHL and lowers the chance of a successful response to chemotherapy. Moreover, chemotherapeutic agents are often responsible for HCV flares. Should these results be confirmed in larger and prospective studies, an obvious consequence would be to combine pIFN-a plus RBV antiviral therapy with the chemotherapeutic regimen adopted (regardless of its composition) when treating chronically HCVinfected patients with B-NHL.

41.4 Pathogenetic Links Between HCV Infection and NHL: A Persistent Conundrum The etiopathogenetic relationships between HCV infection and the development of NHL are a fascinating, though still unclear example of viral tumorigenesis in

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humans. A general similarity is that of the role played by Helicobacter pylori in the onset of gastric MALT lymphoma, which involves the mucosa-associated lymphoid tissue and is considered an indolent marginal-zone lymphoma. Eradication of H. pylori infection results in durable remission of the tumor in approximately 80% of the patients, thus serving as convincing evidence of tumor regression following eradication of an infectious, possibly causative agent (reviewed in [27]). It has been shown [28] that H. pylori directly translocates the bacterial protein CagA into B lymphoid cells, where it may undergo tyrosine kinase phosphorylation. Additional steps include the activation of extracellular kinase and mitogen-activated protein kinase in human B-lymphoid cells, as well as up-regulation of Bcl-2 expression, resulting in the inhibition of apoptosis and the development of MALToma. In the case of HCV-related NHLs, available evidence indicates that the malignant transformation is a complex and multifactorial process, as we discussed in Chap. 40 and more extensively in an already mentioned review [10]. The fact that combined pIFN-a plus RBV antiviral therapy is able to achieve a CR in a significant percentage (roughly 75%) of HCV-positive patients with NHL (including those with aggressive histotypes) strongly argues for the oncogenic role of HCV, and thus for a favorable response that cannot be demonstrated in HCV-negative NHL patients. In addition, viral eradication seems to be capable of preventing lymphomagenesis in HCV-infected patients [20]. In those case in which antiviral treatment fails to induce a complete or partial remission of the hematological malignancy, several explanations should be considered. In addition to the heterogeneity of NHLs, which may develop in response to different inciting factors, and discarding the trivial situation of NHL patients who become HCV-infected after tumor onset, a crucial and largely unexplored point is the timeliness of the antiviral treatment; that is, once tumor growth has reached a “no-return threshold,” it may no longer be sensitive to HCV eradication. A matter of intensive debate is the nature of the mechanisms that enable HCV, likely with the intervention of genetic and environmental factors, to trigger complex host phenomena such as lymphomagenesis. Among the hypotheses more commonly considered are chronic antigenic stimulation by HCV-E2,

41  Antiviral Therapy in HCV-Positive Non-Hodgkin’s Lymphoma: Pathogenetic Implications

high-affinity interactions of HCV-E2 with the tetraspanin CD81 resulting in the activation and chronic proliferation of B cells, and direct HCV infection and replication inside discrete, permissive B cells [10]. A possible step in the elucidation of the pivotal role of HCV in a subset of NHLs will hopefully derive from an experimental model involving transgenic mice with B-lineage-restricted HCV gene expression [29]. A 25% incidence of diffuse large B-cell NHLs was observed in these animals. Although 32 chemokines, cytokines, and growth factors were investigated, in addition to serum transaminase levels, the only correlation for the occurrence of B-NHLs in the mice was the level of the a-subunit of the soluble interleukin-2 receptor (IL-2Ra). Accordingly, HCV may increase the expression of IL-2Ra on the B-cell surface, possibly as a consequence of IL-10 expression induced by HCV core protein. This, in turn, would lead to up-regulation of IL-2Ra expression on the membranes of normal and leukemic B cells. In this scenario, the HCV gene would be capable of inducing B-NHLs in the absence of a host immune response against the HCV gene product.

References 1. Schultz DR, Yunis AA (1975) Immunoblastic lymphadenopathy with mixed cryoglobulinemia. A detailed case study. N Engl J Med 292:8–12 2. Monteverde A, Rivano MT, Allegra GC et al (1988) Essential mixed cryoglobulinemia, type II: a manifestation of a lowgrade malignant lymphoma? Clinical-morphological study of 12 cases with special reference to immunohistochemical findings in liver frozen sections. Acta Haematol 79:20–25 3. Monteverde A, Sabattini E, Poggi S et al (1995) Bone marrow findings further support the hypothesis that essential mixed cryoglobulinemia type II is characterized by a monoclonal B-cell proliferation. Leuk Lymphoma 20:119–124 4. Ferri C, Caracciolo F, Zignego AL et al (1994) Hepatitis C virus infection in patients with non-Hodgkin’s lymphoma. Br J Haematol 88:392–394 5. Mazzaro C, Zagonel V, Monfardini S et al (1996) Hepatitis C virus and non-Hodgkin’s lymphomas. Br J Haematol 94: 544–550 6. Silvestri F, Pipan C, Barillari G et al (1996) Prevalence of hepatitis C virus infection in patients with lymphoproliferative disorders. Blood 87:4296–4301 7. Pozzato G, Mazzaro C, Crovatto M et al (1994) Low-grade malignant lymphoma, hepatitis C virus infection, and mixed cryoglobulinemia. Blood 84:3047–3053 8. Luppi M, Ferrari MG, Bonaccorsi G et al (1996) Hepatitis C virus infection in subsets of neoplastic lymphoproliferations

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not associated with cryoglobulinemia. Leukemia 10: 351–355 9. Rasul I, Shepherd FA, Kamel-Reid S et al (1999) Detection of occult low-grade b-cell non-Hodgkin’s lymphoma in patients with chronic hepatitis C infection and mixed cryoglobulinemia. Hepatology 29:543–547 10. Marcucci F, Mele A (2011) Hepatitis viruses and non-Hodgkin lymphoma: epidemiology, mechanisms of tumorigenesis, and therapeutic opportunities. Blood 117:1792–1798 11. Dammacco F, Sansonno D, Piccoli C et al (2000) The lymphoid system in hepatitis C virus infection: autoimmunity, mixed cryoglobulinemia, and overt B-cell malignancy. Semin Liver Dis 20:143–157 12. Zignego AL, Giannini C, Ferri C (2007) Hepatitis C virusrelated lymphoproliferative disorders: an overview. World J Gastroenterol 13:2467–2478 13. Giordano TP, Henderson L, Landgren O et al (2007) Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus. JAMA 297:2010–2017 14. Saadoun D, Landau DA, Calabrese LH et al (2007) Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation. Rheumatology (Oxford) 46:1234–1242 15. Libra M, Polesel J, Russo AE et al (2010) Extrahepatic disorders of HCV infection: a distinct entity of B-cell neoplasia? Int J Oncol 36:1331–1340 16. Gisbert JP, Garcia-Buey L, Pajares JM et al (2003) Prevalence of hepatitis C virus infection in B-cell non-Hodgkin’s lymphoma: systematic review and meta-analysis. Gastroenterology 125:1723–1732 17. Negri E, Little D, Boiocchi M et al (2004) B-cell non-Hodgkin’s lymphoma and hepatitis C virus infection: a systematic review. Int J Cancer 111:1–8 18. Fiorilli M, Mecucci C, Farci P et al (2003) HCV-associated lymphomas. Rev Clin Exp Hematol 7:406–423 19. McColl MD, Singer IO, Tait RC et al (1997) The role of hepatitis C virus in the aetiology of non-Hodgkins lymphoma–a regional association? Leuk Lymphoma 26:127–130 20. Kawamura Y, Ikeda K, Arase Y et al (2007) Viral elimination reduces incidence of malignant lymphoma in patients with hepatitis C. Am J Med 120:1034–1041 21. Weng WK, Levy S (2003) Hepatitis C virus (HCV) and lymphomagenesis. Leuk Lymphoma 44:1113–1120 22. Hermine O, Lefrère F, Bronowicki JP et al (2002) Regression of splenic lymphoma with villous lymphocytes after treatment of hepatitis C virus infection. N Engl J Med 347:89–94 23. Gisbert JP, García-Buey L, Pajares JM et al (2005) Systematic review: regression of lymphoproliferative disorders after treatment for hepatitis C infection. Aliment Pharmacol Ther 21:653–662 24. Vallisa D, Bernuzzi P, Arcaini L et al (2005) Role of antihepatitis C virus (HCV) treatment in HCV-related, lowgrade, B-cell, non-Hodgkin’s lymphoma: a multicenter Italian experience. J Clin Oncol 23:468–473 25. Harris NL, Jaffe ES, Diebold J et al (2000) Lymphoma classification–from controversy to consensus: the R.E.A.L. and WHO Classification of lymphoid neoplasms. Ann Oncol 11(Suppl 1):3–10

334 26. Dammacco F, Tucci FA, Lauletta G et al (2010) Pegylated interferon-alpha, ribavirin, and rituximab combined therapy of hepatitis C virus-related mixed cryoglobulinemia: a longterm study. Blood 116:343–353 27. Stathis A, Bertoni F, Zucca E (2010) Treatment of gastric marginal zone lymphoma of MALT type. Expert Opin Pharmacother 11:2141–2152 28. Lin WC, Tsai HF, Kuo SH et  al (2010) Translocation of Helicobacter pylori CagA into human B lymphocytes, the

F. Dammacco et al. origin of mucosa-associated lymphoid tissue lymphoma. Cancer Res 70:5740–5748 29. Kasama Y, Sekiguchi S, Saito M et  al (2010) Persistent expression of the full genome of hepatitis C virus in B cells induces spontaneous development of B-cell lymphomas in vivo. Blood 116:4926–4933

Active or Indolent Cutaneous Ulcers in Cryoglobulinemia: How Should They Be Treated?

42

Maurizio Pietrogrande

Cutaneous involvement characterizes almost all cases of cryoglobulinemic syndrome [1], with purpura, reflecting the vasculitic aspect of the disease, as the most frequent presentation. In 10% of cases, cutaneous ulcers appear on the distal third of the legs or in the perimalleolar area. In patients exhibiting Raynaud phenomena, digital ulcers may be present. The appearance, often with rapid onset, of an ulcer may involve isolated areas of normal skin or represent a complication of purpura. In some cases, the ulcers are symmetrical. The spontaneous recovery of ulcers is rare; instead, in the majority of patients they persist chronically, over periods of years. Patients describe pain, frequently very intense, that coincides with the appearance of the ulcer or, in the case of an indolent chronic ulcer, that arises suddenly. The pathogenesis of cryoglobulinemic ulcers is not completely understood [2]. Massive leukocytoclastic capillaritis diffusing to the arteriolar and venular borders is believed to be the primary mechanism. Thrombotic vessel occlusion is also observed. Moreover, autonomic neuropathy may play a pathogenetic role in cryoglobulinemic ulcers, as is the case in diabetic ulcers. Personal preliminary data suggest that peripheral autonomic dysfunction is present and is more intense in patients suffering from ulcers than in controls [3]. A cutaneous ulcer should always be evaluated with care, especially if the site and extension are atypical.

M. Pietrogrande Dipartimento di Medicina, Chirurgia e Odontoiatria, University of Milan, Milan, Italy e-mail: [email protected]

History of cigarette addiction, hypercholesterolemia, and atherosclerotic cardiopathy suggest arterial involvement that is independent of cryoglobulinemic disease. An ultrasound study of the vessels of the legs is useful, and in selected patients angiographic computed tomography (CT) or magnetic resonance imaging (MRI) study may be necessary [4]. Leg ulcer is a serious complication of cryoglobulinemia as it causes a major deterioration of the patient’s quality of life, due to the continuous pain, which compromises nocturnal sleep, and to the need for frequent refills of medications and thus multiple trips from the house to the hospital when walking is difficult. Careful analgesia is mandatory, with analgesics recommended even in the context of combination therapy. The first step in evaluating an ulcer is to clean it with room-temperature saline. All tissue debris and necrotic material must be removed. Scars, if present, may require surgical debridement. Swabs from different sites of the ulcer burden and bed should be taken, especially from ulcers that are undermined, erythematous, edematous, or cyanotic, and if a bloody secretion is observed or the ulcer bed is red. Systemic signs of inflammation, such as erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and leukocytes, have to be evaluated [5]. The therapy of cryoglobulinemic ulcer is based upon two cornerstones: treatment of the underlying disease and local and systemic cure of the ulcer. Plasma exchange [6], followed by cytotoxic drugs or provided in association with high-dose intravenous IgG [7], has been used in patients with cryoglobulinemia, with satisfactory results in most cases. Improvement and healing of ulcers during antiviral therapy in patients with

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HCV-related cryoglobulinemia has been described [8]. In our experience, chronic ulcers are resistant to antiviral therapy whereas the use of anti-CD20, either alone or in association with antiviral therapy, represents a promising approach [9]. Less aggressive treatments, such as long-term colchicine or low-antigen-containing (LAC) diet, are effective in many cases [10–13]. However, small ulcers of recent onset seldom respond to LAC diet alone. Nonetheless, in patients treated with a LAC diet and/or colchicine, there is a lower incidence of vasculitis (purpura and ulcer). In patients with ulcers who cannot be treated with the approaches described above or in whom treatment fails, re-epithelialization and scarring are favored, with a variety of topical and systemic approaches available. Important factors in the promotion of ulcer healing include a regular and sufficient intake of calories, proteins, and vitamins. Dietary supplementation products can be used in patients with poor nutritional status. Patients with cryoglobulinemic ulcers are often cirrhotic: hypoalbuminemia or edema of the lower limbs can worsen the skin lesions and prevent healing. Swabs are of enormous relevance and must be taken not only from the obviously infected ulcer, but also from those that are even slightly erythematous or edematous. Antibiotic systemic treatment based on the culture results is the “golden rule” to obtain rapid improvement of the ulcer and a reduction of pain. Since multiresistant bacteria (pseudomonas, staphylococci, enterococci) are often isolated from cutaneous ulcers, the choice of antibiotic and combination therapy at adequate doses is very important [5]. Infected ulcers can be dressed with alginate products or with foam, so that a more efficacious cleaning is possible. The use of hydrocolloids facilitates the removal of necrotic tissue. The ulcer bed must be kept moist and histotoxic antiseptics such as hydrogen peroxide should be absolutely avoided [14]. An important approach not only in the treatment of ulcers but also in their prevention is anti-aggregation or even anticoagulation. Granulation can be induced with local application of growth-inducing agents or neuroelectrical stimulation [15]. We are currently evaluating transdermal electrical stimulation in cryoglobulinemia patients with cutaneous ulcers, as this approach has proved to be very useful in the control of diabetic ulcer and neuropathy. The apparatus under study (from Lorenz Biotech) produces very short electrical impulses with a frequency and intensity that are regulated by ad hoc

M. Pietrogrande

software. Our results thus far show that, when used in the treatment of cryoglobulinemic ulcers, there is rapid induction of granulation and good control of pain. Ulcer healing can require many months, but the ulcer often becomes chronic. In this case, therapy of the underlying disease should be reconsidered. Information and counseling of the patient and his or her family members may improve compliance when long-term treatment is required [14].

References 1. Invernizzi F, Pietrogrande M, Sagramoso B (1995) The cryoglobulinemic syndrome. Clin Exp Rheumatol 13(s13):123–128 2. Auzerie V, Chiali A, Bussel A et al (2003) Leg ulcers associated with cryoglobulinemia: clinical study of 15 patients and response to treatment. Arch Dermatol 139:391–393 3. Pietrogrande M, Meroni M (2006) Dysautonomy in the cryoglobulinemic neuropathy: results of a pilot, case-control study. Ann Rheum Dis 65(II):377 4. Gardner SE, Frantz RA, Doebbeling BN (2001) The validity of clinical signs and symptoms used to identify localized chronic wound infection. Wound Repair Regen 9:178–186 5. Sibbald RG, Orsted HL, Coutts PM, Keast DH (2006) Best practice recommendations for preparing the wound bed: update. Wound Care Canada 4:15–29 6. Ferri C, Gremignai G, Bombardieri S et al (1986) Plasmaexchange in mixed cryoglobulinemia. Effects on renal, liver and neurological involvement. Ric Clin Lab 16(2):403–411 7. Pietrogrande M, Trolese L, Vozzo N, Invernizzi F (2001) Treatment with plasma-exchange and intravenous immunoglobulin for severe cryoglobulinemic syndrome alpha-interferon unresponsive. Reumatismo 53:35–40 8. Mazzaro C, Pozzato G, Moretti M et al (1994) Long-term effects of alpha-interferon therapy for type II mixed cryoglobulinemia. Haematologica 79:342–349, [published erratum appears in Haematologica 1994 Sep-Oct, 79(5):486] 9. Sansonno D, De Re V, Lauletta G et al (2003) Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20. Blood 101:3818–3826 10. Ferri C, Pietrogrande M, Cecchetti R et al (1989) Lowantigen-content diet in the treatment of patients with mixed cryoglobulinemia. Am J Med 87:519–524 11. Pioltelli P, Maldifassi P, Vacca A et al (1995) GISC protocol experience in the treatment of essential mixed cryoglobulinaemia. Clin Exp Rheumatol 13(S13):S187–S190 12. Invernizzi F, Monti G (1993) Colchicine and mixed cryoglobulinemia. Arthritis Rheum 36:722–723 13. Pietrogrande M, Meroni M, Fusi A, Amato M (2006) Therapeutical approach to the mild cryoglobulinemic syndrome: results from a retrospective cohort study. Ann Rheum Dis 65(II):70 14. RNAO (registered nurses’ association of Ontario) (2005) Ostomy Care & Management. http://www.RNAO.org/ bestpractices 15. Pietrogrande M, Meroni M (2006) Treatment of cryoglobulinemia-related neuropathic pain, with FREMS neurostimulation: results of a preliminary study. Ann Rheum Dis 65(II):376

Double Filtration Plasmapheresis: An Effective Treatment of Cryoglobulinemia

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Alfonso Ramunni and Paola Brescia

43.1

Introduction

Cryoglobulins are aggregates of circulating blood proteins that precipitate below 37°C. Their pathogenic effect is due to their ability to trigger a vasculitis by causing circulating immune complexes to accumulate within the vessel walls. This, in turn, results in activation of the complement system, and thus the release of chemotropic factors for leukocytes, vascular damage due to the release of oxygen radicals by polymorphonuclear lymphocytes, proteases, and lysosomal enzymes, and, finally, small- and in some cases medium-sized vessel occlusion [1, 2]. The specific histological picture is that of a leukocytoclastic vasculitis of the medium-sized and small vessels, caused by intravascular deposits of cryoprecipitable and non cryo-precipitable circulating immune complexes. This vasculitis is responsible for severe skin lesions, the most frequent being purpura, followed by skin ulcers as the second most common form of damage [3]. Visceral lesions, affecting the kidneys (in the form of proliferative membranous glomerulonephritis) [4], or the peripheral nervous system [5], are less frequent.

43.2

Apheresis Therapy

The therapy chosen for mixed cryoglobulinemia must address at least three different levels of treatment: (1) etiological, with the aim of eradicating hepatitis C A. Ramunni (*) Division of Nephrology, Department of Internal and Public Medicine, University of Bari, Bari, Italy e-mail: [email protected]

virus, now recognized as the main cause of mixed cryoglobulinemia; (2) pathogenic, by blocking the formation and/or removing cryoglobulins; and finally (3) symptomatic, to control the clinical symptoms. The goal of apheresis, the elimination of toxins through extracorporeal circuits, is to remove harmful molecules, in this case cryoglobulins and, as discussed below, viral particles. The removal of circulating immune complexes using therapeutic apheresis procedures elicits a number of positive results, such as restoring the function of the blocked reticulo-endothelial system, previously unable to efficiently perform normal clearance of the cryoglobulins. This triggers a process of immunomodulation and the subsequent production of immune complexes with important physicochemical characteristics, as well as the proliferation of immunocompetent cells, which makes these complexes more vulnerable to cytostatic treatment. Besides inducing remission, this response of the immune system is also essential to prevent the rebound effect, which occurs when treatment is reduced or suspended [6]. Unfortunately, practical demonstration of the benefits of plasma exchange has been described only in a few isolated cases and controlled studies are still lacking. For example, Spanish authors reported a case involving a cryoglobulinemic patient with recalcitrant ulcers associated with renal failure. The cryoglobulinemia improved following the administration of plasmapheresis in association with immunosuppressive agents [7]. The authors concluded that periodic plasmapheresis can control the level of cryoglobulins, thereby preventing renal deterioration and allowing the dose of immunosuppressor drugs to be tapered, thereby reducing their side effects.

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In an early work, a patient with essential cryoglobulinemia and cutaneous and renal involvement underwent a 50% plasma exchange (plasmapheresis) each week for 12 weeks. An improvement in renal function, healing of the ulcerations, and disappearance of the purpura were reported [8]. It might seem reasonable to ask why alternative apheresis procedures, such as double filtration plasmapheresis, are needed if plasma exchange has proven to be efficacious to treat, e.g., cryoglobulinemia and its related complications. There are essentially two reasons: first of all, a better understanding of the pathogenic mechanisms underlying cryoglobulinemia (and many other diseases) has allowed a more precise identification of the harmful molecules that need to be depleted. Second, advances in technology have provided us with more versatile equipment, capable of removing only what is necessary from the blood while preserving its other essential components. What does plasma exchange allows us to do? The first part of the procedure consists of separation of the plasma from the cellular elements of the blood. Initially, this was achieved with a centrifuge but today it is done with a filter, such as those used in dialysis but with a very different cut-off membrane that allows the plasma to pass through. The large pores of the filter block only the blood cells, from the very smallest, the platelets, to the remaining, larger red and white blood cells. Following this plasmapheresis step, in the therapeutic apheresis procedure known as plasma exchange, the collected plasma is eliminated and replaced with an equivalent volume of a solution. This replacement solution must obviously be sterile, allergen- and pyrogen-free, isotonic, isovolemic and, according to the particular needs of the patient, a plasma expander, or plasma obtained from donors, or an albumin solution. A 4% albumin solution is most frequently used; it is prepared by adding 400 mL of a 20% albumin solution to 1,600 mL of a physiological saline solution. Clearly, during plasma exchange, discarding the plasma portion means the elimination of both good and bad molecules. This is like blind fishing, simply lowering the net and removing everything that is caught. Plasma exchange is therefore a completely non-selective method. While this approach was acceptable in the past, when it was not known exactly which molecules should be removed, today, based on a better understanding of the pathophysiology of the various diseases and the identification of pathogenic molecules,

A. Ramunni and P. Brescia

selective subtraction is not only more logical but also in many cases possible. Another major disadvantage of plasma exchange is the fact that there are doubts about the safety of the plasma substitutes, as well as problems of immunization against foreign proteins. Finally, by removing many of the nutrients and other substances that are essential for survival (antibodies, coagulation factors, etc.) plasma exchange is clearly not suitable for long-term applications. Accordingly, following plasmapheresis using a centrifuge or a plasma filter, rather than discarding the plasma it can be processed using a secondary system that allows the selective removal of harmful molecules. The simplest secondary system currently available is plasma filtration. The extracorporeal circuit that can achieve plasma filtration is known as double filtration plasmapheresis (DFPP) and is schematically shown in Fig. 43.1. This semi-selective apheresis procedure consists of separating the plasma from the blood cells through a conventional plasma filter with a pore size of 0.4 mm. The plasma is subsequently processed in a secondary filter with a pore size of 0.01–0.03 mm, in which highmolecular-weight proteins are separated from the rest of the plasma elements and discarded. Unlike nonselective plasma exchange, which, as described above, removes all the plasma components, in DFPP only high-molecular-weight substances are eliminated, because their size prevents them from passing through the secondary filter, such that they are trapped and can thus be eliminated from the bloodstream. The membrane of the secondary filter is depicted in Fig. 43.2, which shows how the membrane-permeable components can be returned to the patient, while those that are impermeable, mainly globulins and lipoproteins, remain trapped in the filter. To summarize the advantages of DFPP over plasma exchange and to answer the question why the latter has been replaced by DFPP in our clinical practice, we can state that while plasma exchange is non-selective, DFPP is semi-selective and therefore allows a certain margin of choice regarding the molecules to be removed. Moreover, while plasma exchange requires a reinfusion solution that poses risks of contamination or intolerance, this is not the case with DFPP as it merely removes elements from the blood circulation, without adding foreign components or solutions. Finally, if we look at costs, as a few sporadic detractors of DFPP have done, it should be considered that the cost of the

43

Double Filtration Plasmapheresis: An Effective Treatment of Cryoglobulinemia

339

Dead-End Procedure

Heater PV

PPL

BLD

SAC

V

Cascade Filter

Heparin

V

Plasma Filter

SAD

PA

Retinate Pump

Retinate Bag

Fig. 43.1 Schematic depiction of double filtration plasmapheresis (DFPP)

Permeable components

A G E

L G

E

A

E

Electrolytes

43.3

Experiences with DFPP

Impermeable components

G L

Albumin

A

E A

A E

secondary filter for DFPP is offset by the absence of the replacement solution in plasma exchange; consequently the final cost of the two apheresis procedures is more or less the same.

A

G

Gamma Globulins

L

Lipoproteins

Filtrate

G

Plasma

Fig. 43.2 Scheme of the membrane of the secondary filter in DFPP

On the strength of the above considerations, we carried out DFPP in a patient with HCV RNA-positive type I cryoglobulinemia who had leg ulcers refractory to conventional treatment [9]. In this patient, it had not been possible to eradicate the HCV infection due to intolerance of interferon. Moreover, although a previous cycle of therapy with anti-CD20 monoclonal antibodies had brought about some relief of the burning pain in the lower limbs and improvement of the ulcerative lesions, the clinical picture worsened after a few months and was unresponsive to steroid treatment and

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a

A. Ramunni and P. Brescia Baseline

b

II week

c

IV week

d

VI week

e

VIII week

dx

sx

Fig. 43.3 Gradual scarring of the ulcers until complete healing during the course of apheresis. (a) Baseline, (b) after 2 weeks, (c) after 4 weeks, (d) after 6 weeks, (e) after 8 weeks. (From [9], with permission)

painkillers. We concluded that therapeutic apheresis was the best approach to remove the cryoglobulins. A cycle of DFPP was administered (one plasma volume treated per session), according to a schedule consisting of two sessions a week for the first 2 months, tapered to once a week in the third month, and then, based on the observation of a gradual improvement of the ulcers, to once every 10 days. Thus, in total, 30 sessions were administered. The ulcers gradually healed during the course of the apheresis cycle and completely resolved by the end of the cycle (Fig. 43.3). It must be stressed that no other therapy was administered together with the DFPP. In fact, from the onset of disease until the apheresis treatment, classic steroids treatment had been progressively reduced because no result had been elicited; indeed, before the start of apheresis, there had been a last exacerbation of the skin lesions. It was also possible to progressively reduce the administration of analgesic drugs during the course of the DFPP, due to the gradual relief of pain. Notably, the viral load of the post-apheresis cryoprecipitate was significantly reduced compared with the pre-apheresis value, confirming the capacity of DFPP to remove HCV [10]. DFPP was also proven effective in bringing about an immediate, long-term remission of severe peripheral neuropathy and neurogenic muscular atrophy in patients with essential cryoglobulinemic vasculitis [11]. The authors concluded that early use of DFPP is warranted in this type of disease, when traditional therapy proves ineffective, because of its excellent risk/benefit ratio. In general, one plasma volume is processed per session. In fact, the longer the procedure times, the less

efficient the removal of cryoglobulins with each successive plasma volume removed at a single DFPP session. This finding discourages processing more than one plasma volume per treatment. As is the case in plasma exchange, in DFPP, the number and periodicity of the procedures are not well established but seem to depend upon the rate of cryoglobulin synthesis. In fact, treatment duration cannot be established a priori as evidenced by cases of persistent disease and the frequent relapses. In view of the speed of re-synthesis of cryoglobulins and the fact that five treatments in 9 days results in the apheresis of 95% of these immunoglobulins, the treatment regimen generally consists of an initial period of intensive treatment, with ten sessions in the first 4 weeks (three sessions per week for the first 2 weeks and two sessions for the next 2 weeks), followed by a period of maintenance therapy in which the sessions are gradually tapered. The total number of sessions and overall duration of the treatment depend on the evolution of the clinical picture, and the patient’s response. Either adenosine citrate dextrose (ACD) or heparin is used as the anticoagulant. Blood flow ranges from 60 to 100 mL/min, depending on the vascular access. The apheresis procedure lasts from 90 to 180 min, depending on the plasma volume. Intensive apheresis procedures can cause a transient decrease in coagulation factors. such as antithrombin III, factor VIII, and fibrinogen. These factors are slowly restored to normal levels, whereas immunoglobulin titers take longer to return to preapheresis levels [12].

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Double Filtration Plasmapheresis: An Effective Treatment of Cryoglobulinemia

43.4

Conclusions

When vasculitis and organ damage complicate cryoglobulinemia, removing the cryoglobulins by therapeutic apheresis is a logical approach with demonstrated efficacy. In fact, a number of studies have reported good results, although controlled studies have yet to be conducted. Apheresis has already been used in association with steroid and immunosuppressive therapy in many trials, but it is difficult to establish to what extent the positive effects should be specifically attributed to the apheresis procedures, rather than to the pharmacological therapy, or the combination thereof. In recent experiences with cryoglobulinemia, as in other pathologic conditions that require the removal of immunological components, the classic plasma exchange procedure has been replaced by DFPP, which allows selective removal of the pathogenic substances from the plasma [13]. In these cases, therapeutic apheresis was used as rescue therapy, when traditional therapy had failed, and was shown to be efficacious even in these more difficult cases. It is therefore to be hoped that therapeutic apheresis, especially in the evolved, more selective form of DFPP, will be more widely applied in the treatment of cryoglobulinemic vasculitis. Treatment can begin already in the early stages of the disease and in association with pharmacological treatment. This will target both the pathogenic agent, by removing the cryoglobulins, and the etiological agents, by removing the virus particles. This integrated approach should act synergically, boosting the action of classic pharmacological approaches, and thereby bring about a faster and more efficacious resolution of the clinical picture.

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References 1. Ferri C, Zignego AL, Pileri SA (2002) Cryoglobulins. J Clin Pathol 55:4–13 2. Gorevic PD, Frangione B (1991) Mixed cryoglobulinemia cross-reactive idiotypes: implication for relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 28:79–94 3. Cohen SG, Pittelkow MR, Su WPD (1991) Cutaneous manifestations of cryoglobulinemia: clinical and histopatologic study of seventy-two patients. J Am Acad Dermatol 25: 21–27 4. Tarantino A, Banfi G, Confalonieri R et al (1995) Long term predictors of survival in essential mixed cryoglobulinemic uremic nephritis. Kidney Int 47:618–623 5. Savage COS, Harper L, Cockwell P et al (2000) Vasculitis. BMJ 320:1325–1328 6. Dominguez JH, Sha E (2002) Apheresis in cryoglobulinemia complicating hepatitis C and other renal diseases. Ther Apher 6:69–76 7. Vila AT, Barnadas MA, Ballarin J et al (2004) Cutaneous ulcers with type 1 cryoglobulinemia treated with plasmapheresis. Eur J Dermatol 14:186–189 8. Delaney VB, Fraley DS, Segal DP et al (1984) Plasmapheresis as sole therapy in a patient with essential mixed cryoglobulinemia. Am J Kidney Dis 4:75–77 9. Ramunni A, Lauletta G, Brescia P et al (2008) Doublefiltration plasmapheresis in the treatment of leg ulcers in cryoglobulinemia. J Clin Apher 23:118–122 10. Fujiwara K, Kaneko S, Kakumu S et al (2007) Double filtration plasmapheresis and interferon combination therapy for chronic hepatitis C patients with genotype 1 and high viral load. Hepatol Res 37:701–710 11. Strunk J, Taborski U, Neeck G (2002) Essential cryoglobulinemic vasculitis with severe peripheral neuropathy and neurogenic muscolar atrophy- inducing remission by cascade filtration. Z Rheumatol 61:733–739 12. Wood L, Jacobs P (1986) The effect of serial therapeutic plasmapheresis on platelet count, coagulation factors, plasma immunoglobulin, and complement levels. J Clin Apher 3:124–128 13. Ramunni A, De Robertis F, Brescia P et al (2008) A case report of double filtration plasmapheresis in an acute episode of multiple sclerosis. Ther Apher Dial 12:250–254

Emergency in Cryoglobulinemia: Clinical and Therapeutic Approach

44

Francesco Saccardo, Laura Castelnovo, and Giuseppe Monti

44.1

Introduction

Cryoglobulinemia can sometimes manifest as an emergency. Apart from the classical triad of Meltzer and Franklin (purpura, weakness and arthralgias), kidney involvement, typically presenting as membranoproliferative glomerulonephritis, cutaneous ulcers, peripheral neuropathy, and liver damage, constitutes the main organ involvement in cryoglobulinemic syndrome. Central nervous system (CNS) involvement, including transient ischemic attacks and strokes, has also been reported [1]. The pathological basis for organ damage in cryoglobulinemic syndrome has largely been attributed to leukocytoclastic vasculitis and, in some patients, hyperviscosity syndrome. In large series, the clinical evolution of cryoglobulinemic syndrome was found to be typically very slow [2]. However, in recent years, rapid worsening in clinical course has frequently been reported [3]. In some cases, the emergency cannot be controlled and the evolution is fatal, especially when kidney, gut, lung, and CNS are involved. In this chapter, we discuss the nature of the organ and CNS damage in emergency cryoglobulinemic syndrome and then briefly describe the respective therapeutic options.

F. Saccardo (*) Internal Medicine Unit, Ospedale di Saronno, Saronno, Italy e-mail: [email protected]; [email protected]

44.2

Renal Failure

Cryoglobulinemic glomerulonephritis with serum creatinine >2 mg/dl may become an emergency. It is the most frequent type of life-threatening involvement and significantly influences the prognosis and survival. Both nephrotic and nephritic syndrome develops as a consequence of membranoproliferative glomerulonephritis although in a few cases there may be mesangial proliferative glomerulonephritis and focal proliferative glomerulonephritis [4].

44.3

Gastrointestinal Involvement

Vasculitic involvement of the esophagus, stomach, small intestine, colon, or any intra-abdominal viscera can appear, resulting in gastrointestinal hemorrhage, intestinal ischemia, acute pancreatitis, or acute cholecystitis. Systemic vasculitis is seldom considered as a possible diagnosis by clinicians, because of its low prevalence compared with other, more common diseases. Gastrointestinal signs or symptoms of vasculitis are rare occurrences and the manifestation is often nonspecific. However, if there is significant involvement of the major vessels of the gastrointestinal system, lifethreatening complications, including perforation and bowel ischemia, may occur. This makes early management crucial to improve long-term morbidity and mortality. Gastrointestinal involvement is often seen in the context of multi-organ failure (catastrophic syndrome), which has a high mortality rate [5].

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Table 44.1 Organ involvement responsible for death, as determined from data in the GISC case file (199 patients) Organ involvement Hepatopathy (cirrhosis, hepatorenal syndrome, liver failure) Hepatocarcinoma Sepsis Nephropathy Heart failure Lymphoproliferative disorders Neoplasm CNS vasculitis Gastrointestinal vasculitis Lung vasculitis Hyperviscosity syndrome Multi-organ failure

% 31 4 25 3 8 6 2 8 2 2 7 2

The Gruppo Italiano per lo Studio delle Crioglobulinemie (GISC) collected data regarding the cause of death of cryoglobulinemic patients in the last 20 years. It found three cases of certain death due to gastrointestinal vasculitis (unpublished data, Table 44.1). Other rare emergency situations have been recently described, such as ischemic jejunal vasculitis [6] and spontaneous rectus sheath hematoma [7].

44.4

44.6

Hepatic Failure and Other Critical Events

Liver failure and hepatocellular carcinoma may occur in the course of cryoglobulinemic syndrome. Other emergency events include heart failure, non-Hodgkin’s lymphoma, and sepsis, with their development favored by immune impairment. In addition, in these patients, renal failure, hypertensive crisis, and coronary heart disease may precipitate acute heart failure.

Pulmonary Involvement

Lung involvement may consist of hemorrhage leading to respiratory failure. While the clinical manifestations (asthma, pleural effusion, hemoptysis, or pulmonary fibrosis) are uncommon, lung involvement is very frequent, as determined retrospectively from diagnostic signs and autopsy findings. Several reports of lung involvement in mixed cryoglobulinemia were recently reported [8–10]. In their retrospective analysis, the GISC recorded three cases of death due to pulmonary vasculitis (unpublished data, Table 44.1).

44.5

HCV infection. Involvement may be acute or subacute and characterized by diffuse or focal lesions (transient ischemic attack-like syndromes and cerebrovascular accidents). The main pathophysiologic mechanism of cerebral involvement is ischemia (or, rarely, hemorrhage) due to diffuse or segmental vasculitis of the small cerebral vessels. Occasional occlusive vasculopathy without vasculitis has been documented histologically. In these patients, ischemia may be initiated or enhanced following engorgement of the microvasculature by clumps of red cells as well as aggregates of cryoglobulins. In the same patients, vasculitis and hemorheological abnormalities can alter the clinical picture of cerebral involvement in mixed cryoglobulinemia [1].

CNS Involvement

Cerebral ischemia (in the absence of hypercoagulation or cardiovascular disease) as well as spinal or cranial nerve involvement may occur in patients with cryoglobulinemic syndrome [1, 11]. However, there are only a few well-documented cases of CNS involvement in patients with mixed cryoglobulinemia and/or

44.7

Catastrophic Syndrome

This is a term borrowed from anti-phospholipid syndrome and it occurs when, in less than a week, three or more organ systems are affected by thromboses. However, multi-organ failure is also a consequence in systemic vasculitis related to diseases other than cryoglobulinemic syndrome. Catastrophic syndrome is often life-threatening. Autopsy findings show vasculitis of the cholecystic, pancreatic, gastroinstestinal, and coronary vessels [3, 12, 13]. It remains unclear why severe vasculitis develops in only a few patients with cryoglobulinemic syndrome. Ferri et al. [10] found that in 35% of their patients with cryoglobulinemic vasculitis, the disease had a moderate-to-severe clinical course, with the prognosis strongly affected not only by cryoglobulinemia but also by associated conditions such as HCV-related liver failure.

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Emergency in Cryoglobulinemia: Clinical and Therapeutic Approach

In the study of Ramos Casals et al. [3], life-threatening cryoglobulinemia was found in 14% of patients with cryoglobulinemic syndrome.

44.8

Therapy

44.8.1 Renal Failure Treatment includes corticosteroids, immunosuppressive agents (cyclophosphamide, azathioprine or mycophenolate mofetil) and plasma exchange [14]. Other options include antiviral therapy [15] and rituximab [16]. There are limited data regarding antiviral treatment of HCV-associated glomerulonephritis. The recommended therapeutic strategy depends on the severity of the kidney disease. The optimal strategy for HCV-associated nephritis in cryoglobulinemic syndrome is still undefined. Interferon (IFN)-a treatment has been used increasingly in the past decade. Currently, the first-line treatment for patients with mild to moderate clinical and histological kidney damage is antiviral therapy with IFN-a plus ribavirin. Standard IFN doses are more effective than immunosuppressive agents in lowering proteinuria in patients with HCV-related cryoglobulinemic glomerulonephritis [15]. However, during acute immunological flare-ups, antiviral treatment is usually insufficient to control renal disease. Steroids, immunosuppressive drugs (usually cyclophosphamide), and plasma exchange are advocated in these cases, although this therapeutic approach may cause a substantial increase in viremia, thus exacerbating chronic hepatitis C disease. Nevertheless, immunosuppression is still regarded as the first-line intervention if renal involvement is severe. The rationale is further reinforced if peripheral B-cell expansion and lymphoid infiltrates in bone marrow are detected [16]. These infiltrates are viewed as early lymphomas and are largely indistinguishable from small lymphocytic lymphomas and immunocytomas. Rituximab has raised hopes for a new therapeutic approach in patients with severe vasculitic manifestations and active cryoglobulinemic nephropathy. Promising results were reported recently with a B-lymphocyte depletion protocol in patients with active cryoglobulinemic syndrome. However, treatment options for patients with cryoglobulinemic glomerulonephritis remain very limited [16].

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44.8.2 Gastrointestinal Involvement A combination of corticosteroids, immunosuppressive agents (cyclophosphamide, azathioprine, or mycophenolate mofetil), and plasma exchange may be effective as initial therapy Quartuccio et al. described five patients with gastrointestinal cryoglobulinemic HCV-related vasculitis treated with rituximab 375 mg/m2/week for 4 weeks, with clinical improvement in all patients. One of them developed a cytomegalovirus-related colitis that required a delay in therapy. Two out of five patients eventually relapsed and one died of hepatorenal syndrome. The authors concluded that rituximab could be used as first-line therapy in gastrointestinal HCVrelated vasculitis, (unpublished data presented at the 16th National Congress of GISC Milano 2009). However more data are necessary.

44.8.3 Pulmonary Involvement There are no general guidelines regarding treatment. Some patients may benefit from corticosteroids or immunosuppressive agents but the mortality rate is still high. Combination therapy with corticosteroids, immunosuppressive agents (cyclophosphamide, azathioprine or mycophenolate mofetil), and plasma exchange could be suggested in severe cases.

44.8.4 Neurologic Involvement Plasma exchange is the treatment of choice in patients with acute severe peripheral neuropathy. In this setting, neuropathy might also be successfully treated with high doses of corticosteroids or plasma exchange plus intravenous immunoglobulins [17]. The therapy of cryoglobulnemic peripheral neuropathy during the non-acute phase could include different options, such as cyclophosphamide, either alone or in combination with corticosteroids and other cytotoxic agents, such azathioprine, methotrexate, and mycophenolate mofetil. The use of IFN is debated and careful evaluation must be made before its administration. Thus far, there are only anecdotal findings concerning the use of rituximab in treating peripheral and CNS involvement [1].

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44.8.5 Catastrophic Syndrome There are several treatment options for catastrophic cryoglobulinemic syndrome, but no definitive regimens. As the number of patients with catastrophic syndrome is small, prospective, randomized, controlled trials on therapeutic strategies have not been possible. First-line therapies include immunosuppressant and cytotoxic agents, plasma exchange, and high-dose corticosteroids. Plasma exchange plus intravenous immunoglobulins comprise the second-line treatment for catastrophic syndrome. Other therapeutic strategies have been described, such as the use of rituximab [18].

44.8.6 Hepatic Failure and Other Critical Events In hepatic failure, a conservative approach, consisting of diet, diuretics, the prevention and control of hemorrhage, and the discontinuation of hepatotoxic drugs, may allow patients to overcome the critical phase. If evolution to hepatocellular carcinoma occurs, surgical treatment or radiofrequency ablation is indicated. Targeted antibiotic therapy may be provided to patients with sepsis. If this treatment is not feasible, then the alternative is empirical broad-spectrum antibiotic therapy. Lee et al. [17] reported on two cryoglobulinemic patients with heart failure and pulmonary edema who were successfully treated with pulse therapy of intravenous methylprednisolone and a course of plasma exchange.

44.9

Conclusion

In view of these results, the best therapeutic strategy for life-threatening cryoglobulinemia remains to be systematically defined. Currently, a combination of corticosteroids, immunosuppressive agents, and plasmapheresis is reasonable as initial therapy. In HCV-positive patients, the addition of a combined antiviral treatment is indicated. However, while antiviral agents might play a beneficial role [3] they are not life-saving during mixed-cryoglobulinemiarelated emergencies. Moreover, since the response to antiviral treatment is generally slow, a combination of

corticosteroids with cytotoxic agents is initially useful for the control of life-threatening organ involvement. Recently, data on the efficacy of anti-CD20 monoclonal antibody (rituximab) have been reported in patients with HCV-MC vasculitis. It appears that rituximab is very efficacious against cryoglobulin production and the clinical consequences thereof, such as inflammatory vascular lesions. A complete clinical response is achieved in 60–70% of patients, with cryoglobulin clearance in one-third of patients. The absence of efficacy following HCV viral clearance and, furthermore, the potential increase in HCV viral load suggest the use of combined antiviral treatment to block the HCV infection trigger. In fact, up to the 30% of patients treated with rituximab evidenced vasculitis relapse after therapy, during B-cell recovery [19]. A practical conclusive intensive approach that reflects our best current knowledge is: (a) plasma exchange thrice weekly together with cytotoxic agents, such as cyclophosphamide (150 mg/day) or with intravenous immunoglobulins; (b) pulse therapy with intravenous methylprednisolone (1 g daily for 3 days); and (c) high doses of corticosteroids (prednisone 1–1.5 mg/ kg/day). The therapeutic approach is the same for cryoglobulinemic and non-cryoglobulinemic patients in nonvasculitic emergencies. However, the complexity of cryoglobulinemic syndrome and the fragility of these patients must be considered, with therapeutic procedures started as soon as possible [18].

References 1. Filippini D, Colombo F, Jann S et al (2002) Central nervous system involvement in patients with HCV-related cryoglobulinemia: literature review and a case report. Reumatismo 54(2):150–155 (Review) 2. Monti G, Galli M, Invernizzi F et al (1995) Cryoglobulinemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC, Italian Group for the Study of Cryoglobulinemias. Q J Med 88(2):115–126 3. Ramos-Casals M, Robles A, Brito-Zerón P et al (2006) Lifethreatening cryoglobulinemia: clinical and immunological characterization of 29 cases. J Semin Arthritis Rheum 36(3):189–196 4. Tarantino A, Campise M, Banfi G et al (1995) Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis. Kidney Int 47(2):618–623 5. Garas G, Morgan CA, Aust NZ (1996) Hepatitis C and mixed cryoglobulinaemia: a case of primary gastrointestinal necrotising vasculitis. J Med 26(1):110–111

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6. Kay J, Mc Cluskey RT (2005) Case records of the Massachusetts General Hospital. Case 31–2005. A 60-yearold man with skin lesions and renal insufficiency. N Engl J Med 353(15):1605–1613 7. Pompili M, Pizzolante F, Larocca LM et al (2006) Ischaemic jejunal vasculitis during treatment with pegylated interferon alpha-2b and ribavirin for hepatitis C virus related cirrhosis. Dig Liver Dis 38(5):352–354 8. Ødegård IL, Høgåsen K, Paulsen D et al (2009) A 52-year old woman with fever and respiratory failure. Tidsskr Nor Laegeforen 129(22):2369–2372 9. Witte L, Rupp J, Heyer P et al (2008) Fibrosing alveolitis with hepatitis C-related cryoglobulinemia. Dtsch Med Wochenschr 133(14):709–712 10. Ferri C (2008) Mixed cryoglobulinemia. Orphanet J Rare Dis 3:25 (Review) 11. Cacoub P, Saadoun D, Limal N et al (2005) Hepatitis C virus infection and mixed cryoglobulinaemia vasculitis: a review of neurological complications. AIDS 19(Suppl 3):S128–S134 12. Saccardo F, Novati P, Sironi D et al (2007) Causes of death in symptomatic cryoglobulinemia: 30 years of observation in a Department of Internal Medicine. Dig Liver Dis 39(Suppl):S52–S54 13. Au WY, Kwok JS, Chu KM et al (2005) Life-threatening cryoglobulinemia in HCV-negative Southern Chinese and a

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novel association with structural aortic abnormalities. Ann Hematol 84(2):95–98 Ben Fatma L, Ben Hamida F, Aoudia R et al (2007) Membranoproliferative glomerulonephritis in patients with cryoglobulinemia complicating hepatitis C virus: report of 11 cases. Tunis Med 85(3):220–224 Fabrizi F, Lunghi G, Messa P et al (2008) Therapy of hepatitis C virus-associated glomerulonephritis: current approaches. J Nephrol 21(6):813–825 (Review) Roccatello D, Baldovino S, Rossi D et al (2004) Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinemic glomerulonephritis. Nephrol Dial Transplant 19(12):3054–3061 Agarwal A, Clements J, Sedmak DD et al (1997) Subacute bacterial endocarditis masquerading as type III essential mixed cryoglobulinemia. J Am Soc Nephrol 8(12): 1971–1976 Monti G, Saccardo F (2007) Emergency in cryoglobulinemic syndrome: what do to? Dig Liver Dis 39(Suppl 1): S112–S115 Saadoun D, Resche-Rigon M, Sene D et al (2008) Rituximab combined with Peg-interferon-ribavirin in refractory hepatitis C virus-associated cryoglobulinaemia vasculitis. Ann Rheum Dis 67(10):1431–1436

Novel Therapeutic Approaches to Cryoglobulinemia: Imatinib, Infliximab, Bortezomib, and Beyond

45

Giampaolo Talamo and Maurizio Zangari

45.1

Introduction

Cryoglobulinemia is a rare disorder characterized by the presence of immunoglobulins that precipitate at temperatures below 37°C and by the development of leukocytoclastic necrotizing vasculitis, involving small and medium-sized arteries in multiple body systems [1]. Clinical manifestations include skin lesions (e.g., leg ulcers and purpura), arthralgias, weakness, liver involvement, peripheral neuropathy, and potentially life-threatening involvement of vital organs, such as the kidneys [2, 3]. Several disorders are associated with cryoglobulinemia, including infections (e.g., hepatitis C virus), autoimmune diseases (e.g., systemic lupus erythematosus, rheumatoid arthritis, scleroderma, and Sjögren’s syndrome), and neoplastic diseases (e.g., lymphoplasmacytic lymphoma, chronic lymphocytic leukemia, and monoclonal gammopathy of undetermined significance). The Brouet classification [4] distinguishes three types of cryoglobulinemia. Type I consists of monoclonal immunoglobulins, usually IgM. In type II, there is a mixture of a polyclonal component, usually IgG, and a monoclonal component, usually IgM. Type III consists of polyclonal IgG and polyclonal IgM. In type II and type III cryoglobulinemias, IgM acts as rheumatoid factor, with binding to polyclonal IgG. In this chapter, the term “mixed cryoglobulinemia” refers to the type II and III cryoglobulinemias of the Brouet classification.

G. Talamo (*) Division of Hematology-Oncology, Penn State Hershey Cancer Institute, Hershey, PA, USA e-mail: [email protected]

Conventional treatment of cryoglobulinemia in the absence of viral infection typically involves corticosteroids, immunosuppressives, and plasma exchange [5–9]. The molecular revolution that has characterized the last few decades of medical advances has made available to clinicians an armamentarium of novel biological agents that have emerged as effective therapies for a wide spectrum of diseases. Due to the rarity of cryoglobulinemia, the medical literature does not provide consistent evidence-based treatment guidelines, especially when the disease becomes refractory to conventional treatment. No randomized controlled trials have been conducted with biological agents, and available data are contained in case reports or small series, often with brief follow-up. Therefore, it is likely that publication bias confers a favorable view for some of the drugs discussed in this chapter, overestimating their efficacy and understating their risks, so that the use of novel agents in relapsing/refractory cryoglobulinemias cannot be definitively advocated.

45.2

Novel Agents for the Treatment of Mixed Cryoglobulinemia

A biological agent commonly used in patients with mixed cryoglobulinemia refractory to standard therapy is rituximab [10]. This is a genetically engineered chimeric murine-human monoclonal antibody directed against the CD20 antigen, a surface protein primarily found on B lymphocytes. The B-cell depletion induced by rituximab has clinical applications in the treatment of many types of lymphoma, leukemia, and autoimmune diseases, as well as in mixed cryoglobulinemia, which is mainly characterized by the proliferation of

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B-cell clones producing pathogenic IgM. The role of rituximab in the therapy of cryoglobulinemia has been reviewed in detail elsewhere in this book. The use of rituximab has been associated with an improvement in the vasculitic aspect of mixed cryoglobulinemia, either HCV-induced or essential, with complete and durable responses [11–14].

45.2.1 TNF Inhibitors Infliximab is a chimeric monoclonal antibody against tumor necrosis factor a (TNFa), a cytokine involved in the regulation of immune cells and inflammation. Infliximab is routinely used for the treatment of several immune-mediated diseases, such as psoriasis, rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, and ulcerative colitis [15]. The rationale for the use of TNFa blockade in mixed cryoglobulinemia is based on the observation that the soluble TNFa receptor concentration is increased in this disorder, suggesting an important role for this cytokine in the pathogenesis of mixed cryoglobulinemia [16]. Clinical experience with infliximab in non-cryoglobulinemic vasculitides, such as rheumatoid arthritis, Wegener’s granulomatosis, and other ANCA-associated vasculitides, has been overall successful [17, 18]. A retrospective study by Bartolucci et al. in ten patients with refractory systemic vasculitis showed a positive clinical effect of infliximab treatment. All patients experienced rapid improvement that was still evident at 6 months. A patient with mixed cryoglobulinemia that manifested as purpura, painful cutaneous ulcers, polyneuropathy, and glomerulonephritis experienced ulcer attenuation and resolution of neuropathic pain within 2 weeks of therapy; however, the disease relapsed 4 months later, and the patient subsequently died of progressive disease [18]. The same authors reported results from a study with longer follow-up on the effect of infliximab therapy in a group of 15 patients with refractory vasculitides, but they did not include additional cases of cryoglobulinemia [19]. The remission rate was 73%, comparable to the 83–100% reported in similar trials of infliximab for the treatment of refractory systemic necrotizing vasculitides [17, 20, 21]. Two patients with mixed cryoglobulinemia, reported by Chandesris et al., received treatment with singleagent infliximab, but no significant clinical improvement was observed [22]. Koukoulaki et al. described a

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case of HCV-negative mixed cryoglobulinemia resistant to corticosteroids, cyclophosphamide, and plasma exchange; treatment with a combination of rituximab and infliximab produced a complete clinical recover, evident for more than 10 months [23]. In summary, the experience with infliximab in cryoglobulinemia is currently limited. Clinicians should be aware that the use of infliximab can be associated with a variety of side effects, such as hepatotoxicity, secondary lymphoma and other cancers, drug-induced lupus, demyelinating central nervous system disorders, and infections, especially reactivation of hepatitis B and tuberculosis [24]. Two other TNF antagonists are currently available: adalimumab, a fully human monoclonal antibody, and etanercept, a soluble protein engineered by fusing part of the TNF-receptor with the Fc portion of an IgG antibody. Etanercept is merely a TNFa-neutralizing agent, whereas infliximab not only blocks TNFa by binding to the receptor, but also induces apoptosis of TNFaexpressing activated T lymphocytes [25]. These different mechanisms of action could explain why in some diseases, such as Crohn’s disease, only infliximab has demonstrated efficacy [26]. Inconsistent results were obtained in patients with HCV-related cryoglobulinemia treated with singleagent etanercept. Only one of the six treated patients demonstrated clinical improvement [27]. In a prospective study of etanercept in six patients with active chronic hepatitis C and rheumatoid arthritis, cryoglobulinemia persisted in two patients diagnosed at baseline and developed in two of four patients who did not have the disease before treatment [28]. This experience suggests that TNF inhibition with etanercept has no role in the treatment of patients with mixed cryoglobulinemia.

45.2.2 Imatinib Imatinib is a tyrosine kinase (TK) inhibitor that blocks the activity of c-Abl, c-Kit, and PDGF TK receptors. The development of imatinib has, at least to date, been the most successful achievement of molecular biology applied to hematologic malignancies [29]. In a transgenic mouse model of mixed cryoglobulinemia, imatinib was able to suppress the production of cryoglobulins, and to dramatically improve both systemic and organ side effects, which translated into

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Novel Therapeutic Approaches to Cryoglobulinemia: Imatinib, Infliximab, Bortezomib, and Beyond

improvement of overall survival in mice [30]. The suppression of cryoglobulin was attributed to the inhibition of antibody-secreting peripheral B cells, by direct imatinib suppression of early B-cell development. In fact, both c-Abl and c-Kit are known to be involved in B-cell development [31, 32]. These pre-clinical observations suggest imatinib as a potentially effective agent in the treatment of cryoglobulinemia.

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Type I cryoglobulins are monoclonal IgM, IgG, or IgA antibodies typically produced by hematologic malignancies, such as lymphoplasmacytic lymphoma (Waldenstrom’s macroglobulinemia), multiple myeloma, and chronic lymphocytic leukemia. Type I cryoglobulinemia can also be observed in the absence of overt hematologic malignancies, with monoclonal gammopathy of undetermined significance (MGUS). The management of type I cryoglobulinemia is usually directed toward the underlying hematologic neoplasm and it traditionally involves the use of corticosteroids and chemotherapeutic agents.

addition to the small number of patients, clear conclusions on the efficacy of rituximab in type I cryoglobulinemia cannot be drawn. In fact, a review article describing 57 patients with cryoglobulinemia who were treated with rituximab included only two patients with type I cryoglobulinemia [12]. Alemtuzumab is a humanized monoclonal antibody directed against CD52, a protein present on the surface of mature T and B lymphocytes. After cell surface binding, alemtuzumab induces apoptosis of CD52+ cells. This specific effect has been successfully utilized for the treatment of chronic lymphocytic leukemia, T-cell lymphoma, graft-vs.-host-disease prophylaxis in allogeneic stem cell transplantation, and in several other autoimmune diseases [38]. A patient with severe type I cryoglobulinemia associated with IgGl MGUS, refractory to traditional therapies (plasmapheresis, methylprednisolone, cyclophosphamide, and rituximab), was successfully treated with alemtuzumab [37]. No other experiences with alemtuzumab in cryoglobulinemias have been reported in the literature. Toxicities associated with alentuzumab include fever, cytopenias, and opportunistic infections, especially reactivation of cytomegalovirus. Antimicrobial and antiviral prophylaxis is usually recommended.

45.3.1 Anti-lymphocyte Monoclonal Antibodies

45.3.2 Thalidomide and Immunomodulatory Drugs

While clinical experiences indicate rituximab as the biological agent of choice in the treatment of refractory mixed cryoglobulinemia, no strong evidence supports its use in type I cryoglobulinemia [33]. The activity of rituximab in type I cryoglobulinemia can be questioned, based on two main arguments. First is the expression of CD20 in target cells. Cryoglobulinemiaassociated B-cell neoplasms, such as Waldenstrom’s macroglobulinemia and hairy cell leukemia, are CD20positive and usually responsive to rituximab therapy [34]. However, in patients with myeloma or non-IgM MGUS, the neoplastic plasma cells do not usually express CD20 [35], and rituximab monotherapy has produced disappointing results [36]. Second is the possibility of “flare” reactions, as rituximab can induce a transient exacerbation of symptoms in patients with Waldenstrom’s macroglobulinemia. Such reactions have been associated with massive B-cell apoptosis and cryoglobulin release [34]. For these reasons, in

Thalidomide and its derivatives represent a new class of antineoplastic drugs called immunomodulatory drugs (IMiDs). The immunomodulatory, anti-inflammatory, and anti-angiogenic properties have shown efficacy in multiple myeloma and other hematologic malignancies. IMiDs target tumor cells both by direct cytotoxicity and indirectly, by interfering with several components of the bone marrow microenvironment [39]. Thalidomide, initially introduced in Germany in 1957 as a sedative, was withdrawn from the market in 1961 because it was associated with severe fetal malformations. The renewed interest in thalidomide is related to the discovery of its activity in patients with multiple myeloma [40]. Thalidomide induces apoptosis of myeloma cells and down-regulates the expression of several cytokines involved in cell proliferation and survival, such as TNFa, interleukin-6, and vascular endothelial growth factor (VEGF). However, the precise mechanism of action has not been fully

45.3

Novel Agents for the Treatment of Type I Cryoglobulinemia

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elucidated [41]. The main toxicities of thalidomide include sedation, peripheral neuropathy, bradycardia, hypotension, constipation, venous thromboembolism, and fetal malformations. Due to the increased risk of venous thromboembolism, antithrombotic prophylaxis is recommended. Two cases of type I cryoglobulinemia successfully treated with thalidomide have been reported. The first consisted of a patient with cutaneous cryoglobulinemic vasculopathy associated with IgGk MGUS. Treatment with low-dose thalidomide (50 mg daily) produced healing of skin ulcers within 2 months of therapy [42]. In the second case, a patient with IgGk myeloma and cryoglobulinemic vasculitis was treated with pulse dexamethasone and thalidomide, 100– 200 mg daily. After 2 months of therapy, serum cryoglobulins became undetectable, with a parallel resolution of the vasculitic picture. The myeloma continued to remain in complete remission after months of thalidomide monotherapy. The authors believed that dexamethasone did not significantly contribute to the clinical response, because of the failure of earlier treatment with single-agent corticosteroids [43]. Obviously, more experience is needed before thalidomide can be recommended for use in type I cryoglobulinemia. Moreover, thalidomide should be used with caution in the treatment of cryoglobulinemic vasculitis, because cutaneous purpura with vasculitis can indeed be one of its adverse reactions [44]. The thalidomide analogues lenalidomide and pomalidomide are second-generation ImiDs, developed with the intent to enhance anticancer properties and to improve tolerability [39]. While no clinical experience has been reported with pomalidomide, a patient with myeloma associated with cryoglobulinemia was successfully treated with lenalidomide [45].

45.3.3 Bortezomib Bortezomib was the first proteasome inhibitor agent approved in the USA for the treatment of multiple myeloma. In 2008, we published the case of a patient with type I cryoglobulinemia that responded successfully to single-agent bortezomib treatment. The patient had developed an IgGk smoldering myeloma complicated by type I cryoglobulinemia, which was refractory to multiple lines of therapy, including

G. Talamo and M. Zangari

plasma exchange, corticosteroids, cyclophosphamide, and autologous stem cell transplantation [46]. Based on the lack of clinical response of myeloma to rituximab therapy [35], we decided to start treatment with bortezomib [47]. After two months of treatment, the patient achieved complete clinical remission of the cryoglobulinemia, with resolution of the hemorrhagic acrocyanosis and vasculitic lesions (Fig. 45.1). Response to therapy was also documented by the progressive decline and normalization of tumor markers, best represented in this patient by the serum free k light chain levels (quantitative IgG in the serum was within normal limits even at baseline). Our report highlights the importance of selectively targeting the clonal disorder responsible for the type I cryoglobulinemia. We subsequently treated with bortezomib two additional patients with type I cryoglobulinemia (data not published), and we observed again a positive clinical response. Unfortunately, in both cases, bortezomib was discontinued prematurely, due to the emergency of peripheral neuropathy. While the incidence of peripheral neuropathy in type I cryoglobulinemia has not been clearly determined, in mixed cryoglobulinemia it varies from 7% to 100% of cases [48–50]. Since peripheral neuropathy is a common adverse reaction associated to bortezomib therapy, we advise particular caution when this agent is used in patients with pre-existing cryoglobulinemic polyneuropathy [47].

45.4

Conclusions

In the absence of controlled clinical studies, the treatment of cryoglobulinemia refractory to standard therapies is highly challenging. The two main types of cryoglobulinemia (i.e., mixed and type I) have different pathophysiologies and different responses to treatments. In this chapter, we reviewed the published experiences of novel therapies in the treatment of both types of cryoglobulinemia (see Table 45.1 for a synopsis), but, since the available medical literature provides only small case series or single case reports, conclusions cannot be reached and specific recommendations cannot formulated. Thus, at least for now, the treatment of relapsing/refractory cryoglobulinemia should be individualized and based on the best clinical judgment of the treating physician.

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Novel Therapeutic Approaches to Cryoglobulinemia: Imatinib, Infliximab, Bortezomib, and Beyond

Fig. 45.1 Hemorrhagic acrocyanosis in the external ear (a) and purpuric vasculitis with confluent areas in a leg (b) of a patient with type I cryoglobulinemia. Improvement of the same lesions after treatment with bortezomib (c and d)

a

c

b

d

353

354

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Table 45.1 Summary of the published clinical experiences of novel agents in the treatment of refractory/relapsing cryoglobulinemia (see text for detailed discussion) Type of cryoglobuAgent linemia Infliximab Mixed Mixed Mixed Etanercept Mixed Alemtuzumab I Thalidomide I I Lenalidomide I Bortezomib I

Number of patients 1 2 1 6 1 1 1 1 1

Response to therapy Yes No Yes No Yes Yes Yes Yes Yes

Reference [18] [22] [23] [27] [37] [42] [43] [45] [46]

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30. Iyoda M, Hudkins KL, Becker-Herman S et al (2009) Imatinib suppresses cryoglobulinemia and secondary membranoproliferative glomerulonephritis. J Am Soc Nephrol 20:68–77 31. Lam QL, Lo CK, Zheng BJ et al (2007) Impaired V(D)J recombination and increased apoptosis among B cell precursors in the bone marrow of c-Abl-deficient mice. Int Immunol 19:267–276 32. Rolink A, Streb M, Nishikawa S et al (1991) The c-kitencoded tyrosine kinase regulates the proliferation of early pre-B cells. Eur J Immunol 21:2609–2612 33. Nehme-Schuster H, Korganow AS, Pasquali JL et al (2005) Rituximab inefficiency during type I cryoglobulinaemia. Rheumatology (Oxford) 44:410–411 34. Gertz MA, Anagnostopoulos A, Anderson K et al (2003) Treatment recommendations in Waldenstrom’s macroglobulinemia: consensus panel recommendations from the Second InternationalWorkshoponWaldenstrom’sMacroglobulinemia. Semin Oncol 30:121–126 35. Kapoor P, Greipp PT, Morice WG et al (2008) Anti-CD20 monoclonal antibody therapy in multiple myeloma. Br J Haematol 141:135–148 36. Moreau P, Voillat L, Benboukher L et al (2007) Rituximab in CD20 positive multiple myeloma. Leukemia 21:835–836 37. Chu D, Stevens M, Gladstone DE (2007) Severe, refractory, non-malignant type I cryoglobulinemia treated with alemtuzumab. Rheumatol Int 27:1173–1175 38. Gribben JG, Hallek M (2009) Rediscovering alemtuzumab: current and emerging therapeutic roles. Br J Haematol 144:818–831 39. Knight R (2005) IMiDs: a novel class of immunomodulators. Semin Oncol 32:S24–S30 40. Palumbo A, Facon T, Sonneveld P et al (2008) Thalidomide for treatment of multiple myeloma: 10 years later. Blood 111:3968–3977

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41. Paravar T, Lee DJ (2008) Thalidomide: mechanisms of action. Int Rev Immunol 27:111–135 42. Sampson A, Callen JP (2006) The cutting edge: thalidomide for type 1 cryoglobulinemic vasculopathy. Arch Dermatol 142:972–974 43. Cem Ar M, Soysal T, Hatemi G et al (2005) Successful management of cryoglobulinemia-induced leukocytoclastic vasculitis with thalidomide in a patient with multiple myeloma. Ann Hematol 84:609–613 44. Witzens M, Moehler T, Neben K et al (2004) Development of leukocytoclastic vasculitis in a patient with multiple myeloma during treatment with thalidomide. Ann Hematol 83:467–470 45. Lin RJ, Curran JJ, Zimmerman TM et al (2010) Lenalidomide for the treatment of cryoglobulinemia and undifferentiated spondyloarthropathy in a patient with multiple myeloma. J Clin Rheumatol 16:90–91 46. Talamo G, Claxton D, Tricot G et al (2008) Response to bortezomib in refractory type I cryoglobulinemia. Am J Hematol 83:883–884 47. Cavo M (2006) Proteasome inhibitor bortezomib for the treatment of multiple myeloma. Leukemia 20:1341–1352 48. Chad D, Pariser K, Bradley WG et al (1982) The pathogenesis of cryoglobulinemic neuropathy. Neurology 32:725–729 49. Zaltron S, Puoti M, Liberini P et al (1998) High prevalence of peripheral neuropathy in hepatitis C virus infected patients with symptomatic and asymptomatic cryoglobulinaemia. Ital J Gastroenterol Hepatol 30:391–395 50. Roccatello D, Fornasieri A, Giachino O et al (2007) Multicenter study on hepatitis C virus-related cryoglobulinemic glomerulonephritis. Am J Kidney Dis 49:69–82

Index

A Acrocyanosis, 190, 192 Acute disseminated encephalomyelitis (ADEM), 218 Acute hepatitis C (AHC), 69–71 Adhesion molecules, 113–116 Adverse events, 320, 324 Alemtuzumab, 355, 358 Ann Arbor classification, 330 Antibiotic therapy, 340 Antibodies, 28, 29 Anti-CD20, 302, 304 Anti-CD81 antibodies, 64, 66 Antigen-driven disease, 278 Antigenic stimulation, 109 Antimalarials, 190, 192 Antivirals, 21–26, 29, 55, 57–58, 60 Antiviral therapy, 233, 291–298, 329–337, 339–340 Arthralgias, 291, 295 Arthritis, 159–161, 163, 190–192 Autoantibodies, 190–192 Autoimmune hepatitis, 5, 158, 161–163 Autoimmune thyroid diseases (AITD), 44–46, 49 Autoimmunity, 43–49, 120, 122, 123 Autonomic neuropathy, 339 Azathioprine, 297

B BCA–1 immunodetection, 131 BCA–1 mRNA, 130–132 B cells, 12–15, 37–40, 69, 70, 72, 73 activation, 191 attracting chemokine–1 (BCA–1), 5, 129–135 expansion, 129 lymphoma, 319 non-Hodgkin’s lymphoma (B-NHL), 38, 271–275, 329–337 receptor (BCR), 37–40, 264–266 traffic, 133 Belimumab, 123 BLyS/BAFF, 119–123 Boceprevir, 325 Bortezomib, 285, 353–358 Burkitt’s lymphoma, 271, 273 F. Dammacco (ed.), HCV Infection and Cryoglobulinemia, DOI 10.1007/978-88-470-1705-4, © Springer-Verlag Italia 2012

C C3, 3, 320, 321, 331 C4, 3, 320–322, 331 Cancer, 162 Castelman’s disease, 271 Cause of death, 176, 348 CCL2, 139–141 CD81, 38, 39, 58, 59, 63–66, 274 CD20-positive cells, 323 CD81 tetraspanin, 4, 6 Chemokine CXCL13, 5 Chemotherapy, 330, 331, 333–336 Chromosomes, 257–260 Chronic active hepatitis, 331–333 Chronic antigenic stimulation, 329, 336 Chronic hepatitis C (CHC), 69–73 Chronic infection, 26–27, 29 Chronic inflammatory state, 189 Cirrhosis, 11, 13, 14, 17, 331–335 Classification, 167–170 Clathrin, 65, 66 Claudin, 64–66, 274 Claudin–1 (CLDN1), 58, 59 Clinical manifestations, 182 Clinical response, 292, 295–297 Clonal B-cell expansion, 273, 322, 323 Clonal B-cell proliferation, 329 Co-infection, 231–233 Colchicine, 340 Combined therapy, 291–298 Complement, 85–88, 148–151 Connective tissue diseases, 244, 245 Corticosteroids (Cs), 291, 296, 319, 325, 349, 350 C1q, 114–116 protein, 3, 4 receptor, 3, 4, 91–95 C-reactive protein (CRP), 339 Criteria, 167–170 Cross-reactive idiotypes (CRI), 2, 99–103 Cryocrit, 293, 320, 321 Cryoglobulinemia, 79–82, 85–88, 91–95, 139–141, 147–153, 157–163, 167, 168, 179–184, 186, 249–253, 339–340, 353–358 Cryoglobulinemic syndrome, 232, 233 Cryoglobulinemic ulcer, 339, 340 Cryoglobulinemic vasculitis, 129–135, 311–316, 320, 323–325, 331, 336, 344, 345 357

358 Cryoglobulins, 86–88, 107–110, 167–169, 213–220 Cryoprecipitation, 86, 87 CXC chemokine superfamily, 129 CXCL, 129, 133 CXCL10, 139–141 Cyclophosphamide, 291, 297, 325 Cytotoxic drugs, 291

D DC-SIGN. See Dendritic-cell-specific intercellular adhesion molecule–3 grabbing non-integrin (DC-SIGN) Demographic and survival findings, 173–176 Dendritic cells (DCs), 22–25, 27, 28 Dendritic-cell-specific intercellular adhesion molecule–3 grabbing non-integrin (DC-SIGN), 65 Detection of HCV in immune cells, 69 Diabetes, 141, 195–197 Diagnosis, 168, 169 Differential diagnosis, 184–185 Double filtration plasmapheresis (DFPP), 341–345

E Endocytosis, 65, 66 Epidemiology, 173, 174 Epstein-Barr virus (EBV), 271 Essential MC, 1–3, 243–246 Etanercept, 354, 358 Etiology, 175 Etiopathogenesis, 109 Extrahepatic manifestations, 11, 12, 14–16, 320

F Fatigue, 191 Fc receptors (FcRs), 148–151, 153 Follicle-like structures, 129, 131, 133 FR and CDR determinants, 99

G Genome, 257–258, 260 Germinal centers, 129, 130 Glomerulonephritis, 5, 87, 88, 249, 250, 253 Glycosaminoglycans, 65

H HCV, 257–260, 263–267, 277–278, 311–316 associated NHL, 273–275 cell entry, 63–67 compartmentalization, 69, 72 core protein, 3, 4 E2, 4, 6, 38–40 entry inhibitors, 65–67 genotypes, 273, 319, 321, 325, 330, 331 in cerebrospinal fluid (CSF), 218

Index infection, 43–49, 129, 133–135, 139–141, 190, 192, 193, 320, 329–337 of hematopoietic cells, 69–73 in immune cell cultures, 69, 73 lymphotropism, 69–71, 73 negative mixed cryoglobulinemia (MC), 243–246 NS3, 40 positive B-NHL, 329 receptors or co-receptors, 64, 66 RNA, 2, 3, 5, 7, 38, 40, 292, 295, 297, 320–323, 330–335 in peripheral nerves, 217 Helicobacter pylori, 275, 336 Heparan sulfate, 65 Hepatitis, 170 Hepatitis B virus (HBV), 231–233, 243–245, 271 Hepatitis C, 179, 180, 183, 184, 195 complications, 278 virus (HCV), 2–7, 11–17, 21–29, 37–40, 55–60, 69–73, 79–82, 99–103, 113–116, 120–123, 157–163, 167, 169, 170, 173–176, 199–208, 213–220, 223–229, 231–233, 237–240, 249–253, 271–275, 291–298, 301, 303, 304, 306–307, 319–325, 341, 343, 344, 348–350 Hepatocellular carcinoma (HCC), 11, 271 HIV–1 infection, 273 Hodgkin’s lymphoma, 273, 274 Human herpes virus type 8 (HHV–8/KSHV), 271 Human immunodeficiency virus (HIV), 237–240, 244, 245 Human leukocyte antigen (HLA), 40 Human papilloma-virus, 271 Human scavenger receptor SR-BI, 274 Human T-cell leukemia virus type I (HTLV-I), 271 Hypothyroidism, 195, 196

I Ibritumomab tiuxetan, 325 Idiotypes, 103 IgM monoclonal gammopathy, 281 IgM RF, 40 IL–28B polymorphism, 324 Imatinib, 152, 153, 353–358 Immune complexes, 1, 3, 38, 40, 85–87, 189, 192, 291, 292, 297 Immune-mediated demyelination, 218 Immunocomplex, 108–110 Immunoglobulin, 107, 108, 110 Immunologic response, 292, 295 Immunology, 349 Immunosuppressive therapy, 291, 292, 296 Infection, 119–123 Infliximab, 353–358 Innate immunity, 5, 24, 29, 149, 151 Interferon (IFN), 21, 22, 119, 123 Interferon-a (IFN-a), 2, 6, 7, 292, 312 Interferon (IFN)-b, 292 Interferon therapy, 45–48 Interleukin–2 receptor (IL–2Ra), 337 Intravenous IgG, 291, 339

Index K Kaposi’s sarcoma, 271 Kidney transplantation, 250–251

L Large-fiber sensory neuropathy (LFSN), 215 Laser capture microdissection (LCM), 131–132 LDL receptor (LDL-R), 58, 59 Lenalidomide, 356, 358 Leukocytoclastic capillaritis, 339 Leukocytoclastic vasculitis, 189–191, 203, 204, 207, 291 Liver transplantation, 249–253 Long term course, 223–229 Low and high grade lymphoma, 260 Low-antigen-containing (LAC) diet, 340 Low-grade malignant lymphoma, 329 L-SIGN, 65 Lymphoma, 14, 15, 120, 122, 158–162, 223, 244, 245 Lymphomagenesis, 6, 273–275, 330, 336 Lymphoplasmacytic lymphoma, 353, 355 Lymphoproliferation, 122, 123, 263–267 Lymphoproliferative disease, 99, 101–103 Lymphoproliferative disorders, 273, 281, 324, 329, 330

M MALT lymphoma, 336 Manifestation, 245 Membranoproliferative glomerulonephritis (MPGN), 147–153, 179–186 Memory, 37 Mixed cryoglobulinemia (MC), 2–6, 14, 15, 38–40, 55, 60, 113–116, 119–123, 129–135, 173–176, 195–197, 199–202, 205–208, 223–229, 231–233, 237–240, 258, 273–275, 277–278, 291–298, 301–307, 319–325, 348, 350 Monoclonal B-cell proliferation, 329 Monoclonal gammopathy of undetermined significance (MGUS), 355 Monocytes, 69, 72 Mortality, 176, 347, 349 Myelitis, 218, 219

N Naïve, 37 T cells, 5 Nasopharyngeal carcinoma, 271 Natural history, 11–17 Natural killer (NK) cells, 5, 24–26 Neurostimulation, 340 Non- Hodgkin’s lymphoma (NHL), 2, 5–7, 39, 55, 277–278, 296, 297, 323, 324 Nucleos(t)ide analogs, 233, 285

359 O Occludin (OCLN), 58, 59, 64–66, 274 Occult HBV infection, 231, 232 Occult HCV infection (OCI), 3, 69, 71–72, 245, 246 Ofatumumab, 2, 7, 325 Organ-specific autoimmunity, 43–49 Osteosclerosis, 190, 192

P Pathogenesis, 11–17 Pegylated IFN-a/ribavirin (RBV) combination, 319, 321, 325 Pegylated interferon (PEG-IFN), 295–297 Pegylated interferon-a (pIFN-a), 233, 319, 321, 325, 330, 331, 336 Peripheral neuropathy, 213–220, 353, 356 pIFN-a plus RBV antiviral therapy, 331, 336 pIFN-a plus RBV plus RTX (PIRR), 319–325 PIRR therapy, 2, 331 Plasma-exchange, 296, 297, 339, 349, 350 Plasmapheresis, 291, 296, 297 Plasmapheresis/plasma exchange, 284, 285 Polyneuropathy, 291, 296, 297 Post-transplant lymphoma, 271 Prevention, 11–17 Prognosis, 175, 176, 185 Progressive multifocal leukoencephalopathy, 324 Protease inhibitors, 2, 7 Purpura, 2, 167, 170, 291, 292, 295, 339, 340 Purpura/arthralgia/asthenia, 3, 320

Q Quasispecies, 22, 59–60 Questionnaire, 167–170

R Raynaud phenomena, 339 REAL classification criteria, 330 Rheumatoid factor (RF), 1–4, 6, 38–40, 87, 107–110, 120–123, 129, 130, 291, 320, 321, 324, 331 Ribavirin (RBV), 2, 7, 233, 294–297, 314–316, 319–325, 330–334, 336 Rituximab (RTX), 2, 6, 7, 123, 220, 250, 253, 285, 296–297, 301–307, 311–316, 320–325, 331, 332, 335, 336, 349, 350, 353–356 RT-PCR, 57, 60

S Scavenger receptor BI (SR-BI), 58, 59, 64–66 Serum sickness syndrome, 324 Sicca syndrome, 190–192 Sjögren’s syndrome (SS), 5, 101, 120–123, 158, 160–162, 244, 245 Small-fiber sensory neuropathy (SFSN), 215–216 Small vessels, 189, 191 Solubilization, 87

360

Index

Somatic hypermutation, 101 Splenic lymphoma with villous lymphocytes, 275, 333–335 Survival, 173–176, 245 Sustained biochemical response (SBR), 331, 332 Sustained virological response (SVR), 292–295, 297, 319–321, 323–325, 330–334, 336 Systemic lupus erythematosus, 244

TSLP See Thymic stromal lymphopoietin (TSLP) TSLP tg, 148–153 Tumor necrosis factor (TNF), 3, 354, 355 Type 1 diabetes mellitus, 46–49 Type I cryoglobulins, 281–283 Type II cryoglobulins, 281, 282 Type 2 mixed cryoglobulins, 103

T T-cell NHL, 330, 333 T cells, 25–29 Telaprevir, 325 Tetraspanin CD81, 337 Tetraspanins, 63, 64 Thalidomide, 355, 356, 358 Therapeutic apheresis, 341, 342, 344, 345 Thrombosis, 192 Thymic stromal lymphopoietin (TSLP), 148 Thyroid autoimmunity, 195 Thyroid cancer, 196 Thyroiditis, 5, 140–141 Tight junction proteins, 64–65 TLR4, 150, 151, 153 TLR7, 38, 40 T lymphocytes, 70, 71 Toll like receptors (TLRs), 37 Transgenic mice, 337 Transinfection, 265 Treatment, 185, 245, 302–307 Treg cells, 28

U Ulcer, 339–340

V Vasculitic neuropathy, 216 Vasculitis, 2, 3, 5–7, 113–116, 139–141, 157–163, 167–170, 199–208, 237–240 complications, 175, 176, 347 VCAM–1, 113–116 Very-low-density lipoproteins (VLDL), 59 Viral interplay, 231 Virus, 108, 109 Virus entry, 58–60 Virus-related cancers, 271 V-region genes, 99–103

W Wa cross idiotypes, 2, 6 Waldenström’s macroglobulinemia (WM), 281–285, 355