Immune-Related Disorders and Extrahepatic Diseases in Chronic HCV...

Clinical and Developmental Immunology

Guest Editors: Domenico Sansonno and Jürg Schifferli

Immune-Related Disorders and Extrahepatic Diseases in Chronic HCV Infection

Immune-Related Disorders and ExtrahepaticDiseases in Chronic HCV Infection

Clinical and Developmental Immunology

Immune-Related Disorders and ExtrahepaticDiseases in Chronic HCV Infection

Guest Editors: Domenico Sansonno and Jurg Schifferli

Copyright © 2012 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Clinical and Developmental Immunology.” All articles are open access articles distributed under theCreative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

Editorial Board

B. D. Akanmori, GhanaR. Baughman, USAStuart Berzins, AustraliaBengt Bjorksten, SwedenK. Blaser, SwitzerlandFederico Bussolino, ItalyNitya G. Chakraborty, USARobert B. Clark, USAMario Clerici, ItalyEdward P. Cohen, USARobert E. Cone, USANathalie Cools, BelgiumMark J. Dobrzanski, USANejat Egilmez, USAEyad Elkord, UKSteven Eric Finkelstein, USABernhard Fleischer, GermanyRichard L. Gallo, USALuca Gattinoni, USADavid E. Gilham, UKRonald B. Herberman, USAD. Craig Hooper, USA

H. Inoko, JapanDavid Kaplan, USAW. Kast, USATaro Kawai, JapanMichael H. Kershaw, AustraliaHiroshi Kiyono, JapanShigeo Koido, JapanGuido Kroemer, FranceH. Kim Lyerly, USAEnrico Maggi, ItalyStuart Mannering, AustraliaGiuseppe V. Masucci, SwedenEiji Matsuura, JapanC. J. M. Melief, The NetherlandsJiri Mestecky, USAC. Morimoto, JapanHiroshi Nakajima, JapanTetsuya Nakatsura, JapanT. Nakayama, JapanHans Nijman, The NetherlandsPaola Nistico, ItalyGraham Ogg, UK

G. Opdenakker, BelgiumIra H. Pastan, USABerent Prakken, The NetherlandsNima Rezaei, IranClelia M. Riera, ArgentinaLuigina Romani, ItalyB. T. Rouse, USAAurelia Rughetti, ItalyTakami Sato, USASenthamil R. Selvan, USANaohiro Seo, JapanE. M. Shevach, USAS. Sozzani, ItalyGeorge B. Stefano, USATrina J. Stewart, AustraliaHelen Su, USAJacek Tabarkiewicz, PolandBan-Hock Toh, AustraliaJ. F. Urban, USAYvette Van Kooyk, The NetherlandsY. Yoshikai, JapanQiang Zhang, USA

Contents

Immune-Related Disorders and Extrahepatic Diseases in Chronic HCV Infection, Domenico SansonnoVolume 2012, Article ID 509309, 2 pages

Extrahepatic Manifestations and Autoantibodies in Patients with Hepatitis C Virus Infection,Takashi Himoto and Tsutomu MasakiVolume 2012, Article ID 871401, 11 pages

The Impact of Interferon Lambda 3 Gene Polymorphism on Natural Course and Treatmentof Hepatitis C, F. Bellanti, G. Vendemiale, E. Altomare, and G. ServiddioVolume 2012, Article ID 849373, 9 pages

Restoration of Innate and Adaptive Immune Responses by HCV Viral Inhibition with an InductionApproach Using Natural Interferon-Beta in Chronic Hepatitis C, Y. Kishida, N. Imaizumi, H. Tanimura,Y. Haruna, S. Kashiwamura, and T. KashiwagiVolume 2012, Article ID 582716, 15 pages

Indolent B-Cell Lymphomas Associated with HCV Infection: Clinical and Virological Features and Roleof Antiviral Therapy, Luca Arcaini, Michele Merli, Stefano Volpetti, Sara Rattotti, Manuel Gotti,and Francesco ZajaVolume 2012, Article ID 638185, 10 pages

Rheumatoid Factor, Complement, and Mixed Cryoglobulinemia, Peter D. GorevicVolume 2012, Article ID 439018, 6 pages

Molecular Signature in HCV-Positive Lymphomas, Valli De Re, Laura Caggiari, Marica Garziera,Mariangela De Zorzi, and Ombretta RepettoVolume 2012, Article ID 623465, 9 pages

The Place of Immunotherapy in the Management of HCV-Induced Vasculitis: An Update,Laurent Chiche, Stanislas Bataille, Gilles Kaplanski, and Noemie JourdeVolume 2012, Article ID 315167, 8 pages

Rituximab-Based Treatment, HCV Replication, and Hepatic Flares, Evangelista Sagnelli,Mariantonietta Pisaturo, Caterina Sagnelli, and Nicola CoppolaVolume 2012, Article ID 945950, 5 pages

Morphologic Features of Extrahepatic Manifestations of Hepatitis C Virus Infection, Huaibin M. Ko,Juan C. Hernandez-Prera, Hongfa Zhu, Steven H. Dikman, Harleen K. Sidhu, Stephen C. Ward,and Swan N. ThungVolume 2012, Article ID 740138, 9 pages

HCV-Related Nervous System Disorders, Salvatore Monaco, Sergio Ferrari, Alberto Gajofatto,Gianluigi Zanusso, and Sara MariottoVolume 2012, Article ID 236148, 9 pages

HCV and Lymphoproliferation, Anna Linda Zignego, Carlo Giannini, and Laura GragnaniVolume 2012, Article ID 980942, 8 pages

Pathogenetic Mechanisms of Hepatitis C Virus-Induced B-Cell Lymphomagenesis, Fabio Forghieri,Mario Luppi, Patrizia Barozzi, Rossana Maffei, Leonardo Potenza, Franco Narni, and Roberto MarascaVolume 2012, Article ID 807351, 9 pages

Immunological HCV-Associated Thrombocytopenia: Short Review, Dimitrios Dimitroulis,Serena Valsami, Paraskevas Stamopoulos, and Gregory KouraklisVolume 2012, Article ID 378653, 5 pages

Hepatitis C Virus Infection and Mixed Cryoglobulinemia, Gianfranco Lauletta, Sabino Russi,Vincenza Conteduca, and Loredana SansonnoVolume 2012, Article ID 502156, 11 pages

HCV Proteins and Immunoglobulin Variable Gene (IgV) Subfamilies in HCV-Induced Type II MixedCryoglobulinemia: A Concurrent Pathogenetic Role, Giuseppe Sautto, Nicasio Mancini, Laura Solforosi,Roberta A. Diotti, Massimo Clementi, and Roberto BurioniVolume 2012, Article ID 705013, 11 pages

Autoimmunity and Extrahepatic Manifestations in Treatment-Naıve Children with ChronicHepatitis C Virus Infection, Giuseppe Indolfi, Elisa Bartolini, Biagio Olivito, Chiara Azzari,and Massimo RestiVolume 2012, Article ID 785627, 4 pages

Cytokines and HCV-Related Disorders, Poupak Fallahi, Clodoveo Ferri, Silvia Martina Ferrari,Alda Corrado, Domenico Sansonno, and Alessandro AntonelliVolume 2012, Article ID 468107, 10 pages

Hindawi Publishing CorporationClinical and Developmental ImmunologyVolume 2012, Article ID 509309, 2 pagesdoi:10.1155/2012/509309

Editorial

Immune-Related Disorders and Extrahepatic Diseases inChronic HCV Infection

Domenico Sansonno

Liver Unit, Department of Biomedical Sciences and Human Oncology, Medical School, University of Bary,Piazza G. Cesare, 11 70124 Bari, Italy

Correspondence should be addressed to Domenico Sansonno, [email protected]

Received 30 August 2012; Accepted 30 August 2012

Copyright © 2012 Domenico Sansonno. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Hepatitis C virus (HCV) represents one of the most impor-tant causes of chronic active liver disease worldwide, poten-tially resulting in cirrhosis, and hepatocellular carcinoma.

HCV is mainly characterized by two major immunologicfingerprints, namely, escape of immune response in morethan 80% of infected patients and production of autoanti-bodies in almost half of them.

Within the infected host, HCV exists as a quasispecies ora flock of related viral sequences. HCV can evolve rapidlyas its replicative cycle yields 1012 HCV particles per day inthe liver, whereas in the serum the newly produced viralparticles have an estimated halflife of about 3 hours. Thishigh rate of virus production coupled with the lack ofproofreading capacity of the HCV’s NS5B RNA-dependentRNA polymerase lead to its rapid evolution and escapefrom immune recognition in each host. In addition, HCVprofoundly deranges the functions of immune system. Itderegulates both innate and adaptive antiviral response. Ithas been shown that HCV-encoded proteins subvert typeI interferon (IFN) receptor signal transduction and thefunction of downstream IFN effector pathway. The virusdecreases the ability of NK cells to promote dendritic cellmaturation modifying antigen presentation and productionof soluble mediators including IL-10 and IL-12. Resolutionof HCV infection is mainly dependent on adaptive immunityand is associated with a robust and sustained specific T-cellresponse targeting multiple epitopes and intrahepatic IFN-γproduction. Defective CD4+ T cells in both acute and chronicHCV infection lead to CD8+ T-cell exhaustion.

While HCV evolves under immunological pressure,the cellular immune response remains focused on viral

sequences encountered early in the course of the infection.Indeed, humoral immune response seems more flexible inthat the continued emergence of new antibody specificitiessharply contrasts with the static T-cell response. Indubitable,interaction of HCV with B cells may promote favourableconditions for lymphocyte proliferation. Viral replicativeintermediary was found in the B cell from patients withmixed cryoglobulinemia (MC), whereas no traces of HCVproductive particles were demonstrable in HCV-infectedindividuals without cryoglobulin production, supporting thenotion that HCV is not a genuine lymphotropic virus,but its entry and replication are largely dependent on hostselective interactions. HCV binding to BCR triggers both theinitiation of signaling cascade and internalization of the BCRand bound antigen into the cell. These interactions result inin vivo polyclonal activation and expansions of CD19+ CD5+

cells.Extrahepatic disease manifestations, which include

autoimmune phenomena and frank autoimmune and/orrheumatic diseases, may complicate the clinical features ofchronic HCV infection and sometimes dominate its coursein almost half of chronic HCV carriers. Possibly, progressionto frank B-cell lymphoid malignancy may also be superposedas additional stochastic oncogenic event. However, there aremany dark areas in the comprehension of several aspects oftheir pathogenetic mechanisms. The nature of the processduring which B cells expand with preferential involvementof rheumatoid factor- (RF-) producing B cells is undefined.Whether B-cell clonal expansion of a particular specificityoccurs as a result of distinct selection is not clear. The processof B-cell clonal expansion occurring in an environment

2 Clinical and Developmental Immunology

favourable to the immortalization of one specific clone mustbe clarified. Predisposing factors for transforming eventsmust be identified.

Against this background, it was felt that times wereripe to produce a state-of-the-art survey of the multifacetedpictures of HCV-related immune disorders. This special issueis therefore devoted to expand our knowledge on the featuresand mechanisms underlying the relationship between B-cell immune response and extrahepatic manifestations ofchronic HCV infection. It comprises 16 review articles and1 clinical study.

The paper by T. Himoto and T. Masaki is a generaloverview on HCV-related extrahepatic manifestationsincluding cryoglobulinemia, Sjogren’s syndrome, autoim-mune thyroid disease, lichen planus, CREST syndrome,IFN-induced autoimmunity, and autoimmune cytopenia.Few data are available on this topic in children. The paperby G. Indolfi et al. deals with the clinical significance ofnon-organ specific autoantibodies in the course of paediatricchronic hepatitis C. Subclinical hypothyroidism and mem-branoproliferative glomerulonephritis have been described.

Dysregulation of the cytokine/chemokine network,involving proinflammatory and Th1 chemokines, isaddressed by P. Fallahi et al. Both the humoral and viralcounterparts at the basis of cryoglobulins production inHCV-induced type II MC, with particular attention to themost frequently involved single IgV subfamilies have beenanalyzed by G. Sautto et al. Neurological complications thatoccur in a large number of patients and range from periph-eral neuropathy to cognitive impairment are discussed by S.Monaco et al. Autoimmune thrombocytopenia is a frequentcomplication of HCV chronic infection. D. Dimitrouliset al. estimated the epidemiological characteristics of thedisease and discussed the potential treatment strategies.Extrahepatic manifestations of HCV infection include amultitude of disease processes affecting the small vessels,skin, kidneys, salivary gland, eyes, thyroid, and immunologicsystem. The majority of these conditions are thought tobe immune-mediated. H. M. Ko et al. highlighted thehistomorphologic features of these pathologic conditions.G. Lauletta et al. described epidemiological, clinical, andpathogenetic mechanisms underlying the cryoglobulinemicvasculitis. In MC, consumption of complement component4 may be due to activation of complement cascade. Potentialactivators include monoclonal IgM-RF, IgG antibodies, andthe complexing of the two in the cold, perhaps modulated bythe rheology and stoichiometry of cryocomplexes in specificmicrocirculations. P. D. Gorevic elucidated the role of the RFand the complement in the vessel damage.

The association between HCV infection and B-cell non-Hodgkin’s lymphomas has led to search for molecularsignatures capable of predicting patients’ characteristics. V.De Re et al. underscored that HCV-related lymphomas aresubject to specific deregulation induced by the virus. Epi-demiology and mechanisms of HCV-induced lymphoprolif-eration have been elucidated by F. Forghieri et al. Differentbiological mechanisms, namely, chronic antigen stimulation,high-affinity interaction between HCV-E2 protein and itscellular receptors, direct HCV infection of B cells, and

“hit and run” transforming events, may cooperate in amultifactorial model of HCV-associated lymphomagenesis.A comprehensive review of molecular mechanisms involvedin HCV-related lymphomagenesis has been resumed by A. L.Zignego et al. It is concluded that HCV lymphomagenesisis a complex, multistep, multifactorial process. L. Arcainiet al. illustrated the relationship between HCV infectionand different subtypes of indolent B-cell lymphomas. Theysummarized the data from the therapeutic studies reportingthe use of antiviral treatment as hematological therapy. L.Chiche et al. provided an updated overview on the placeof immunotherapy, especially biologics, in the managementof HCV-induced cryoglobulinaemic vasculitis. Rituximabhas been used to treat oncohaematological diseases, B-cell-related autoimmune diseases, rheumatoid arthritis, and,more recently, HCV-associated mixed cryoglobulinaemicvasculitis. E. Sagnelli et al. discussed rituximab-based treat-ment and conclude that this drug is capable of enhancingHCV replication with potential liver failure. Y. Kishidaet al. explored the hypothesis that an induction approachwith nIFN-beta followed by PEG-IFN-alpha plus ribavirinwould increase the initial virologic response in chronichepatitis C. They concluded that this combined therapeuticscheme results in high rate of sustained virologic responseand improvement of innate and adaptive immunity. F.Bellanti et al. accomplished an overview about the biologicalactivity and clinical applications of interferon lambda 3,summarizing the available data on its impact on HCVinfection. The potential usefulness of this type of interferonin the treatment of HCV infection has been also discussed.

Domenico Sansonno


Review Article

Extrahepatic Manifestations and Autoantibodies inPatients with Hepatitis C Virus Infection

Takashi Himoto1, 2 and Tsutomu Masaki1

1 Department of Gastroenterology and Neurology, Kagawa University School of Medicine, Kagawa, Japan2 Department of Integrated Medicine, Kagawa University School of Medicine, 1750-1, Ikenobe, Miki-Cho,Kita-Gun, Kagawa 761-0793, Japan

Correspondence should be addressed to Takashi Himoto, [email protected]

Received 27 April 2012; Revised 13 June 2012; Accepted 13 June 2012

Academic Editor: Domenico Sansonno

Copyright © 2012 T. Himoto and T. Masaki. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Patients with chronic hepatitis C virus (HCV) infection frequently have many extrahepatic manifestations, as persistent HCVinfection often triggers lymphoproliferative disorders and metabolic abnormalities. These manifestations primarily include auto-immune disorders such as cryoglobulinemia, Sjogren’s syndrome, and autoimmune thyroid disorders. It has been well establishedthat chronic HCV infection plays important roles in the production of non-organ-specific autoantibodies, including antinuclearantibodies and smooth muscle antibodies, and organ-specific autoantibodies such as thyroid autoantibodies. However, the clinicalsignificance of autoantibodies associated with the extrahepatic manifestations caused by HCV infection has not been fully recog-nized. In this paper, we mainly focus on the relationship between extrahepatic manifestations and the emergence of autoantibodiesin patients with HCV infection and discuss the clinical relevance of the autoantibodies in the extrahepatic disorders.

1. Introduction

Persistent hepatitis C virus (HCV) infection has been wellcharacterized as having a preferential evolution which oftenevokes lymphoproliferative disorders [1] and metabolicabnormalities [2]. Therefore, patients with chronic HCVinfection frequently develop extrahepatic manifestations [3–5]. Previous studies have revealed that 38–76% of patientswith chronic HCV infection develop at least one extrahep-atic manifestation [6–8]. These extrahepatic manifestationsmainly include autoimmune disorders such as mixed cryo-globulinemia, Sjogren’s syndrome, and thyroid autoimmunedisorders.

On the other hand, persistent HCV infection is responsi-ble for the production of a variety of autoantibodies includ-ing non-organ-specific autoantibodies and organ-specificautoantibodies, as a virus-induced autoimmune pheno-menon. The diversity of autoantibodies in the sera of patientswith HCV-related chronic liver disease (CLD) [9–13] hasbeen shown. Some autoantibodies in chronic HCV infection

have biochemical, histological, or genetic characteristics,while other autoantibodies may predict the response to anti-viral treatments, concomitant disorders, or prognosis inpatients with HCV-related CLD [14].

Various mechanisms for the production of autoantibod-ies in patients with HCV-related CLD have been proposed.Molecular mimicry between a component of a virus and a“self” protein may account for the production of autoanti-bodies in chronic HCV infection [15]. A sequence homologybetween the HCV polyprotein and cytochrome p450 2D6(CYP 2D6), which was identified as the antigenic target ofantibodies to liver-kidney microsome type 1 (anti-LKM1),was previously reported [16]. The reactivity against theviral protein induces the production of anti-LKM1 in HCV-related CLD. Polyclonal B-cell activation by persistent HCVinfection has been proposed as another mechanism for theproduction of autoantibodies [17]. B-cell proliferation seemsto be essential for the development of autoimmune disordersincluding Sjogren’s syndrome and mixed cryoglobulinemia(MC). Genetic predisposition is also strongly related to


the presence of autoantibodies in chronic HCV infec-tion [18]. The susceptibility to develop non-organ specificautoantibodies (NOSA) appears to be restricted to a specifichuman leukocyte antigen (HLA) in patients with HCVinfection [19].

The presence of NOSA including antinuclear antibodies(ANAs) and smooth muscle antibodies (SMAs) is associatedwith the severity of necroinflammation and fibrosis in theliver of patients with HCV-related CLD [20–24]. It is notablethat the titers of these autoantibodies seem to be independentof HCV genotypes or loads of HCV-RNA [21–25]. Theemergence of these autoantibodies did not affect antiviraltreatments. [23]. However, we have to exclude concomitantautoimmune hepatitis (AIH) from patients with HCV infec-tion seropositive for NOSA, because antiviral treatmentoccasionally exacerbates AIH in those patients [26].

The clinical significance of autoantibodies in the extra-hepatic manifestations caused by HCV infection has beenrarely discussed. This paper highlights the aspects of autoan-tibodies in extrahepatic manifestations by HCV infectionand elucidates their clinical and therapeutic implications.

2. Extrahepatic Manifestations andTheir Associated Autoantibodies

2.1. Cryoglobulinemia. Cryoglobulinemia is one of the mostcommon extrahepatic diseases in patients with HCV infec-tion and is detected in 19–54% of those patients [8, 27–30].Cryoglobulins are immunochemically classified into threetypes according to the method by Brouet and his colleagues[31]. Type I cryoglobulins are composed of a monoclonalimmunoglobulin and are often associated with hematolog-ical disease. Type II cryoglobulins are immune complexesconsisting of polyclonal IgG with monoclonal rheumatoidfactor (RF) activity, while type III cryoglobulins are char-acterized by polyclonal IgG with polyclonal RF. Therefore,type II and type III cryoglobulins are referred to mixedcryoglobulins. Persistent HCV infection is strongly associ-ated with types II and III mixed cryoglobulinemia (MC) andoccasionally associated with type I cryoglobulinemia. Cry-oprecipitates contain HCV core proteins, IgG moleculeswith specific anticore activities, and IgM molecules withRF activities. C1q proteins and C1q binding activity wereenriched in this immune complex [32], and were related tothe wide expression of C1q receptor on the surface of bloodcells and endothelial cells [33–35].

MC secondary to HCV infection often involves otherorgan systems in, for example, cutaneous manifestations,peripheral neuropathy, and glomerular disease [1, 36, 37].There are interesting issues in the relationship between theemergence of cryoglobulin and more advanced hepaticfibrosis in patients with chronic HCV infection [27, 30, 38,39]. However, all patients with HCV-related MC do notsuffer from these involvements [8, 25, 30]. Overt vasculitisis observed in only 2-3% of patients with HCV-related MC[7, 40, 41]. The circumstances predisposing HCV-infectedpatients to develop these manifestations remain obscure.

It is noteworthy that cryoglobulins are usually found atlow concentrations in patients with chronic HCV infection[25, 28, 29]. Patients with HCV-related cryoglogulinemicvasculitis had higher cryocrit levels than those without vas-culitis [42]. Patients with cryoglobulinemic vasculitis hadclinical characteristics of female-predominance, older ageand longer disease duration [42]. The natural history andprognosis of cryoglobulinemic vasculitis was highly depen-dent on renal involvement or the severity of vasculitis lesion.

The precise mechanism by which HCV infection involvesMC has not been well established. However, one hypothesisof a possible role played by HCV in polyclonal B-cell res-ponse has been proposed as follows: HCV has a strong affin-ity to the tetraspanin (CD81) ligand on the surface of B lym-phocytes via the E2 protein (the second proportion of theHCV envelope) [43]. CD81 forms a costimulatory complexwith CD19 and CD21 [44]. The ligation of CD81 on B cellsresults in the activation of this complex, which lowers theantigen threshold necessary for antibody production andeventually causes the formation of cryoglobulins [33]. Thecryoglobulins initially produced are polyclonal IgG (type IIIMC), but as a dominat B-cell clone emerges, it may producemonoclonal immunoglobulins (type II MC) [9].

On the other hand, B-cell-activating factor (BAFF), amember of the tumor necrosis factor-alpha (TNF-α) familythat plays crucial roles in B-cell differentiation, survival, andimmunoglobulin secretion, is considered to be associatedwith the development of autoimmune disorders [45]. Theelevation of serum BAFF levels was observed in patientswith HCV-related lymphoproliferative disorders [46, 47]which represents a link between infection and autoimmunity.Quantitative decrease in regulatory T cells may be involved inpatients with HCV-related MC [48].

Serological hallmarks reflecting autoimmunity inpatients with HCV-related MC have been fully recognized.Table 1 summarizes the clinical characteristics of auto-antibodies in patients with HCV-related MC. Non-organ-specific autoantibodies including ANA and SMA have beenobserved in 12–65% [8, 29, 36, 49–52] and 33–37% [8, 53] ofpatients with HCV-related MC, respectively. It is noteworthythat the immunofluorescence pattern of ANA on HEp-2cells in HCV-related MC was speckled [47, 51]. Rheumatoidfactor (RF), which recognizes the Fc portion of IgG mole-cules as their antigens, often appears in sera of patients withHCV-related MC, at frequencies of 14 to 99% of thosepatients [30, 36, 49, 52, 54, 55]. However, the titers of ANAand RF in sera of HCV-related MC were less than 1 : 80and 50 UI/mL, respectively [51], which appeared to be low.Antineutrophil cytoplasmic antibodies (ANCAs), which aredivided into two groups by immunofluorescence patterns:pANCA and cANCA [56], are also present in the sera ofpatients with HCV-related MC [8, 57, 58]. However, theoccurrence of these autoantibodies was not necessarilyrelated to HCV-related cryoglobulinemic vasculitis [57].

Some types of autoantibodies are available for predictivemarkers of HCV-related MC. The presence of circulatingautoantibodies to C-reactive protein antibodies (anti-CRP),which are directed against monomeric CRP [64], was depen-dent on HCV-related MC [59, 60]. In addition, antibodies

Clinical and Developmental Immunology 3

Table 1: Autoantibodies detected in sera of patients with HCV-related MC.

Autoantibodies Frequency (%) Clinical significance References

ANA 12–65%Low titer

[8, 30, 37, 49–52]Speckled pattern on HEp-2 cells

SMA 33–37% Low titer [8, 53]

RF 14–99% Low titer [30, 37, 49, 52, 54, 55]

ANCA (pANCA, cANCA) 4–26% No association with vasculitis [57, 58]

Anti-CRP 75% Predictive marker of cryoglobulinemia [59, 60]

Anti-C1q 39% Predictive marker of type III cryoglobulinemia [61]

AECA 50% Predictive marker of vasculitis [62]

Anti-GM1 ganglioside antisulfatide 52% Predictive marker of peripheral neuropathy [63]

to C1q, which is closely associated with immune complexdiseases including hypocomplementaemic urticarial vas-culitis and systemic lupus erythematosus (SLE) [65], wasdetected in 38% of patients with chronic HCV infection [66],implying that the emergence of anti-C1q represented con-current cryoglobulinemic vasculitis. However, Saadoun andhis colleagues revealed that anti-C1q was not associated withcryoglobulinemic vasculitis, but indicated the susceptibilityto type III MC in those patients [61].

Other types of autoantibodies are closely linked to theorgan involvements in patients with HCV-related MC. Posi-tivity for RF seems to reflect the development of cutaneousvasculitis in patients with HCV-related MC. Karisbergand his colleagues demonstrated that all HCV-related MCpatients with cutaneous vasculitis had RF and liver involve-ment [67]. The detection of Type II cryoglobulins containingRF (type II-RF) appeared to monitor cryoglobulinemic vas-culitis in those patients [68]. IgMκRF was strongly associatedwith membranoproliferative glomerulonephritis (MPGN) intype II MC [69]. On the other hand, a recent study reportedby Knight and his colleagues elucidated that the monoclocalRFs that bear the WA cross-idiotype are responsible for vas-culitis in patients with HCV-related MC [70].

Antiendothelial cell autoantibodies (AECAs) wererecently identified as a serum parameter for an autoantibodyagainst a variety of antigen determinations on endothelialcells and their titers represented the activity of vasculitis [71].Cacoub and his colleague revealed that AECA was presentin 50% of patients with HCV-related MC and that sero-positivity for AECA was associated with the prevalence ofvasculitis and serum cryoglobulin levels in those patients[62]. However, the authors did not describe the correlationbetween the AECA titers and the activity of vasculitis. Eleva-ted soluble vascular cell adhesion molecule-1 (VCAM-1)was likely to contribute to the involvement of vasculitisin HCV-related MC. Therefore, AECA-induced activationof endothelial cells may initiate an upregulation in theexpression of endothelial adhesion molecule.

The analysis of antineuroual antibodies [72] includinganti-ganglioside GM1 and anti-sulfatide antibodies was per-formed using the sera of HCV-related MC [63]. The asso-ciation between those titers and the involvement of theperipheral nervous system was apparent in patients withHCV-related MC.

Genetic susceptibilities may be related to the develop-ment of MC in patients with chronic HCV infection. HLADRB1∗11 (DR11) was found to predict cryoglobulinemicvasculitis in patients with HCV infection, whereas HLA DR7seemed to protect from the development of type II MC[73, 74].

2.2. Sjogren’s Syndrome. Sjogren’s syndrome (SS) is anothercommon extrahepatic manifestation caused by HCV infec-tion. 6–26% of patients with chronic HCV infection com-plain of sicca syndrome (xerostomia and/or xerophthalmia)[6–8, 33, 75, 76]. The pathogenesis of HCV-associated SS isnot well established. The virus is unlikely to have a directeffect, because HCV has not been proven in glandular tissue[77, 78]. The proposed mechanism includes cross-reactivitybetween the HCV envelope and host salivary tissue orHCV envelope-mediated salivary glands. Koike and his col-leagues elucidated the resemblance of salivary lesions from atransgenic model overexpressing the HCV envelope protein[79].

SS secondary to HCV infection can be distinguished, tosome extent, from primary SS by analyzing several types ofautoantibodies (Table 2). The seropositivities for antibodiesto SS-A/Ro and to SS-B/La are much lower in HCV-asso-ciated SS than those in primary SS patients [75, 80]. Theprevalences of cryoglobulin and RF were higher in the seraof patients with HCV-associated SS than in those of primarySS patients [75, 80, 81]. It is of interest that the coexistenceof cryoglobulinemia in HCV-related SS may favor thedevelopment of lymphoproliferative diseases including B-cellNHL [82]. There has been recent discussions on the rele-vance of antibodies to alpha-fodrin in patients with HCV-related SS seronegative for antibodies to SS-A/Ro [83].

Apart from the antibody status, HCV-related SS hasseveral clinical characteristics distinct from primary SS. Bio-chemical analysis revealed a higher frequency of hypocom-plementemia in HCV-SS than primary SS [80, 84]. Theimbalance of Th1/Th2, namely, poor Th1 response andenhanced Th2 response, was apparent in HCV-related SS[80]. A recent study revealed that the detection of monoclo-nal gammopathy (IgMκ) might help to distinguish HCV-related SS from primary SS [85]. De Vita and his colleaguesdocumented higher prevalence of monoclonal gammopathy


Table 2: Comparisons of immunological and histological findings between primary SS and HCV-related SS.

Variable Primary SS HCV-related SS

Autoantibodies High frequency of ANA, anti-Ro, and anti-La Low frequency of ANA, anti-Ro, and anti-La

Cryoglobulin Rare Common

Hypocomplementemia Rare Common

Lymphocytic capillaritis Moderate to severe Mild to moderate

Th1/Th2 balance Th1 predominant Th2 predominant

Monoclonal gammopathy Low High

Association of HLA DR-3 High Low

in HCV-related SS than that in primary SS [86]. Histologicalexamination of the salivary gland in HCV-related SS showedmilder pericapillary and nonpericanalary lymphocytic infil-tration than primary SS [87]. The prevalence of liver invol-vement was far higher in HCV-related SS than in primary SS[84].

Patients with the HLA haplotype of HLA DQB1∗02 had asusceptibility to the development of HCV-related SS [88]. Onthe other hand, the prevalence of HLA DR 3 [89], a haplotypespecific to primary SS, was far lower in patients with HCV-related SS [75].

2.3. Autoimmune Thyroid Disease. Thyroid disorders arecommon in patients with chronic HCV infection. Approx-imately 10–25% of patients with persistent HCV infec-tion have thyroid autoantibodies, including thyroid micro-some autoantibodies (TMAs), thyroglobulin autoantibodies(TGAs), and antibodies to thyroid peroxidase autoantibodies(anti-TPO), regardless of the liver involvement severity [90–92]. TMAs are frequently useful to detect latent autoimmunethyroiditis in patients with CH-C prior to antiviral treatment[91]. The presence of TMAs also may predict thyroid dys-function including hyperthyroidism and hypothyroidism[93]. Therefore, the detection of these thyroid autoantibodiesis considered useful for the clinical diagnosis of concurrentautoimmune thyroid diseases in patients with HCV infec-tion. However, HCV-associated thyroid disorders cannot bedistinguished from primary thyroid disorder by the existenceof these thyroid autoantibodies. The possible role of HCVin the development of thyroid disorders has not been fullyunderstood.

HCV-related thyroid disorders include Graves’ diseaseand Hashimoto’s thyroiditis induced by interferon (IFN)treatment [94, 95]. The discovery of anti-TPO at base-linemay be regarded as a predictive factor for IFN-inducedthyroid autoimmunity in patients with CH-C. A strongcorrelation between thyroid disorders and the presence ofanti-LKM1 at base-line in patients with chronic hepatitisC was also observed [96, 97]. HCV poly protein, CYP2D6and thyroperoxidase are likely to share epitopes encodinghomologous amino acid sequences [97]. Therefore, seropos-itivity for anti-LKM1 is a susceptibity factor for IFN-inducedautoimmune thyroid disorders in patients with HCV-relatedCLD.

2.4. Lichen Planus. Lichen planus is well known to be a skinlesion associated with persistent HCV infection, althoughthe pathogenesis remains uncertain [98]. Approximately1–6% of patients with chronic HCV infection have beenestimated to be afflicted with oral lichen planus [7, 8, 49].The existence of concurrent lichen planus was associatedwith chronic active hepatitis [99], suggesting that the chronicHCV infection alone did not cause lichen planus. However,the severity of hepatic fibrosis and necroinflammation wasindependent of the severity of lymphocytic infiltration in theoral lichen planus [100].

Nagao and her colleagues revealed that the emergenceof antibodies to cardiolipin (anti-CL), the hallmark of anti-phospholipid syndrome [101], might reflect concurrent orallichen planus in patients with chronic HCV infection [102],although the association seems to be controversial [103].Surprisingly, these patients with anti-CL did not fulfill thecriteria for antiphospholipid syndrome [102]. The emer-gence of TMA may be associated with oral lichen planussecondary to HCV infection [104], although the putativemechanism was not described in detail. Clinical relevance ofantibodies to epithelial components in HCV-associated orallichen planus was also reported [105]. To the contrary, Car-rozzo and his colleagues elucidated no relationship betweenthe emergence of autoantibodies including ANA, SMA,parietal cell antibodies, and anti-epithelial antibodies andconcomitant lichen planus in patients with HCV infection[106]. Another study revealed that cryoglobulin-positivepatients with CH-C had higher prevalence of lichen planusthan cryoglobulin-negative patients [6].

2.5. CREST Syndrome. CREST syndrome is a rare concur-rent autoimmune disease in patients with persistent HCVinfection [3]. We previously elucidated that approximately1% of patients with HCV-related CLD have anticentromereantibodies (ACAs) [107], the serological hallmark of CRESTsyndrome [108]. However, all patients seropositive for ACAdid not have symptoms of CREST syndrome [107], like thecases of primary biliary cirrhosis (PBC) patients with ACA[109]. The putative role of persistent HCV infection in theproduction of ACA remains uncertain, although the mole-cular mimicry between the HCV core antigen and CENP-A[110], one of the major centromere proteins [111], has beenshown.


2.6. IFN-Induced Autoimmunity. Treatment with IFN-α, anantiviral drug, can precipitate or exacerbate autoimmuneendocrine diseases as well as autoimmune thyroid disordersin patients with CH-C [101]. Treatment with IFN-α resultedin the upregulation of major histocompatibility complex(MHC) class I expression on thyroid epithelial cells andswitching the immune response to the Th1 pattern and sub-sequent cytokine release (IFN-γ and interleukin-2) [93–95, 112]. The thyroid was the organ most susceptible to treat-ment with IFN-α in patients with CH-C [113].

The association of the IFN treatment with the develop-ment of type 1 diabetes mellitus (DM) has been shown inpatients with CH-C patients. Of all CH-C patients, 1.4–2.8%had antibodies to glutamic acid decarboxylase (anti-GAD),the serological hallmark of type 1 DM [114], prior to thetreatment with IFN-α [115, 116]. Most CH-C patients withanti-GAD developed type 1 DM by the IFN therapy. Theonset of type 1 DM was closely restricted to the genetic sus-ceptibility demonstrated by the presence of the HLA DRB1-DQB1 haplotype in those patients [117].

Antibodies to 21-hydroxylase were observed in 4.8% ofpatients with CH-C receiving IFN treatment. However, thepresence of this autoantibody was independent of adrenalfailure [118].

The existence of non-organ-specific autoantibodiesincluding ANA at end of the treatment or the increase intiters of SMA can predict unfavorable outcomes of antiviraltreatments in patients with CH-C [119].

It is of interest that Covini and his colleagues identifieda novel autoantigen, which appeared like distinct rods andrings (RRs) in the cytoplasm of HEp-2 cells, in patients withCH-C under treatment with pegylated IFN-α and ribavirin[120].

Genetic backgrounds may trigger the development ofIFN-induced thyroid disorders. There was a close correlationbetween HLA A2 and IFN-induced autoimmune thyroiditisin Japanese patients with CH-C [121]. Another studyrevealed the association of DRB1∗11 with IFN-induced auto-immune thyroiditis in a Caucasian population [122].

2.7. Malignant Transformation. An oncogenic role in chronicHCV infection has been widely shown by the development ofhepatocellular carcinoma (HCC) and B-cell NHL [123]. Theclose relationship between carcinogenesis and autoimmunityhas been also well recognized.

A previous issue revealed that the minority of patientswith HCV-related MC (5–10%) develop a frank malignantlymphoma in long-term follow-up [124]. The clonal B-cellexpansion by HCV infection may account for the malignanttransformation in patients with HCV-related MC [125]. Thet (14 : 18) translocation with overexpression of bcl-2 anti-apoptotic protein [126] in B cells leading to extension ofB-cell survival [127, 128] and the subsequent mutations ofoncogenes including c-myc and p53 [129, 130] seemed toplay essential roles in the development of B-cell NHL.

A strong linkage between HCV-related SS and B-cellNHL has been widely documented. Ramos-Casals and his

colleagues recently demonstrated that patients with HCV-related SS who developed NHL had the immunological fea-tures of higher prevalence of RF and type II MC than thosewith HCV-related SS without NHL [82].

Monoclonal gammopathy of undetermined significance(MGUS) has been recognized as another lymphoproliferativedisorder in HCV-related MC [131]. The monoclonal gam-mopathies of IgGκ and IgMκ are frequently observed inHCV-related MC [132]. The monoclonal gammopathy inthose patients should be monitored to exclude the possibilityof an evolution to multiple myeloma [133].

Raedle and his colleagues revealed that three of 7 (43%)patients with HCV-related HCC had autoantibodies to p53[134], one of the tumor-associated antigens [135], whilenone of the patients with HCV-related CLD did, suggestingthat HCV-induced carcinogenesis resulted in the productionof these autoantibodies. On the other hand, the prevalenceof autoantibodies to survivin [136], a protein which belongsto the inhibitor-of-apoptosis protein (IAP) family [137], washigher in patients with HCV-related HCC than in those withHBV-related HCC. We previously reported 8 of 86 (9%)patients with HCC had autoantibodies to tumor-associatedantigens including p53, insulin-like growth factor II mRNA-binding proteins (IMPs), c-myc, and survivin [138, 139]. Itis of note that seven of 8 HCC patients with those antibodieswere HCV related [138, 139].

A few types of autoantibodies frequently observed inautoimmune diseases are generally present in sera of patientswith HCC. There is an interesting issue on the relationshipbetween autoantibodies to Golgi, which are usually seen inthe patients with rheumatoid arthritis and SLE [140] andHCV-related HCC [141]. Our present report revealed thatpatients with HCV-related CLD seropositive for ACA fre-quently developed HCC [107]. Antibodies to CENP-B, themost common target antigen of ACA, were regarded as apotential biomarker for small-cell lung cancer [142].

2.8. Autoimmune Cytopenia. As another extrahepatic man-ifestation, reports of HCV-related autoimmune cytopeniaincluding autoimmune hemolytic anemia (AHA), autoim-mune thrombocytopenia, and autoimmune neutropenia arerecently increasing in number [143]. It is of interest thatpatients with HCV-related AHA are often associated withthe emergence of ANA and MC [143]. Patients with HCV-related autoimmune thrombocytopenic purpura also hadmore immunological markers [144].

On the other hand, 66–88% of patients with chronicHCV infection and thrombocytopenia had antiplatelet anti-bodies [145, 146], although the development of antiplateletsis not associated with thrombocytopenia. The most commontarget antigen of anti-platelet antibodies is glycoprotein (GP)IIb/IIIa [146].

2.9. Atherosclerosis. Atherosclerosis has been described asa metabolic abnormality caused by HCV infection [147].Autoantibodies to oxidized low-density lipoprotein (anti-ox-LDLs) have been identified as a serological hallmark of


atherosclerosis [148]. We recently revealed that anti-ox-LDLs were associated with the severity of hepatic steatosisin patients with CH-C [149]. Further examination willbe required whether anti-ox-LDL become a biomarker foratherosclerosis in patients with chronic HCV infection ornot.

Abbreviations

ACA: Anticentromere antibodiesAECA: Antibodies to endothelial cellsAHA: Autoimmune hemolytic anemiaAIH: Autoimmune hepatitisanti-CRP: Antibodies to C-reactive proteinanti-GAD: Antibodies to glutamic acid decarboxylaseAMA: Antimitochondrial antibodiesANA: Antinuclear antibodyanti-CL: Antibodies to cardiolipinanti-IMPs: Antibodies to insulin-like growth factor II

mRNA-binding proteinsanti-LKM1: Antibodies to liver kidney microsome type 1anti-ox-LDL: Antibodies to oxidized low-density

lipoproteinanti-TPO: Antibodies to thyroid peroxidaseBAFF: B-lymphocyte activating factorc-ANCA: Antineutrophil cytoplasmic antibody with

cytoplasmic pattern,CH-C: Chronic hepatitis CCLD: Chronic liver diseaseCYP2D6: Cytochrome p450 2D6DM: Diabetes mellitusHCC: Hepatocellular carcinomaHLA: Human leukocyte antigensIAP: Inhibitor of apoptotic proteinsIFN: InterferonMHC: Major histocompatibility complexNHL: Non-Hodgkin’s lymphomaNOSA: Non-organ specific autoantibodiespANCA: ANCA with perinuclear patternMC: Mixed cryoglobulinPBC: Primary biliary cirrhosisRF: Rheumatoid factorSLE: Systemic lupus erythematosusSMA: Smooth muscle antibodyTGA: Thyroglobulin antibodyTMA: Thyroid microsomal antibodyVCAM-1: Vascular cell adhesion molecule-1.

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Review Article

The Impact of Interferon Lambda 3 Gene Polymorphism onNatural Course and Treatment of Hepatitis C

F. Bellanti,1 G. Vendemiale,1, 2 E. Altomare,1 and G. Serviddio1

1 Department of Medical and Occupational Sciences, C.U.R.E. Centre for Liver Disease Research and Treatment,University of Foggia, Italy

2 IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia, Italy

Correspondence should be addressed to G. Serviddio, [email protected]

Received 28 April 2012; Accepted 2 July 2012


Copyright © 2012 F. Bellanti et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Host genetic factors may predict the outcome and treatment response in hepatitis C virus (HCV) infection. Very recently,three landmark genome-wide association studies identified single nucleotide polymorphisms near the interleukin 28B (IL28B)region which were more frequent in responders to treatment. IL28B encodes interferon (IFN)λ3, a type III IFN involved in hostantiviral immunity. Favourable variants of the two most widely studied IL28B polymorphisms, rs12979860 and rs8099917, arestrong pretreatment predictors of early viral clearance and sustained viral response in patients with genotype 1 HCV infection.Further investigations have implicated IL28B in the development of chronic HCV infection versus spontaneous resolution of acuteinfection and suggest that IL28B may be a key factor involved in host immunity against HCV. This paper presents an overviewabout the biological activity and clinical applications of IL28B, summarizing the available data on its impact on HCV infection.Moreover, the potential usefulness of IFNλ in the treatment and natural history of this disease is also discussed.

1. Introduction

More than 20 years after the discovery of the hepatitis Cvirus (HCV) in 1989, it is now well established that HCVinfection affects all countries, leading to a major global healthproblem that requires widespread active interventions for itsprevention and control [1]. Prevalence data estimate that130–170 million persons, or 2-3% of the world populationare infected with HCV [2]. HCV infection is characterizedby two distinct outcomes: in about 30% of cases, innateand adaptive immune responses achieve a permanent controlof infection, referred to as spontaneous HCV clearance;however, most frequently, the host immune responses failand chronic infection is established [3]. Chronic hepatitisC is highly heterogeneous in clinical presentation and out-comes. This heterogeneity may be dependent on virus geno-type but is also largely related to host factors that have beenclearly proven to affect the severity and rapidity of diseaseprogression [4]. The successful eradication of HCV in chron-ically infected patients, defined as a sustained virologicalresponse (SVR), is associated with a reduced risk of disease

progression. Currently, pegylated interferon (PEG-IFN) plusribavirin (RBV) is considered the standard of care for chronichepatitis C, but the rate of SVR is around 50% in patientswith HCV genotype 1, the most common genotype [5, 6].Because PEG-IFN/RBV therapy is costly and often accompa-nied by several adverse effects, pre-treatment predictions ofthose patients who are unlikely to benefit from this regimenenables ineffective treatment to be avoided. Viral load andviral genotypes and the stage of liver disease strongly predictthe response to HCV treatment [7, 8]. Moreover, host geneticdifferences may influence the response to HCV treatment.Recently, through a genome-wide association study (GWAS)of patients infected by genotype 1 HCV, it has been reportedthat single nucleotide polymorphisms (SNPs) linked tothe cytokine IFNλ3 (also known as IL28B) are stronglyassociated with a response to PEG-IFN/RBV therapy [9–12]. There has subsequently been rapidly increasing dataregarding the significance of the IL28B polymorphism notonly in response to therapy but also in spontaneous clearanceof acute HCV infection. Clinically available tests then madeIL28B genotype testing become part of the standard of care.


Genetic analysis of the host may thus predict which patientsare more likely to respond to treatment, taking into accountthat IL28B genotype is only one of many factors that caninfluence response rates to PEG-IFN/RBV therapy in HCVinfection and should be interpreted in the context of otherclinical factors predicting SVR.

2. The IFNλ Family

IFNλ was identified during the recent years and classified as anew group, type III IFN. The IFNλ gene family is composedof three distinct genes: IFNλ1 (IL29), IFNλ2 (IL28A), andIFNλ3 (IL28B) [13, 14]. The members of this IFN familyinteract through unique receptors that are distinct from typeI (IFNα/β) and type II (IFNγ) IFN receptors. The receptorfor type III IFN is composed of the unique IFNλR1 chain,also called IL28Rα and the IL10R2 chain, which is sharedwith IL10, IL22, and IL26 receptor complexes (Figure 1) [15].IL28A, IL28B, and IL29 are clustered together on chromo-some 19 (19q13.13 region) and are coexpressed together withother type I IFNs (IFNα and IFNβ) by virus-infected cells[13, 14]. IFNλRs are expressed at variable levels on mostcell types, and they mediate signalling resulting in inductionof many of the same genes that are induced by signallingthrough IFNα/β Rs [13]. Very interestingly, hepatocytes fromliver biopsy specimens have a high IFNλ receptor expression(IL28Rα), while no expression is evidenced on fibroblasts,endothelial cells, adipocytes, or primary central nervoussystem cells [16–18].

Signalling through type I (IFNα/β) or type III (IFNλ)IFN receptor complexes results in the formation of atranscription factor complex known as IFN-stimulated genefactor 3 (ISGF3), which consists of three proteins: STAT1,STAT2 and IRF-9 (also known as ISGF3γ or p48). After itis fully assembled, ISGF3 translocates to the nucleus whereit binds to IFN-stimulated response elements (ISREs) inthe promoters of various IFN-stimulated genes classicallyassociated with the antiviral phenotype, including OAS1,MX1, IRF7, and EIF2AK2 [double-stranded RNA-activatedprotein kinase (PKR)] [19]. The proteins encoded by thesegenes mediate the antiviral activity induced by the IFNs [20].As a result, the downstream biological activities induced byeither IFNα or IFNλ are very similar, including induction ofantiviral and antiproliferative activity in many cell types.

There are currently very limited available data comparingthe different biological activity of the three IFNλs. Biologicalactivity ultimately depends on the receptor cytoplasmicdomains, which are not related, and these may trigger over-lapping but different biological functions. This, in additionto the pattern of receptor distribution among different celltypes, means that the different IFNλs are functionally distinct[21]. The effects of IFNλs on immune cell function appearto be complex and diverse, while the antiviral effects areclearer. In fact, IFNλ-induced antiviral activity has beendemonstrated against many different viruses but has alsobeen shown to inhibit in vitro replication of HBV and HCV[22–26]. In particular, it has been demonstrated that IFNλ3inhibits HCV replication in three independent HCV modelsby the JAK-STAT pathway [27]. The first use of IFNλ in

clinical setting has started for hepatitis C: pegylated rHuIFNλ (PEG-IFNλ) has now been evaluated in two phase1 clinical trials [28, 29]. The initial phase 1A study wasdesigned to evaluate the safety, tolerability, pharmacokinetic,and pharmacodynamic activity of a single dose of PEG-IFNλin healthy volunteers; a few participants developed reversible,dose-related increases in liver transaminases, but PEG-IFNλdid not induce fever, fatigue, or any overt haematologicalchanges [28]. The phase 1B study has been conducted inpatients with chronic genotype 1 HCV infection, mostly nonresponders to PEG-IFN/RBV therapy; PEG-IFNλ inducedsignificant decreases in the levels of HCV, was well tolerated,and did not induce any significant haematological toxicitiessuch as neutropenia, thrombocytopenia, or anemia [29].However, this study lacks a direct comparison between IFNλand IFNα and the influence of viral and patient genotypes,and it is not clear whether the antiviral effects are mediateddirectly or through the stimulation of immune cells orboth. Since the presumed effects of IFNλ on viral clearanceobserved in the GWAS are mediated through treatment withexogenous IFNα, it is conceivable that although IFNλ maybe less potent than IFNα as a direct antiviral, taken togetherthese cytokines may have an additive effect [24].

3. The Effect of IFNλ3 Polymorphism onIFNλ Biology

IL28B/IFNλ3 may harbour the ability to induce potentinnate antiviral responses in vitro via signalling throughthe IL28 receptor complex, whose expression has beenverified on a variety of cells, including lymphocytes [24,30, 31]. Moreover, studies performed in vivo showed thatIL28B/IFNλ3 had the potential to induce helper T-cell type1-biased adaptive cellular immune responses [32]. IFNλ3has significant influence on antigen-specific CD8+ T-cellfunction, especially in regards to cytotoxicity, since it is apotent effector of the immune system with special emphasison CD8+ T-cell killing functions [32].

IFNλ3 is to date the only member of type III interferonswith genetic variations associated with differential expressionprofiles for downstream genes involved in the immuneresponse and outcome for a disease that exhibits symptomsof dysregulation of the immune response. In 2009, threegenome-wide association studies reported an associationbetween SVR and two SNPs located near the gene regionencoding IFNλ3 (IL28B; rs12980275 and rs8099917) inHCV-infected patients treated with PEG-IFN/RBV com-bination therapy [9–11]. Further subsequent studies wellestablished that the response to IFNα or the natural clearanceof HCV infection is dependent on SNPs, upstream of IFNλ3,which could be used as biomarkers to help determine thetreatment outcome [21]. However, the exact mechanisms bywhich IFNλ3 polymorphisms are identified in the GWASaffect immune function or exert specific antiviral effects inHCV-infected patients are still unclear.

In 2010, two studies performed on HCV-infectedpatients revealed that both the SNPs of IL28B linked withSVR were strongly associated with lower hepatic expressionof interferon-stimulated genes (ISGs) [33, 34]. These results


Extracellular space

Cytoplasm

STAT2P P

IRF9

Nucleus

JAK1 JAK1 JAK2

STAT1 STAT1

P P

GAS

TYK2

P

ISGF3

ISRE

Interferon-stimulated genes

Antiviral activity

JAK1JAK2 TYK2 JAK1

ISRE

STAT1 STAT2

IRF9ISGF3

STAT1

IFNαR1 IFNαR2 IFNγR1

Type III

IFNs (λ )

IL10R2 IFNλR

Type IIIFN (γ)

Type I IFNs

(α-β-κ-

ε-δ-τ)

Figure 1: Types I, II, and III IFN receptors and their downstream signalling pathways.


imply that IFNλ3 polymorphism may explain the relation-ship between hepatic ISG expression and HCV treatmentoutcome but do not reveal any specific mechanism. However,the increase of infected hepatocyte death rate for specificIFNλ3 polymorphism suggests that an immune-mediatedmechanism may be responsible [35].

Further investigations reported some relationshipsbetween IL28B genetic variation and allergic disease inchildren and susceptibility to develop hepatocellular carci-noma in HBV-infected patients [36, 37]. However, eventhough screening of these polymorphisms and functionalstudies would be useful to clinical practice for identifyinggroups at high risk and might help to modify the designof surveillance programs, these studies do not provide anymechanism that explains this association.

In summary, the identity of the functional variantsunderlying the observed associations is still unknown, andfurther studies are encouraged to clarify the biologicalimpact of IFNλ3 polymorphism.

4. The Impact of IFNλ3 Polymorphisms onHCV Infection

4.1. rs12979860 Polymorphism. The SNP on chromosome19q13 (rs12979860) strongly associated with SVR in geno-type 1 HCV-infected subjects, identified by the study of Ge etal., results in three possible genotypes: the C/C genotype wasassociated with 2.5 or greater rate (depending on ethnicity)of SVR compared to the T/T genotype, and the C allelewas overrepresented in a random multiethnic population ascompared to the chronically infected study cohort, raisingthe possibility that the C allele may favour spontaneousclearance of HCV [11]. According to this report, thers12979860 polymorphism also may explain much of thedifference in response between different population groups:in fact, the genotype leading to better response presents withgreater frequency in European than in African populations[11]. Moreover, the C allele was found associated with moresubjective appetite, energy, and sleep complaints, as well aslower serum triglycerides and higher serum LDL cholesterolbut less hepatic steatosis [38–40]. This latter report wasconfirmed by a subsequent study, which showed a lowersteatosis severity grade in HCV-infected patients with C/Cgenotype [41]. This association was then observed in HCVgenotypes 2, 3, and 4, and also in HCV/HIV-infected patientseven though prior non-responders [42–46]. The C allele alsoappears to affect positively early viral kinetics in patientswith chronic hepatitis C receiving interferon-free treatment[47, 48]. However, the C allele is associated with more pro-nounced liver histopathology damage in patients chronicallyinfected with HCV genotype 3, which may be secondaryto higher viral load but has no impact among genotype2 infected patients, implying that IL28B may differentiallyregulate the course of genotype 2 and 3 infection [49, 50]. Inan analysis of HIV-infected patients with acute hepatitis C,the C/C genotype was associated with higher serum levels ofhepatitis C virus RNA, and lower γGT and CD4 cell count,but not significantly associated with treatment response

rates, suggesting that its effects would be different in HIV-infected patients with chronic and acute hepatitis C [51].The C/C genotype predicts SVR in chronically coinfectedpatients, but it is also associated with higher all-causemortality [52, 53]. When this variant was genotyped in HCVcohorts comprised of individuals who spontaneously clearedthe virus or had persistent infection, the C/C genotypestrongly enhanced resolution of HCV infection amongstindividuals of both European and African ancestry, showingthat IL28B plays a determinant role in natural clearance ofHCV and spontaneous resolution of HCV infection [54].This report was also observed in women affected by acutehepatitis C [55]. However, rs12979860 homozygosity is notassociated with resistance to HCV infection in exposeduninfected patients [56]. Studying the impact of donorand recipient genotypes of IL28B rs12979860C>T SNP onhepatitis C virus (HCV) liver graft reinfection revealed adominant but not exclusive impact of the donor rather thanthe recipient genetic background on the natural course andtreatment outcome [57]. Interestingly, the risk of developingposttransplant diabetes mellitus is significantly increased inrecipients carrying the IL28B rs12979860C>T SNP [58].

4.2. rs8099917 Polymorphism. The study by Rauch et al.demonstrated that the rs8099917 minor allele (T/G or T/T)was associated with progression to chronic HCV infectionand also with failure to respond to therapy, with the strongesteffects in patients with HCV genotype 1, 2, or 4 [12, 59].However, an analysis performed on Taiwanese patients witha lower daily viral production rate than Western patients,demonstrated that the T/T genotype may contribute to theincreased viral clearance rate and better virological responsesin these patients [60]. Even though there are no studiesconsidering multiethnic cohorts, a recent meta-analysisevidenced that rs8099917 T/T had slight predictive value inAsian patients [61]. A further study reported that combina-tion analyses of SNP of rs8099917 in recipient and donortissues and mutations in HCV RNA allowed prediction ofSVR to PEG-IFN/RBV therapy in patients with recurrentHCV infection after orthotopic liver transplantation [62].Another study revealed the high prevalence of the rs8099917G allele in HCV/HIV-1-coinfected patients as well as itsstrong association with treatment failure in HCV genotype1-infected patients [63]. The rs8099917 T/T genotype is asso-ciated with higher levels of apoB-100 and LDL cholesterol ingenotype 1-HCV-infected patients [64].

4.3. Combined IFN3λ Polymorphisms. Both favourable geno-types for rs12979860 (C/C) and rs8099917 (T/T) were asso-ciated with spontaneous HCV clearance, possibly interactingin synergy with female sex [65]. On the other side, IL28Bvariants associated with poor response to interferon therapymay predict slower fibrosis progression, especially in patientsinfected with non-1 HCV genotypes [66].

Concerning HCV genotype 3-infected subjects, IL28Bpolymorphisms are associated with RVR but not SVR toPEG-IFN therapy [67]; other studies showed that IL28Bpolymorphisms are strongly associated with the first phase


Table 1: Summary of the impact of different IL28B polymorphisms on HCV infection in several conditions (SVR: sustained virologicalresponse; RVR: rapid virological response).

Polymorphism Genotype Impact on HCV Impact on HCV/HIV Impact on liver graft reinfection

rs12979860 C/C

(i) Higher SVR (genotypes 1,2, 3, 4) [11, 41–45]

(ii) Spontaneous clearance [11](iii) Early viral kinetics [46, 47]

(i) Higher SVR [51](ii) No influence on acute HCV

infection [50](iii) Higher all-cause mortality[52, 53]

(i) Natural course and treatmentoutcome dependent on donorrather than recipient geneticbackground [54]

(ii) Higher frequency ofposttransplant diabetesmellitus [55]

rs8099917T/G or T/T

(i) Treatment failure(genotypes 1, 2, 4) [12, 56]

(ii) Increased viral clearanceand virological response inTaiwanese patients [57]

(i) SVR in patients with recurrentHCV infection [58]

G/G(i) High prevalence [59](ii) Treatment failure (genotype 1)[60]

Combined C/C and T/T

(i) Spontaneous clearance [61](ii) Early viral kinetics [62, 64, 65](iii) RVR but not SVR

(genotype 3) [63]

(i) Early viral kinetics (genotypes 1,4) [65]

viral decline during PEG-IFN/RBV therapy of chronic HCVinfection, irrespective of HCV genotype, and in genotype 1–4 HIV/HCV-coinfected patients [66, 68, 69]. These reportssuggest that IL28B polymorphisms could play a role in block-ing the production or release of virions in the first phase viraldecline [70]. Treatment outcome in HCV-infected patientsmay be also influenced by viral polymorphisms within theviral core and NS5A proteins, even though it has beenclearly demonstrated that IL28B polymorphisms and HCVcore amino acid 70 substitutions contribute independently toan SVR to PEG-INF/RBV therapy [71]. Another independ-ent predictor of RVR and final therapeutic outcome is IFNγinducible protein-10 (IP-10), and the concomitant assess-ment of pretreatment IP-10 and IL28B-related SNPs has beenproposed to improve response or spontaneous clearanceprediction [72–74].

When ten SNPs of IL28B were simultaneously analysedin treatment-naıve patients with genotype 1-HCV chronicinfection who received PEG-IFN/RBV, rs12979860 resultedas the critical predictor for SVR even in patients without RVR[75]. A further study demonstrated that SNPs rs8099917and rs12979860 used alone may be useful for predicting theoutcome of HCV treatment, and in a rational and cost-effective approach, determination of only one of these twoSNPs is sufficient for predicting SVR. Because of the highestpredictive SVR associated with rs12979860 C/C comparedwith the rs8099917 T/T, rs12979860 determination alone issufficient for predicting interferon response [76, 77], evenin HIV/HCV-coinfected patients [78]. However, there is evi-dence that a significant proportion of heterozygous carriersof the rs12979860 T nonresponder allele can profit withrespect to SVR prediction by further determination of thers8099917 SNPs [79].

When the combined effect of variants of IL28B withhuman leukocyte antigen C (HLA-C) and its ligands the

killer immunoglobulin-like receptors (KIRs), which havepreviously been implicated in HCV viral control, wasstudied, prediction of HCV treatment response improved,supporting a role for natural killer (NK) cell activationin PEG-IFN/RBV treatment-induced clearance, partiallymediated by IL28B [80]. Nevertheless, there are conflictingreports on the concept that IFNλ3 might directly act on NKcells to modify their functional activity [81, 82].

4.4. IFN3λ Polymorphisms and Directly Acting Antivirals(DAAs). HCV therapy has been recently updated by theapproval of two DAAs against the NS3/4A serine proteasefor use in genotype 1, the ketoamide inhibitors boceprevirand telaprevir [83, 84]. The impact of IFN3λ polymorphismson therapy outcome has been evaluated in several phase 3clinical trials of boceprevir and telaprevir when used inconjunction with PEG-IFN/RBV. The predictive factors ofSVR to a regimen of PEG-IFN/RBV/telaprevir therapy wasevaluated in 72 Japanese adults infected with HCV genotype1, showing rs8099917 T/T as significant determinant of SVR[85]. In the Phase 3 ADVANCE trial, the addition of telapre-vir to PEG-IFN/RBV improved SVR across all rs12979860genotypes [86]. The retrospective analyses from the SPRINTII and RESPOND-2 trials suggest that patients with thers12979860 C/C genotype are highly likely to be treated forjust 6 months with PEG-IFN/RBV/boceprevir [87].

Several other classes of DAAs are under development,and it is expected that they will increase SVR rates anddecrease the required duration of therapy [88]. The roleof IL28B variations in predicting response to triple-therapyregimens including DAAs different from boceprevir andtelaprevir has very recently been investigated, but it isdifficult to draw firm conclusions owing to the smallnumber of patients in some groups, and additional researchis required before final conclusions can be drawn [89].


Finally, the rs12979860 C/C genotype presented a favourableinfluence on early viral kinetics during treatment with aninterferon-free DAA regimen (mericitabine plus danopre-vir), even though the importance of IL28B genotype onresponse to future interferon-free combination DAA reg-imens remains to be determined with analyses of largerand longer duration studies [47] (refer to the Table 1 for acomprehensive summary of the effects).

5. Conclusion

The IFNλ3 gene polymorphism closely associates with thenatural course and treatment response of chronic hepatitis Cin different populations, irrespective of HCV genotype, thusit can be considered an important predictive pretreatmentfactor. The differential global distribution of IFNλ3 SNPsmay explain the observed clinical differences between ethnicgroups. The identification of these polymorphisms will pro-vide support for clinical decision making in current standardcare. However, in genotype 2/3 patients who achieve RVRor in treatment initiated in acute HCV, IFNλ3 SNPs testingmay have less clinical utility. This raises questions aboutjustifying the cost of incorporating IFNλ3 SNPs testing inroutine practice in such patients. Furthermore, the effect ofthe IL28B genotype is not absolute, and it should not be usedas a criterion for denying therapy when unfavourable.

Future studies are needed to establish the role of IFNλ3genotype using direct antivirals, which rapidly reduce theviral load and may therefore lower the influence of IL28Bgenotyping in predicting SVR. Further functional studies ofIFNλs and the significant SNPs should be investigated toimprove the positive predictive value using the point muta-tion analysis of the targeted polymorphisms. For applying apractical tailor-made therapy, it is also necessary to revealthe cause of exceptional cases that do not follow the IL28Bgenotyping.

Acknowledgments

This work was supported by the Fondazione Banca del Montedi Foggia, Italy.

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Clinical Study

Restoration of Innate and Adaptive Immune Responses byHCV Viral Inhibition with an Induction Approach Using NaturalInterferon-Beta in Chronic Hepatitis C

Y. Kishida,1 N. Imaizumi,1 H. Tanimura,1 Y. Haruna,2 S. Kashiwamura,3 and T. Kashiwagi4

1 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Osaka Kaisei Hospital 1-6-10 Miyahara,Yodogawa-Ku, Osaka City, Osaka 532-0003, Japan

2 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Osaka Prefectural General Medical Center,Osaka City, Osaka 558-858, Japan

3 Laboratory of Host Defenses Institute for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya,Hyogo 663-8501, Japan

4 Department of Nuclear Medicine and PET-Center, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan

Correspondence should be addressed to Y. Kishida, [email protected]


Academic Editor: Jurg Schifferli

Copyright © 2012 Y. Kishida et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chronic hepatitis C (CHC) is a serious medical problem necessitating more effective treatment. This study investigated thehypothesis that an induction approach with nIFN-beta for 24 weeks followed by PEG-IFN-alpha+ribavirin (standard of care:SOC) for 48 weeks (novel combination treatment: NCT) would increase the initial virologic response rate and restore innate andadaptive immune responses in CHC. Seven CHC patients with a high viral load and genotype 1b were treated with NCT. Serumcytokine and chemokine levels were evaluated during NCT. NCT prevented viral escape and breakthrough resulting in persistentviral clearance of HCVRNA. IL-15 was increased at the end of induction therapy in both early virologic responders (EAVRs) andlate virologic responders (LAVRs); CXCL-8, CXCL-10, and CCL-4 levels were significantly decreased (P < 0.05) in EAVR but notin LAVR during NCT, and IL-12 increased significantly (P < 0.05) and CXCL-8 decreased significantly (P < 0.05) after the endof NCT in EAVR but not in LAVR. NCT prevented viral breakthrough with viral clearance leading to improvement of innate andadaptive immunity resulting in a sustained virologic response (SVR). NCT (n = 8) achieved a higher SVR rate than SOC (n = 8)in difficult-to-treat CHC patients with genotype 1 and high viral loads.

1. Introduction

About 180 million people (around 3% of the world’s popu-lation) are infected with the hepatitis C virus (HCV) [1].Chronic hepatitis C (CHC) is a leading cause of chronic hep-atitis, cirrhosis, liver failure, and hepatocellular carcinomaworldwide [2]. CHC is a serious global medical problemnecessitating effective treatment. However, 50% of treatedpatients are not cleared of viremia when treated with pegy-lated- (PEG-) interferon- (IFN-) alpha plus ribavirin (RBV)for 48∼72 weeks (standard of care: SOC) [3, 4]. The triplecombination of PEG-IFN-alpha, RBV, and a protease inhi-bitor (telaprevir or boceprevir) fails to eradicate HCV inapproximately 20∼30% of treatment-naıve and 50∼60% of

treatment-experienced patients [5, 6]. Thus, more effective,more tolerable and/or more tailored therapies are required.

Viral kinetics in response to anti-HCV treatment is animportant factor during treatment. With successful anti-viral treatment, the HCVRNA concentration in serumpromptly decreases to undetectable levels and remainsnegative throughout therapy and thereafter. The faster thevirus becomes undetectable during therapy, the better thechance of achieving a sustained virologic response (SVR).Accumulating evidence suggests that an early response totreatment is best determined by the level of HCVRNA inserum at 4 and 12 weeks of therapy [7, 8]. Because anSVR has been shown to be more likely after favorable earlyviral kinetics (i.e., a more rapid and profound reduction in


HCV RNA levels), a rapid initial clearance augmented byinduction therapy for the first several months was postulatedas an approach to optimizing new therapeutic strategies toachieve SVR [9, 10]. HCV exists as a genetically heterogenousviral population, named quasispecies. Thus, the clinical suc-cess of new HCV therapies will depend on their ability tosuppress all viral variants as well as prevent the emergenceof resistant viruses [11].

Recent advances in the understanding of innate immu-nity show that the activation of the innate immune system isessential for subsequent adaptive immune responses includ-ing specific antibody production and CTL activation whichplay a key role in protection against viral infections [12]. Inaddition to evading the innate immune system, HCV hasevolved effective means of thwarting the adaptive immunesystem [13, 14].

IFNs are key mediators of the host innate antiviralimmune response. IFN-stimulated gene (ISG) products canprevent the translation of viral RNA and thereby limit theinitial viral spread in the liver until viral clearance occurs byHCV-specific T cells [15]. The first response is thought to beIFN-beta production by infected hepatocytes. IFN-beta hasdifferent signaling and biological activities from IFN-alphaand achieved a higher rate of viral clearance than IFN-alpha[16–21]. Contrary to the actions of IFN-alpha, IFN-beta andIFN-lambda signaling in the liver does not become refract-ory during repeated stimulation of the IFN signaling trans-duction pathway. The sustained efficacy of IFN-beta andIFN-lambda could be important for the treatment of patientswho do not respond to PEG-IFN-alpha through a preacti-vated endogenous IFN system [21].

Resolution of an HCV infection may restore impairmentsof innate and adaptive immunity [22–24].

However, the issue of how to increase the initial virologicresponse rate has not been resolved and is complicated byviral breakthrough and adverse effects.

In a previous study, we have shown that cyclic and peri-odic IFN treatment (CPIT) consisting of induction treatment(IT) with natural (n) IFN-beta for 2 weeks followed bymaintenance treatment (MT) with nIFN-alpha for 2 weekscould prevent virologic breakthrough and achieve an earlyvirologic response (EVR) and an end-treatment virologicresponse (ETVR). In addition to the improvement of innateimmunity due to virologic clearance by CPIT during theinitial course of therapy, persistent virologic clearance andrestoration of innate and adaptive immune responses by RBVplus PEG-IFN-alpha were more likely to result in a higherrapid virologic response (RVR), EVR, ETVR, and SVR. Onthe basis of these findings, we conducted a pilot study in 7CHC patients with genotype 1b, high viral loads, and wild orintermediate type IFN sensitivity determining region (ISDR)to assess the efficacy, tolerability, and safety of treatment withRBV plus PEG-IFN-alpha 2b for 48 weeks (SOC) using aninduction approach with initial virologic clearance inducedby CPIT for 24 weeks (novel combination treatment: NCT)[25].

Little is known about the chemokine and cytokine res-ponse to HCV infection before, during, and after IFN treat-ment. Aiming to better understand the immunological

determinants of the protective immune response to HCVinfection, we performed an extensive analysis of the innateand adaptive immune responses in CHC patients with geno-type 1b and high viral load. We have evaluated the serumlevels of cytokines and chemokines that mediate humoraland cellular immunity and inflammation, correlated withdisease activity, and characterize the immunomodulatoryeffects of therapy.

In addition, we compared the efficacy and safety ofNCT versus SOC in CHC patients with genotype 1b andhigh viral loads. The rate of SVR was significantly higheramong patients receiving NCT than those receiving SOC.NCT is beneficial to treat difficult-to-treat CHC patients withgenotype 1b and high viral loads.

2. Patients and Methods

2.1. Study 1

2.1.1. Patients. Seven patients [3 males and 4 females, meanage 53.3 ± 8.5 years (range 39–66)] with CHC, genotype1b (serotype 1), ISDR with 3 wild type, 3 intermediatetype, and 1 not determined, and a viral load of 2144.3 ±1701.2 KIU/mL (range 536–>5000 KIU/mL) were enrolledin this open-label, prospective study. Patients underwent aliver biopsy before the IFN therapy, and the severity [inflam-mation (grade) and fibrosis (stage)] of liver disease [26]was evaluated as chronic hepatitis (grades 1–3, stages 1-2)(Table 1). Serum was collected from five healthy donors,ranging in age from 28 to 58 years. Written informed consentwas obtained from all patients according to the Declarationof Helsinki.

2.1.2. Exclusion Criteria. The following were considered asexclusion criteria: refusal by women of child-bearing ageor by sexually active patients to use a safe contraceptive,pregnancy or breast-feeding, cirrhosis with signs of decom-pensated liver diseases, coronary heart diseases, the presenceof overt psychiatric diseases, active alcohol or drug abuse,uncontrolled diabetes mellitus, uncontrolled hypertension,uncontrolled retinopathy, autoimmune disorders, or anyother unstable medical condition not because of liver disease.All patients were negative for hepatitis B surface antigen, andfrequent causes of chronic liver diseases were excluded.

2.1.3. Study Design. Cyclic and periodic IFN treatment(CPIT): the patients were treated with 6 cycles (24 weeks) ofcyclic and periodic IFN treatment (CPIT). One cycle of CPITconsisted of IT with nIFN-beta (Feron, Toray, Chiba, Japan)at 3–6 MU/day, intravenously by drip infusion in 100 mL ofsaline solution, daily for 2 weeks followed by MT with nIFN-alpha (Sumiferon, Sumitomo, Osaka, Japan) at 6 MU/day,subcutaneously, three times weekly for 2 weeks.

CPIT was followed by treatment with RBV plus PEG-IFN-alpha 2b (SOC) (novel combination treatment: NCT):we investigated the efficacy, tolerability, and safety of CPITfor 24 weeks as induction therapy followed by RBV (Rebetol:Schering Plough, Kenilworth, NJ, USA; 200–800 mg/day,per os, daily) plus PEG-IFN-alpha 2b (Pegintron, Schering


Table 1: Characteristics of chronic hepatitis C with high viral load, serotype-1 (genotype 1b), and wild or intermediate type in ISDR before,during, and after RBV plus PEG-IFN-alpha 2b using an “induction” therapy with cyclic and periodic interferon treatment (CPIT); novelcombination treatment (NCT).

Patient NO.

ALT (lU/mL)

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BMI(kg/m2)

Liverhistology

(Stage/Grade) baseline24 weeks

(end of NCT)72 weeks

(end of NCT)

96 weeks (24weeks after end

of NCT)

Outcome oftreatment

Early virologic responders

1 61/F 46.1 20.4 1/2 197 37 15 13 SBR

2 47/M 67.0 21.1 1/1 37 28 22 22 SBR

3 66/M 78.0 24.5 3/2 57 29 29 17 SBR

4 49/M 60.0 19.8 1/2 50 32 12 11 SBR

5 61/F 48.5 20.9 1/2 38 9 14 10 SBR

Late virologic responders

6 39/F 73.0 27.1 1/2 48 331 264 161 NBR

7 56/F 55.0 20.6 −/− 33 19 13 49 TBR

Mean ± SD. 53.3± 8.5 61.1± 12.1 22.1±2.7 1–3/1-2 65.7± 58.5 69.3± 115.8 52.7± 93.3 39.0± 59.9

IFN: interferon, BMI: body mass index, ISDR: IFN sensitivity determining region, F: female, M: male, SBR: sustained biochemical response, NBR: no bio-chemical response, and TBR: transient biochemical response.

Plough, Kenilworth, NJ, USA; 60–120 micro-g/day, percu-taneously inj., once weekly) (SOC) for 48 weeks (total 72weeks) in a pilot clinical trial as a potential treatment for 7difficult-to-treat CHC patients with genotype 1b, high viralload (a viral load of more than 100 KIU/mL), and wild orintermediate type ISDR [25].

2.1.4. Measurements. All patients were monitored with clin-ical, biochemical, and virologic assessments before and every1 to 4 weeks during the entire 72-week treatment periodand were followed for an additional period of more than 24weeks. The level of HCVRNA in serum was determined usingthe quantitative COBAS AMPLICOR HCV MONITOR test,ver. 2.0 (Roche Diagnostic Systems, Tokyo, Japan; sensitivity<50 IU/mL).

Assessments of serum cytokines and chemokines (mul-tiplex cytokine assay) were done. A multiplex biomet-ric enzyme-linked immunosorbent assay- (ELISA-) basedimmunoassay [27, 28], with dyed microspheres conjugatedto a monoclonal antibody specific for a target protein, wasused according to the manufacturer’s instructions (Bio-PlexHuman Cytokine assay; BioRad Inc., Tokyo, Japan). Cyto-kines measured were (i) Th1 cytokines: IFN-gamma, TNF-alpha, IL-1-alpha, IL-1-beta, IL-2, IL-12 (p70), and IL-15,(ii) Th2 cytokines: IL-4, IL-6, IL-9, Il-10, and IL-13, (iii)hematopoietic cytokines: GM-CSF and G-CSF, (iv) CXCchemokines: CXCL-8 (IL-8) and CXCL-10 (IP-10), (v) CCchemokines: CCL-2 (MCP-1), CCL-3 (MIP-1-alpha), CCL-4(MIP-1-beta), CCL-5 (RANTES), and CCL-11 (EOTAXIN),and (vi) other cytokines: VEGF and PDGF.

2.2. Study 2. We investigated whether induction therapyusing CPIT with natural IFN-beta would increase SVR ratesin patients with CHC genotype 1b and high viral loads. Wecompared the efficacy and safety of NCT (n = 8) versus SOC(n = 8) in CHC patients with genotype 1b and high viral

loads. All patients were monitored with clinical, biochemicalassessments, and virologic responses assessed by TaqManPCR (limit of detection, 15 IU/mL) before and every 1 to 4weeks during the 48∼72-week treatment period, and werefollowed for at least an additional 24 weeks after cessation oftreatment. The primary efficacy end point was achievementof an SVR 24 weeks after cessation of treatment (Table 2).

2.3. Assessment of Safety. Safety was assessed with laboratorytests and an evaluation of adverse events (AEs) every 1–4weeks during and after the end of NCT. A reduction in theRBV dosage from 800 to 200–600 mg per day and reductionin the PEG-IFN-alpha 2b dosage from 60–120 microg to 50–100 microg without virologic breakthrough were allowed tomanage AEs or laboratory abnormalities that had reachedpredetermined thresholds of severity. If the AEs were resolvedor improved, a return to initial dosing levels was permitted.

2.4. Statics. Data were expressed as the mean ± standarddeviation, and a paired-t test was used to evaluate the differ-ences of the means between groups, with a P value of <0.05considered significant.

3. Results

3.1. Study 1. HCV viral titers decreased in all patientsafter 4 weeks of CPIT highlighting the efficacy of thistreatment modality. None of the patients showed viro-logic breakthrough. Serum HCVRNA [2144.3 ± 1701.2(range 536–>500) KIU/mL at baseline] decreased signifi-cantly to 1.5 ± 2.4 KIU/mL (P = 0.0157) at the end of CPIT.The rates of RVR and EVR [partial EVR (pEVR), completeEVR (cEVR), and RVR plus cEVR (extended RVR)] were7/7(100 %) and 7/7 [100%; 4/7 (57.1%), 3/7 (42.9%), and 3/7(42.9%)], respectively. Viral titers dropped below detectablelevels in 5 patients before the end of CPIT, and in 2 patients


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after the end of CPIT (after beginning of RBV plus PEG-IFN-alpha-2b). The rates of ETVR at the end of CPIT and NCTwere 5/7 (71.4%) and 7/7 (100%), respectively. The rate ofSVR was 5/7 (71.4%). Transient virologic response (TVR)was found in 2 patients who showed undetectable HCVRNAin serum after the end of CPIT (Table 3).

To refine our understanding of the heterogeneity oftherapeutic responses, patients were classified into twostatistically distinct groups based on the time of clearanceof viremia. Of note, early virologic responders (EAVR) withundetectable HCVRNA in serum before the end of CPIT,which included 5 patients (pt. NO 1–5), showed SVR. Latevirologic responders (LAVRs), with undetectable HCVRNAin serum after the end of CPIT, which included 2 patients(pt. NO 6-7), showed a TVR. The viral titer values in LAVRwere extremely high (>5000 and 4400 KIU/mL).

Serum ALT decreased at the end of NCT and after the endof NCT. The rate of sustained biochemical response (SBR)was 5/7 (71.4%) (Table 1).

3.1.1. Serum Cytokines and Chemokines at Baseline (Figure 1).CXCL-8, CXCL-10, CCL-4, and CCL-11 levels were signi-ficantly higher (P < 0.05); IFN-gamma, TNF-alpha, IL-1alpha, IL-2, IL-6, IL-9, IL-15, GM-CSF, G-CSF, and CCL-2levels were higher; IL-10, IL-12, and IL-13 levels were signi-ficantly lower (P < 0.05) in all CHC patients than in thecontrols.

IL-6, IL-15, CXCL-8, CXCL-10, and CCL-11 levels weresignificantly higher (P < 0.05), and IFN-gamma, TNF-alpha,

IL-1alpha, IL-2, GM-CSF, G-CSF, CCL-2, and CCL-4 levelswere higher in EAVR than in the controls. IL-10 and IL-13levels, were significantly lower (P < 0.05), and IL-12 levelswere lower in EAVR than in the controls.

GM-CSF, CXCL-10, and CCL-4 levels were significantlyhigher (P < 0.05), and TNF-alpha, IFN-gamma, IL-1alpha,IL-1beta, IL-2, IL-15, IL-6, IL-9, IL-4, G-CSF, PDGF, CXCL-8, and CCL-11 levels were higher in LAVR than in the con-trols.

3.1.2. Serial Values of Serum Cytokines and Chemokines during

the NCT (Figures 2, 3, 4, 4, 5, 6, 7, 8, and 9)

At the End of CPIT. In all CHC patients, the levels of CCL-4decreased significantly (P < 0.05), the levels of IFN-gamma,TNF-alpha, IL-1alpha, IL-beta, IL-2, IL-4, GM-CSF, and G-CSF decreased, and the levels of IL-9, IL-10, IL-13, IL-15,CCL-2, PDGF, and VEGF increased from baseline.

In EAVR, the levels of CCL-4 decreased significantly (P <0.05) from baseline but to a lesser extent than in LAVR. Thelevels of CXCL-8 decreased in EAVR but increased signi-ficantly (P < 0.05) in LAVR. The levels of CXCL-10, CCL-3, and PDGF decreased in EAVR but increased in LAVR atthe end of CPIT.

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CCL-3, CCL-11, PDGF, GM-CSF, and G-CSF decreased frombaseline.

In EAVR, the levels of IFN-gamma, IL-1alpha, CCL-4,and CXCL-8 decreased significantly (P < 0.05), and the levelsof CXCL-10 decreased (P < 0.1) from baseline. In LAVR, thelevels of IFN-gamma, IL-1alpha, and CCL-4 decreased andCXCL-8 increased. The levels of IL-9, G-CSF (P < 0.1), andCXCL-10 (P < 0.1) decreased in EAVR but not in LAVR. Thelevels of IL-6, IL-12, IL-15, and CCL-3 (P < 0.1) decreasedin LAVR but not in EAVR. CPIT induced the upregulation ofIL-15 expression, but RBV/PEG-IFN-alpha 2b did not. IL-10and VEGF levels increased in LAVR but were unchanged inEAVR.

Four Weeks after the End of NCT. In all CHC patients, thelevels of IL-12 and VEGF increased significantly (P < 0.05),and IL-10 (P < 0.1) and CCL-2 levels increased frombaseline. The levels of IFN-gamma, TNF-alpha, IL-1alpha,IL-1beta, IL-2, IL-4, IL-6, CXCL-8, CXCL-10 (P < 0.1), CCL-4 (P < 0.1), CCL-11, GM-CSF, and G-CSF decreased frombaseline.

The levels of IL-12 and VEGF increased (P < 0.05) inEAVR, and to a lesser extent, in LAVR. The levels of IL-15and CCL-2 increased in EAVR but decreased in LAVR. Thelevels of IL-13 increased (P < 0.1) in LAVR and to a lesserextent in EAVR. The levels of CXCL-10 (P < 0.1) decreased

in EAVR and to a lesser extent in LAVR. CXCL-8 and CCL-3levels were unchanged in EAVR but decreased in LAVR.

Correlation of Serum Cytokine and Chemokine Levels toTherapeutic Responses (Figures 2, 3, 4, 5, 6, 7, 8, and 9). TheIL-15 level increased at the end of CPIT in EAVR and LAVRand 4 weeks after the end of NCT in EAVR but not in LAVR.The level of CXCL-8 decreased significantly (P < 0.05) inEAVR but not in LAVR during NCT. After the end of NCT, inEAVR but not in LAVR, the IL-12 level increased significantly(P < 0.05), and the CXCL-8 level decreased significantly(P < 0.05). CXCL-8 increased in LAVR at the end of CPITand NCT.

At the end of NCT and after the end of NCT, the CXCL-10 level significantly decreased (P < 0.05) in EAVR but notin LAVR. At the end of CPIT and the end of NCT, the CCL-4level significantly decreased (P < 0.05) in EAVR but not inLAVR.

3.2. Study 2. HCV viral titers significantly decreased (P <0.05) from the baseline in NCT and SOC after the beginningof treatment. HCV RNA levels decreased more in NCT thanin SOC (Figure 10).

The rates of early virologic response differed in the initial4 and 12 weeks, and ETVR and SVR in CHC patients withgenotype 1b and high viral loads treated with the NCT and


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Figure 5: Effect of RBV plus PEG-IFN-alpha 2b using an “induction” approach with CPIT (NCT) on serum CCL-4 in chronic hepatitis Cpatients with high viral load, genotype 1b (serotype I), and wild or intermediate type of ISDR. RBV: ribavirin, PEG-IFN: pegylated interferon,CPIT: cyclic and periodic interferon treatment, NCT: novel combination treatment. Significant difference: ∗P < 0.05, ∗∗P < 0.1.

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Figure 6: Effect of RBV plus PEG-IFN-alpha 2b using an “induction” approach with CPIT (NCT) on serum CXCL-8 in chronic hepatitis Cpatients with high viral load, genotype 1b (serotype I), and wild or intermediate type of ISDR. RBV: ribavirin, PEG-IFN: pegylated interferon,CPIT: cyclic and periodic interferon treatment, NCT: novel combination treatment. Significant difference: ∗P < 0.05, ∗∗P < 0.1.

the SOC. The rate of RVR in week 4, pEVR in week 12, cEVR(extended RVR) in week 12, virological response in week 24,and ETVR and SVR among CHC patients with genotype1b and high viral loads receiving the SOC and NCT were87.5 versus 100%, 50 versus 25%, 50 versus 75%, 50 versus75%, 50 versus 100% (P = 0.0764), and 37.5 versus 75%(P = 0.0435), respectively, (Figures 10 and 11). Serum ALTlevel decreased after the NCT and SOC in CHC patients withgenotype 1b and high viral loads (Figure 12).

Adverse events: levels of platelets in peripheral blood dur-ing the NCT and SOC (18.9± 5.5 versus 17.8± 6.1× 104/mLat baseline) in CHC patients with genotype 1b and highviral loads were significantly higher among patients receiv-ing the NCT compared with patients receiving the SOC(22.7 ± 6.2 versus 15.3 ± 6.7 × 104/mL, P = 0.0174) 24weeks after cessation of treatment (Figure 13). Levels of Hb(13.2 ± 2.1 versus 14.4 ± 2.0 g/dL at baseline) in peripheral

blood during the NCT and SOC in CHC patients with geno-type 1b and high viral load were significantly higher amongpatients receiving the NCT compared with patients receiv-ing the SOC at week 12 (12.6 ± 1.7 versus 10.8 ± 1.9,P = 0.0767) and at week 24 (12.3 ± 1.2 versus 10.8 ± 1.8,P = 0.0641) (Figure 14).

4. Discussion

This study investigated the hypothesis that an inductionapproach using CPIT with nIFN-beta would increase the ini-tial virologic response rate and restore innate and adaptiveimmune responses in CHC patients with genotype 1b and ahigh viral load.

Study 1 has shown that NCT with an induction approachwith nIFN-beta achieved the prevention of viral escapeand breakthrough resulting in persistent viral clearance of


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Figure 7: Effect of RBV plus PEG-IFN-alpha 2b using an “induction” approach with CPIT (NCT) on serum IL-10 in chronic hepatitis Cpatients with high viral load, genotype 1b (serotype I), and wild or intermediate type of ISDR. RBV: ribavirin, PEG-IFN: pegylated interferon,CPIT: cyclic and periodic interferon treatment, NCT: novel combination treatment. Significant difference: ∗P < 0.05, ∗∗P < 0.1.

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HCVRNA leading to an improvement in innate and adaptiveimmune responses in difficult-to-treat CHC patients withgenotype-1b, high viral loads, and wild or intermediate typesof ISDR.

The current results (Figures 1, 2, 3, and 4) show that(1) the significantly lower levels (P < 0.05) of IL-12 andthe significantly higher levels (P < 0.05) of CXCL-8, IL-10, CXCL-10, CCL-4, CCL-11, and VEGF in CHC patientscompared to the controls at baseline suggested the impair-ment of innate and adaptive immunity in CHC patients,(2) the level of IL-15 was increased at the end of CPIT inboth EAVR and LAVR; levels of CXCL-8, CXCL-10, CCL-4, and CCL-4 were significantly decreased (P < 0.05) inEAVR but not in LAVR during NCT, and (3) the level ofIL-12 increased significantly (P < 0.05), and the level ofCXCL-8 decreased significantly (P < 0.05) after the end ofNCT in EAVR but not in LAVR. Importantly, the current

study suggested that initial early virologic clearance inducedby CPIT before the use of a combination of RBV and PEG-IFN-alpha 2b induced the restoration of DC function andimprovement of activation of NK cells indicated by theupregulation of IL-12 and IL-15 and the downregulation ofCXCL-8, CXCL-10, CCL-4, and CCL-11. These observationssuggested the timing and breadth of innate and adaptiveimmune responses to be important in determining the out-come of HCV infections. Protective immunity against HCVlikely depends primarily on the activation of both innate andcellular immune response [29].

Recent research identified multiple strategies that HCVemploys to attenuate the innate type IFN response [30].Innate immunity is the first line of defense against an invad-ing viral, bacterial, or fungal pathogen, and the hepatitis Cvirus (HCV), a single-strand RNA virus, is no exception.The recognition of a viral pathogen via the a coordinated


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interaction of the cells of the innate immune system leads tothe activation of adaptive immunity targeting viral-specificantigens. HCV interferes with the activation of adaptiveimmune responses by innate immune cells [31].

There appear to be innate immune responses that controlthe levels of viruses and lead to significant decreases in theHCVRNA titer with SVR. The timing of these responses isnot the same for early and late virologic responders. There isa distinct shift at the point at which viral replication beginsto decrease in individual HCVRNA titers. One of the keycharacteristics of an HCV infection is the delayed immuneresponse despite the early increase in the HCV titer and theinduction of ISGs. The delay in the induction of the innate

immune response that caused this decrease results in contin-ued viral replication, which may account for the higher peakHCVRNA titers seen in the non-SVR group. This delay maylead to immune escape or exhaustion of the induced responsedue to high numbers of infected cells [32].

Chemokines and cytokines are critical regulators of liverinflammation, and innate and adaptive immunity to HCV,the complex orchestration of which is suggested to determinethe outcome of HCV infections [30, 33].

Both maturation and functional differentiation of cDCsare altered during an HCV infection with decreased IL-12[34] and increased IL-10 production in vitro [35, 36]. TheHCV core protein has been shown to bind to the globulardomain of the complement receptor of macrophages andDCs and downregulate IL-12 production [37]. Consideringthat IL-12 is a key cytokine in the induction of CD4 T-cell activation, whereas IL-10 has complex inhibitory effects,HCV-induced modulation of these cytokines may have spe-cial importance in altered HCV-specific T-cell responses inchronic HCV infections [30]. IL-12 governs the Th1-typeimmune response, affecting the spontaneous and treatment-induced recovery from HCV infection [38].

Increased levels of IL-15 at the end of CPIT suggested thatinitial viral clearance, induced by CPIT before the beginningof RBV plus PEG-IFN-alpha 2b therapy, improved the innateimmune response to HCV.

IL-15 plays an important role in the innate immune sys-tem and is a stimulatory cytokine for DCs impaired in CHC.IL15 is induced by IFN-alpha and/or IFN-beta and stimu-lates the proliferation and accumulation of NK cells. IL-15 isrequired for the maturation and survival of NK cells. NK cellshave roles in both innate and adaptive immunity [39].Theactivation of NK cells, as well as the timing, breadth, androbustness of the subsequent antigen-specific T cell immu-nity, is likely to be substantially shaped by early events in theinnate response to the pathogen. IL-12 and IL-15 are bio-markers for the innate immune response.


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Weeks

NCT (n = 8)SOC (n = 8)

Figure 12: Changes in ALT level of peripheral blood during theNCT and the SOC in chronic hepatitis C patients with genotype 1and high viral loads. NCT: novel combination treatment; cyclic andperiodic IFN treatment (CPIT) consisting of induction treatmentwith natural interferon-β followed by maintenance treatment withnatural interferon-α for 24 wks as induction approach followed bySOC for 48 wks. SOC: standard of care; ribavirin plus pegylatedinterferon α 2b for 48 wks.

High serum levels of IL-10 are associated with an incom-plete response to IFN therapy. Chronic HCV infection is cha-racterized by poor cellular immune response, which mightbe in part due to the production of immune suppressivecytokines like IL-10 [40–43]. IL-10 inhibits IFN-alpha pro-duction, promotes apoptosis of pDC, and downregulateseffector T-cell responses [30]. IL-10-inhibiting peptides mayhave important applications to enhance anti-HCV immune

0 4 12 24 48 72 96

Weeks

Pla

tele

ts (

104/m

L)

10

15

20

25

30

P = 0.074∗

NCT (n = 8)SOC (n = 8)

Figure 13: Changes in platelets level of peripheral blood during theNCT and the SOC in chronic hepatitis C patients with genotype 1and high viral loads. NCT: novel combination treatment; cyclic andperiodic IFN treatment (CPIT) consisting of induction treatmentwith natural interferon-β followed by maintenance treatment withnatural interferon-α for 24 wks as induction approach followed bySOC for 48 wks. SOC: standard of care; ribavirin plus pegylatedinterferon α 2b for 48 wks.

responses by restoring the immunostimulatory capabilitiesof DCs.

The levels of CXCL-10, CCL-3, and PDGF decreased inEAVR but increased in LAVR at the end of CPIT. The level ofCXCL-8 was significantly higher in CHC than in the controls.Because CXCL-8, the production of which is stimulated byHCV NS5A, is able to directly inhibit the antiviral activity

Clinical and Developmental Immunology 13H

b (g

/dL)

0 4 12 24 48 72 96

NCT (n = 8)SOC (n = 8)

10

11

12

13

14

15

P = 0.0641

P = 0.0767

P = 0.0594

∗∗

∗

Weeks

Figure 14: Changes in Hb level of peripheral blood during theNCT and the SOC in chronic hepatitis C patients with genotype 1and high viral loads. NCT: novel combination treatment; cyclic andperiodic IFN treatment (CPIT) consisting of induction treatmentwith natural interferon-β followed by maintenance treatment withnatural interferon-α for 24 wks as induction approach followed bySOC for 48 wks. SOC: standard of care; ribavirin plus pegylatedinterferon α 2b for 48 wks.

of IFN-alpha, higher levels of CXCL-8 in nonrespondersmay contribute in part to the poor response to IFN-alphatherapy [44, 45]. The levels of CXCL-8 decreased in EAVRbut increased significantly in LAVR. This result suggested therestoration of antiviral activity of type 1 IFN inhibited byCXCL-8 in EAVR but not in LAVR.

Serum CXCL-10 levels at baseline were higher in CHCpatients than in controls. Levels of serum CXCL-10 weresignificantly decreased in EAVR but not in LAVR. CXCL-10 is a chemotactic CXCL chemokine. CXCL-10 targets theCXCR 3 receptor and attracts T lymphocytes, NK cells, andmonocytes. There is a strong association between pretreat-ment expression of intrahepatic CXCL-10 mRNA and plasmaconcentration of the protein, indicating that HCV-infectedhepatocytes are likely the primary source of plasma CXCL-10 in chronic HCV infections. The intrahepatic expressionof CXCL-10 mRNA predicts the HCV viral kinetic response.Low CXCL-10 levels both in the liver and in plasma beforethe onset of treatment are associated with SVR and pro-nounce the first-phase reduction of HCV viral load for allviral genotypes [46–48].

Serum VEGF levels at baseline were significantly higherin EAVR but not in LAVR of CHC patients. Serum VEGF lev-els were associated with SVR [49].

Serum CCL-4 and CCL-11 levels at baseline were higherin CHC patients than in controls. Serum CCL-4 and CCL-11 levels significantly decreased in EAVR but not in LAVR inCHC patients. CCL-4-mediated T-cell infiltration is essen-tial for the delivery of IFN-gamma to mediate protectivedownstream responses against HCV infections in the liver.It has been shown from the intrahepatic gene expressionprofiles of chimpanzees that CCL-4 was upregulated duringacute infection at the time of viral clearance, but not in thosewho failed to eradicate the virus [31]. CCL-11 is a chemokine

that is thought to selectively attract eosinophils by activatingCCR3 receptors. Several studies have shown that CCL-11is involved in the pathogenesis of inflammatory processesduring liver diseases as well [50]. Harvey et al. recently ana-lyzed the association between chemokines and virologic res-ponses to IFN and RBV in HIV and HCV coinfected patients[51]; in patients achieving an SVR, plasma CCL-11 levelsbefore therapy were statistically higher than in nonrespond-ers [31].

Study 2 has shown that NCT was well-tolerated andenhanced RVR, cEVR (extended RVR), ETVR, and SVR ratesin difficult-to-treat CHC with genotype 1b and high viralload and revealed less adverse effects (AEs) than those inSOC. The higher virologic response rates highlight the bene-fit of NCT with an induction approach using nIFN-beta inCHC patients.

These results suggested that (1) early virological clear-ance by CPIT before the beginning of RBV/PEG-IFN-alpha2b treatment induced the restoration of innate immune resp-onses and lead to antiviral responses and (2) persistent viro-logic clearance for more than 48 weeks with the subsequentRBV plus PEG-IFN-alpha 2b therapy-induced restorationof innate immune responses linked to adaptive immuneresponses and resulting in SVR and SBR. These results sug-gested that CPIT improved the innate immune response;however, there was insufficient improvement of the adaptiveimmune response in CHC during NCT. The findings fromthis study support the concept that viral clearance early inthe course of therapy with reduced virologic resistance islinked to restoration of innate and adaptive immune res-ponses, suggesting that agents providing the greatest viralsuppression leading to extended RVR may be preferable asthe initial early induction approach. An initial viral clearanceinduced by more adequate CPIT before beginning RBV plusPEG-IFN-alpha 2b therapy may lead to an improvement ofinnate and adaptive immune responses resulting in a higherrate of SVR in difficult-to-treat CHC patients with genotype1b and a high viral load.

In previous studies, dose reductions or treatment dis-continuations of PEG-IFN-alpha that were often required tomanage adverse hematological events have been associatedwith a reduction in therapeutic efficacy. In NCT, no seriousAEs were found, and good tolerance of NCT was confirmedby the high compliance rates. Indeed, the results observed inthis study agree favorably with other findings on the safety ofIFN-beta treatment in CHC patients and support the use ofnIFN-beta as a safe and alternative option.

5. Conclusion

An induction approach with nIFN-beta for 24 weeks fol-lowed by SOC for 48 weeks (NCT) was well tolerated with-out discontinuation. NCT prevented viral breakthroughwith viral clearance leading to an enhanced early virologicresponse and improved SVR rates in difficult-to-treat CHCpatients with genotype 1b and high viral loads. Early viro-logic clearance (extended RVR) by CPIT for 24 weeksbefore the beginning of RBV plus PEG-IFN-alpha treatmentinduced the restoration of innate immune responses linked


to adaptive immune responses and resulting in SVR and SBR.The higher SVR rates in CHC patients with genotype 1b andhigh viral loads among patients receiving NCT comparedwith SVR rates in those receiving SOC were revealed. NCT isbeneficial to treat difficult-to-treat CHC patients with geno-type 1b and a high viral load.

Conflict of Interests

The authors declare that they have no conflict of interests.

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Review Article

Indolent B-Cell Lymphomas Associated with HCV Infection:Clinical and Virological Features and Role of Antiviral Therapy

Luca Arcaini,1 Michele Merli,2 Stefano Volpetti,3 Sara Rattotti,1

Manuel Gotti,1 and Francesco Zaja3, 4

1 Department of Hematology Oncology, University of Pavia, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy2 Division of Hematology, Department of Internal Medicine, Ospedale di Circolo, Fondazione Macchi, 21100 Varese, Italy3 Department of Hematology, DISM, Azienda Ospedaliero Universitaria S. M. Misericordia, 33100 Udine, Italy4 Clinica Ematologica, Centro Trapianti e Terapie Cellulari “Carlo Melzi”, DISM, Azienda Ospedaliero Universitaria S. M. Misericordia,p.le S. Maria Misericordia 15, 33100 Udine, Italy

Correspondence should be addressed to Francesco Zaja, [email protected]

Received 16 May 2012; Revised 4 July 2012; Accepted 4 July 2012


Copyright © 2012 Luca Arcaini et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The association between hepatitis C virus (HCV) infection and B-cell non-Hodgkin’s lymphomas (NHL) has been demonstratedby epidemiological studies, in particular in highly endemic geographical areas such as Italy, Japan, and southern parts of UnitedStates. In these countries, together with diffuse large B-cell lymphomas, marginal zone lymphomas are the histotypes mostfrequently associated with HCV infection; in Italy around 20–30% cases of marginal zone lymphomas are HCV positive. Recently,antiviral treatment with interferon with or without ribavirin has been proved to be effective in the treatment of HCV-positivepatients affected by indolent lymphoma, prevalently of marginal zone origin. An increasing number of experiences confirmed thevalidity of this approach in marginal zone lymphomas and in other indolent NHL subtypes like lymphoplasmacytic lymphoma.Across different studies, overall response rate was approximately 75%. Hematological responses resulted significantly associatedwith the eradication of the virus. This is the strongest evidence of a causative link between HCV and lymphomas. The aim ofthis paper is to illustrate the relationship between HCV infection and different subtypes of indolent B-cell lymphomas and tosystematically summarize the data from the therapeutic studies that reported the use of antiviral treatment as hematologicaltherapy in patients with HCV-associated indolent lymphomas.

1. Introduction

In the last two decades, evidences from either epidemiolog-ical studies, biological insights, and also therapeuticapproaches provided strong support to the associationbetween hepatitis C virus (HCV) and B-cell non-Hodgkin’slymphomas (NHL). HCV has been associated with B-cellindolent lymphomas, especially marginal zone lymphomas,as well as with aggressive lymphomas, mainly diffuse largeB-cell lymphomas. Indolent lymphomas are defined from aclinical point of view as scarcely symptomatic lymphomas,growing and spreading slowly [1] and encompass the follow-ing histologic subtypes of low-grade lymphoma accordingto the WHO classification [2]: follicular lymphoma, smalllymphocytic lymphoma, marginal zone lymphomas, splenicmarginal zone lymphoma, primary nodal marginal zone

lymphoma and extranodal marginal zone lymphoma ofmucosa-associated tissue (MALT), and lymphoplasmacyticlymphoma. According to the currently more accepted patho-genetic model, the role of HCV infection in lymphomage-nesis may be related to the chronic antigenic stimulation ofB-cell immunologic response by the virus [3], similarly tothe well-characterized induction of gastric MALT lymphomadevelopment by Helicobacter pylori chronic infection [4]. Ina similar way, chronic HCV infection may possibly sustaina multistep evolution from type II mixed cryoglobulinemiato overt low-grade NHL and eventually to high-grade NHL[3, 4]. The most convincing argument in favour of a causativelink between HCV and lymphoproliferation is represented byinterventional studies demonstrating that in HCV-positivepatients affected by indolent NHL eradication of HCV withantiviral treatment (AT) could directly induce lymphoma


regression [5]. Moreover, the upcoming novel antiviral anti-HCV agents as boceprevir and telaprevir, whose addition tostandard treatment has already demonstrated an increasedrate of viral eradication also in more resistant genotypes (i.e.,genotype 1b) [6, 7], will possibly further improve the efficacyof this treatment for HCV-positive indolent NHL in the nearfuture.

2. Methods

The aim of this paper is to systematically summarize theavailable data indolent B-cell NHL associated with HCVinfection and the up-to-now reported experiences with theuse of AT with interferon with or without ribavirin as hema-tologic treatment in patients with HCV-positive indolent B-cell NHL.

To this aim, we performed a systematic PubMed search(http://www.pubmed.gov/) using the keywords “indolentlymphoma,” “marginal zone lymphoma,” “MALT lympho-ma,” “lymphoplasmacytic lymphoma,” “hepatitis C virus,”“interferon,” “ribavirin,” “antiviral therapy.” All relevant arti-cles were included, as well as the most significant abstractspresented at American Society of Hematology (ASH) meet-ings and International Conference on Malignant Lym-phomas (ICML) meetings since 2005. The articles werereviewed with reference to the features of HCV-associatedindolent NHL and were assessed specifically concerningvirological and hematological response in cases treated withAT.

3. HCV Infection, Cryoglobulinemia,and Lymphomas

3.1. HCV and Cryoglobulinemia. The initial finding thatlead to the extensive investigation of the associationbetween HCV and NHL was the very high prevalence(nearly 90–100%) of HCV infection in patients with typeII mixed cryoglobulinemia [8]. Cryoglobulins are serumimmunoglobulins that become insoluble and precipitateat temperatures below 37◦C. The antigenic componentof the immune complexes has been found to be highlyenriched in viral HCV core protein and HCV-RNA. TypeII mixed cryoglobulinemia is characterized by a mixtureof monoclonal and polyclonal immunoglobulins. Themonoclonal component of type II mixed cryoglobulinemiais an IgM/k with a rheumatoid-factor activity (i.e., anti-IgGcross-reactive binding) that reflects the expansion of aB-cell monoclonal population [9]. Overall, up to 50% ofHCV-infected patients exhibit low levels of circulating mixedcryoglobulins, whereas overt cryoglobulinemic vasculitisdevelops in ≤5% of infected patients [10]. Symptoms varyfrom purpura and arthralgia to more severe manifestationslike peripheral neuropathy and glomerulonephritis.Importantly, in HCV-infected patients with symptomaticcryoglobulinemia, the risk to develop an NHL is greatlyincreased with respect to the general population (about 35times according to a multicenter Italian study) [11]. As aresult, approximately 8–10% of patients with type II mixedcryoglobulinemia ultimately progress to a frank NHL after

long-term followup. Type II mixed cryoglobulinemia is oftencharacterized by IgH or Bcl2 rearrangement and by t(14;18) translocation [12]. Treatment of HCV type II mixedcryoglobulinemia with severe organ damage may targeteither the viral trigger (HCV) or the downstream arm of B-cell autoimmunity. AT with pegylated interferon + ribavirinhas been shown to reverse bone marrow B-cell expansion inpatients with HCV-MC, leading to a clinical and virologicresponse in up to 60% of cases [13]. Recent studiesshowed that the combination of AT (PEG-interferon +ribavirin) with the anti-CD20 monoclonal antibody ritux-imab is well tolerated and more effective than AT alone, byincreasing the rate of complete clinical response, immuno-logic (cryoglobulin clearance) and molecular response(eradication of B-cell clonal expansions), and shortening thetime to achieve a complete clinical response [14, 15].

3.2. Epidemiology of HCV and Its Association with Lym-phomas. HCV infection is a global health problem, with upto 170 million persons infected worldwide (3% of globalpopulation) [25]. However relevant regional differences inthe prevalence of infection are described. The lowest preva-lence rates are reported in Northern Europe and Scandinavia(0.01–0.1%) while in Italy, Egypt, Japan, and southern partsof United States, prevalence estimates exceeds 2% [26].HCV infection is a leading cause of chronic hepatitis, cir-rhosis, and hepatocellular carcinoma and has been associatedto extrahepatic manifestations, especially type II mixedcryoglobulinemia and a spectrum of B-cell NHL with orwithout cryoglobulinemia [3]. Several epidemiological stud-ies have been performed beginning from the mid 1990s toinvestigate the link between HCV and NHL. Early studiesbased on relatively small number of cases suggested asignificant increased risk of B-cell NHL in HCV-positivepatients, especially in countries with high prevalence ofHCV infection as Italy [27], Japan, and southern regionsof USA, while studies from areas with low HCV prevalencedid not show any evident association [28]. In 2003, asystematic review of studies evaluating prevalence of HCVinfection in B-cell NHL [29] was published. In this report, 48studies (5,542 patients) were identified. Mean HCV infectionprevalence was 13%. In 10 case-control studies examined,HCV prevalence in B-cell NHL was 17% compared to 1.5%in healthy controls (odds ratio, OR = 10.8). Therefore, itwas concluded that HCV prevalence in patients with B-cellNHL is higher than that reported in general population (15%vs. 1.5%), suggesting a role of HCV in the aetiology of B-cell NHL. Subsequently, in 2006, an updated metaanalysisof 15 case-control studies on the association between HCVinfection and NHL demonstrated a pooled relative risk oflymphoma among HCV-positive subjects of 2–2.5 dependingon study design. Relative risks resulted consistently increasedfor all major B-NHL subtypes. The study, indeed, confirmedthat the risk to develop NHL in the context of HCVinfection is most dramatically evident in populations withhigh HCV prevalence and that consequently the fraction ofNHL attributable to HCV infection varies greatly by country,reaching 10% in highly endemic areas.


Table 1: Clinical pathological studies describing indolent NHL subtypes associated with HCV infection.

Year Diagnosis N◦ ptsN◦ pts tested

for HCVN HCV+ (%) Genotypes

Cryoglobulinemia,N (%)

Arcaini et al. [16] 2006 SMZL 309 255 49 (19)1b (n = 10), 2b (n = 1),

2a/2c (n = 4)13 (10)

Saadoun et al. [17] 2005 SLVL 18 18 18 (100)1 (n = 7), 2 (n = 4),3 (n = 1), 4 (n = 1)

18 (100)

Arcaini et al. [18] 2007 NMZL 47 38 9 (24) NA 2 (14)

Arcaini et al. [19] 2006

MALT-MZLSkin

Salivary glandsOrbit

Other sites

17229322566

17229322566

60 (35%)21 (43)15 (47)9 (36)

15 (22)

1a (n = 11), 1b (n = 1),2a/2c (n = 10)

NA

Ferreri et al. [20] 2006MALT-MZL

orbit55 55 7 (13) NA 2 (29)

Paulli et al. [21] 2009SubcutaneousMALT- MZL

13 13 13 (100)2a/2c (n = 4), 2a (n = 2)

2b, (n = 1)3 (75)

Tedeschi et al. [22] 2009 WM 140 140 21 (15) NA 10 (48)

Arcaini et al. [23] 2011WM

SMZL12298

6692

6 (9)25 (27)

NANA

03

Goldaniga et al.[24]

2008 B-CLPD 156 113 6 (5) NA NA

SMZL: splenic marginal zone lymphoma; NMZL: nodal marginal zone lymphoma; SLVL: splenic lymphoma with villous lymphocytes; MZL: marginal zonelymphoma; FL: follicular lymphoma; LPL: lymphoplasmacytic lymphoma; MCL: mantle cell lymphoma; SLL: small lymphocytic lymphoma; NHL: non-Hodgkin’s lymphoma; B-CLPD: B-cell chronic lymphoproliferative disorders.

The numbers of cases analyzed in these series were toosmall to establish a correlation between HCV and specifichistotypes. In the Epilymph [30] case-control study, the sub-type most clearly associated with HCV infection was diffuselarge B-cell lymphoma, followed by marginal zone lym-phoma and lymphoplasmacytic lymphoma; however theseresults were based on relatively few cases. To obtain a morerobust estimate of the risk to develop specific NHL subtypesafter HCV infection, the International Lymphoma Epidemi-ology Consortium (InterLymph), based in Europe, NorthAmerica, and Australia, performed a pooled case-controlstudy including in the analysis data of 7 previous surveys[31]. Overall, among 4,784 cases of NHL and 6,269 controlsmatched by sex, age, and study centre, HCV infection wasdetected in 172 NHL cases (3.6%) and in 169 (2.7%)controls. In subtype-specific analyses, HCV prevalence wasassociated with diffuse large B-cell lymphoma (OR 2.24),marginal zone lymphoma (OR, 2.47), and lymphoplasma-cytic lymphoma (OR 2.57) whereas risk estimates were notincreased for follicular lymphomas (OR 1.02). Moreover,also retrospective series reported a high HCV prevalenceamong patients with marginal zone lymphoma [4, 16–18, 20,32], lymphoplasmacytic lymphoma, and diffuse large B-celllymphoma [33, 34].

4. HCV and Indolent Lymphomas: Clinical andPathological Studies

As mentioned above, many well-characterized clinical-path-ological studies investigated the association of HCV infectionwith specific indolent NHL subtypes (Table 1).

4.1. Marginal Zone Lymphomas. Within specific indolentNHL subtypes, the association with HCV infection hasbeen best characterized in marginal zone lymphomas. Inthe 2008 edition of WHO classification three marginal zonelymphoma entities were listed [2]: splenic B-cell marginalzone lymphoma, nodal marginal zone lymphoma, andextranodal marginal zone B-cell lymphoma of MALT type.Marginal zone B-cells have been demonstrated to play rolein the immune response to T-cell-independent antigens andfrequently display reactivity to self antigens. Marginal zoneB-cells are involved in various infectious and autoimmuneconditions and marginal zone-related neoplasms often retainthe features of these cells. Many infectious agents are involvedin the pathogenesis of specific types of marginal zone lym-phomas: Helicobacter pylori for gastric MALT lymphoma [4],Campylobacter jejuni for immunoproliferative small intestinedisease [35], Borrelia burgdorferi for MALT lymphoma of theskin [36], and Chlamydia psittaci for MALT lymphoma of theorbit [37, 38]. In all these cases, eradication of the antigenafter antimicrobial therapy may lead to a regression of thelymphoma. For example, eradication of Helicobacter pylorileads to the complete regression of gastric MALT lymphomain most cases, and relapse of the lymphoma is anticipated bythe reoccurrence of the infection [4].

Accordingly to this scenario, also chronic stimulationby HCV may play a role in development of a subgroup ofmarginal zone lymphoma cases. The association betweenHCV and marginal zone lymphomas is demonstrated by epi-demiological studies, as previously summarized, and byclinical-pathological studies of well-characterized marginalzone lymphoma series. However, the major support to the


potential causal role of HCV in marginal zone lymphoma-genesis is represented by the antilymphoma activity of ATevidenced in a subset of HCV-positive MZL.

4.2. Splenic Marginal Zone Lymphoma. Splenic marginalzone lymphoma is a rare indolent lymphoma subtype whichaccounts for less than 2% of all NHL [39]. In some caseslymphocytes with villous projections are found in peripheralblood, and the disease is termed splenic lymphoma withvillous lymphocytes [40, 41]. This entity is considered as theleukemic counterpart of splenic marginal zone lymphoma[42]. In almost all patients a symptomatic splenomegalyis the presenting feature. In a large series, HCV serologywas positive in 49 out of 255 available patients (19%) [16].Among 56 patients tested for HCV-RNA, 25 (45%) werepositive. Cryoglobulins were detected in 13 out of 130patients tested (10%).

In 2005, French authors reported a series of splenic lym-phomas with villous lymphocytes associated with type IIcryoglobulinemia and HCV infection [17]. All 18 patientshad type II mixed cryoglobulinemia, with symptoms ofvasculitis in 13. Clinical symptoms of cryoglobulinemia pre-ceded the diagnosis of lymphoma in 7 patients (at a meantime of 3.5 years before lymphoma diagnosis) and wereconcurrent in the other 6 patients. The authors concludedthat splenic lymphoma with villous lymphocytes could beintegrated in the spectrum of cryoglobulin-associated B-cellproliferations, configuring a new clinical entity.

4.3. Primary Nodal Marginal Zone Lymphoma. Primarynodal marginal zone lymphoma is listed in the WHO lym-phoma classification as a rare but distinct clinical pathologicsubtype characterized by exclusive primary lymph nodelocalization in absence of prior or concurrent extranodal siteof involvement. Primary nodal marginal zone lymphoma isa rare disease accounting for nearly 2% of lymphoid neo-plasms and is frequently associated with HCV infection withpreferential use of specific VH segments [43]. In a large series[18], HCV serology was positive in 9 out of 38 patients (24%)and HCV-RNA was detectable in 4/8 patients studied.

4.4. MALT Lymphoma. MALT lymphomas represent 8% ofall NHL [44–46], behave usually as indolent diseases, anddevelop more frequently in middle and advanced age,with a female predominance [47–50]. Interestingly, somestudies reported an increased prevalence of HCV infection inunselected patients with gastric lymphoma [51]. In an Italianmulticenter study, data on HCV serology were available inall 172 cases of nongastric MALT lymphoma [32]. HCVinfection was documented in 60 patients (35%). A total of22 out of 24 patients tested (92%) had viremia. Interestingly,three specific MALT lymphoma sites showed an elevatedprevalence of HCV infection: salivary glands (47%), skin(43%), and orbit (36%). These data, while confirming thelink between HCV infection and salivary glands lymphoma[52], reveal a possible relationship between HCV and twoother MALT presentations of lymphoma: orbit and skin.Interestingly, a study on B-cell lymphoma in patients with

Sjogren’s syndrome and HCV infection reported an elevatedoccurrence of parotid involvement and a high proportionof MALT lymphomas with primary extranodal involvement(exocrine glands, liver, and stomach) [53]. In 2006, Ferreriet al. found HCV seropositivity in 13% of ocular adnexalymphoma of MALT type with a more aggressive behaviour[20]. Taken together, these data indicate that the typical pre-sentation of HCV-related MALT lymphomas is constitutedby some well-defined forms with a single and peculiar MALTlocalization. At this regard, it has recently reported a seriesof 12 HCV-positive patients presenting with subcutaneousnodules resembling “lipomas” and a typical histology ofextranodal marginal zone lymphoma of MALT [21]. HCV-RNA was detectable in all 10 patients tested. From a clinicalpoint of view, it has to be underlined that the clinical benignappearance of these “lipoma-like” lesions and their indolentclinical behaviour may result in diagnostic delay.

4.5. Lymphoplasmacytic Lymphoma/Waldenstrom Macroglob-ulinemia. Beside marginal zone lymphomas, one of theother indolent B-cell NHL subtypes that has been frequentlyassociated to HCV infection is lymphoplasmacytic lym-phoma, a neoplasm of small B lymphocytes, plasmocytoidlymphocytes, and plasma cells, usually involving bone mar-row and sometimes lymph nodes and spleen. Waldenstrommacroglobulinemia is found in a significant proportionof patients with lymphoplasmacytic lymphomas and isdefined as lymphoplasmacytic lymphoma with bone marrowinvolvement and the detection of a paraprotein of IgM typein the serum. Lymphoplasmacytic lymphoma and Walden-strom macroglobulinemia have been associated with HCVinfection and mixed cryoglobulinemia in some but notall series, perhaps related to geographic differences. Forexample, while a US series did not find any HCV-positivecases among 100 untreated patients affected by Waldenstrommacroglobulinemia [54], an Italian multicentre study [22]reported 21 HCV-positive cases among 140 patients (15%).HCV infection was associated to lower counts of platelets,neutrophil granulocytes, and hemoglobin, and with thepresence of cryoglobulins, splenomegaly, increased LDH,and β2-microglobulin levels. However, the analyses didnot reveal any difference between HCV-positive and HCV-negative patients in terms of “disease progression needingtreatment,” “time from diagnosis to first therapy,” and overallsurvival. Interestingly, a recent report specifically focused onthe comparison between Waldenstrom macroglobulinemiaand splenic marginal zone lymphoma, found that, despitesome common features, splenic marginal zone lymphomadisplayed a clearly higher association with HCV infection (25HCV-positive patients out of 92, 27%) than Waldenstrommacroglobulinemia (6/66 patients, 9%) [55].

4.6. B-Cell Chronic Lymphoproliferative Disorders. Follicularlymphoma and small lymphocytic lymphoma are low-gradelymphoma subtypes rarely associated with HCV-positiveinfection, as evidenced by the above mentioned epidemi-ological studies. Interestingly, these findings have beenconfirmed by a single institution Bayesian analysis that wasperformed to estimate the prevalence of HCV infection


across the different NHL histologies. This approach wasable to demonstrate the association of splenic marginalzone lymphoma and diffuse large B-cell lymphoma withHCV infection, while did not find any correlation withfollicular lymphoma and small lymphocytic lymphoma [56],thus confirming previous findings of classic epidemiologicstudies. Moreover, this study showed that another diseaseentity could be associated with HCV, the so-called “B-cell chronic lymphoproliferative disorders.” B-cell chroniclymphoproliferative disorders are defined as the miscella-neous category of non-CLL leukemic lymphoproliferativedisorders with Royal Marsden Hospital scoring system ≤3[57, 58] and are often reported also as “low-grade B-NHLnot otherwise specified.” Although one series reported alow rate of HCV positivity (5%) in CD5/CD10-negativeB-cell chronic lymphoproliferative disorders [24], furtherinvestigations are needed to elucidate this issue, given theheterogeneity and the small numbers of studies focusing onthis entity now available.

5. Standard Treatment for HCVChronic Hepatitis

The goal of AT in HCV-related chronic hepatitis is to preventdisease complications. This is best accomplished throughvirus eradication, defined as sustained virologic response(SVR), that is, undetectable HCV-RNA by a sensitivepolymerase-chain-reaction- (PCR-) based assay 24 weeksafter discontinuation of treatment. Therapeutic options forHCV-related chronic hepatitis have improved significantlysince the introduction of interferon monotherapy. Thecurrent standard of care is a combination of pegylatedinterferon-α and weight-based ribavirin for 48 weeks forgenotype 1 and 4 and for 24 weeks for genotype 2 and3. Patients with genotype 2 or 3 respond more favourablyto standard AT, obtaining a SVR in 75–90% of cases,whereas in genotype 1 and 4 the likelihood of achievingSVR is considerably lower (45–52%) [59–61]. Recently,however, the introduction of the HCV NS3/4A proteaseinhibitors boceprevir [6] and telaprevir [7], the first twodrugs belonging to a new and promising generation ofdirect-acting antiviral agents, has been demonstrated todramatically improve SVR rates in genotype 1 patients.In particular, in genotype 1 treatment-naive patients, theaddition of boceprevir or telaprevir to standard AT increasedthe rate of SVR to nearly 65–75%, whereas in relapsers ornonresponders, the SVR rate improved to nearly 57–86%.

Other new potent protease inhibitors showing promisingactivity are currently in late phases of development, as wellas other new upcoming classes of direct-acting antiviralagents like polymerase inhibitors: their potential combina-tion seems to herald for the near future the possibility toobtain highly efficacious interferon-free regimens for HCVtherapy [62].

6. Antiviral Treatment of HCV-PositiveIndolent Lymphomas

As previously discussed, case-control epidemiological studiesand clinical-pathological series evidenced the link between

HCV infection and NHL development. However, the mostconvincing evidence to support the causal role of HCVin lymphomagenesis is the possible regression of indolentlymphoma after eradication of HCV infection with AT.Beginning from the seminal work by Hermine et al. in spleniclymphomas with villous lymphocytes in 2002 [5], data fromliterature demonstrate that AT could be considered as first-line approach in HCV-associated indolent lymphomas whenthere is not immediate need of conventional (immuno)-chemotherapy treatment. Among specific NHL subtypes,this treatment modality has been more frequently exploitedin marginal zone lymphomas; however other studies havesupported the validity of this approach for all indolenthistologies when associated to HCV infection. In Table 2 wesummarized results of antilymphoma activity of AT withinterferon with or without ribavirin in low-grade NHL.

Conversely, front-line AT is clearly insufficient in HCV-positive aggressive lymphomas, in which an immediatelyactive therapy is needed; in these cases AT may be arationale recommendation after completion of conventionalimmunochemotherapy with the aim to clear a potentiallymphoma trigger.

6.1. The First Experiences. In 2005, a systematic review con-cerning the efficacy of AT in lymphoproliferative disor-ders was published [76]. Overall, 16 studies reporting theemployment of an antiviral regimen (interferon with orwithout ribavirin) as primary hematologic treatment in65 HCV-infected patients diagnosed with a lymphopro-liferative disorder were identified. Complete remission ofthe lymphoproliferative disorder was achieved in 75% ofthe cases. However, many of these series relied on casereports of few patients and included also patients with mixedcryoglobulinemia with evidence of B-cell monoclonality[77].

Among the studies included in the cited review, thefirst experience of a relatively large cohort of patients withNHL was performed by Hermine et al. in 2002 [5]. In thisreport they described the outcome of 9 patients with spleniclymphoma with villous lymphocytes and HCV infectiontreated with interferon-α2b (3 MU subcutaneously threetimes a week for six months). Six patients had symptomaticcryoglobulinemia. Complete hematological remission andHCV negativity were observed in 7/9 (78%) patients. Twopatients who did not respond were subsequently treated withinterferon plus ribavirin (1,000 to 1,200 mg per day) andobtained the clearance of HCV-RNA as well as lymphomaresponse (one complete remission and one partial remis-sion). On the contrary, none of 6 cases with SLVL withoutHCV infection treated with interferon experienced any gradeof lymphoma regression.

In 2005 a subsequent report from the same groupexpanded these results in 18 patients with chronic HCVinfection, mixed cryoglobulinemia, and splenic lymphomawith villous lymphocytes [17]. All patients were treated withinterferon (+ribavirin in 10). Fourteen patients obtained acomplete hematologic remission after clearance of HCV-RNA. Two patients had a virologic partial response and


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obtained a complete hematologic response. Two virologicnonresponders achieved partial hematologic response. Nota-bly, viral genotype did not seem to correlate with theresponse: in fact 4 out of 7 patients with HCV genotype 1,that is usually associated with poor responses, achieved acomplete lymphoma regression after interferon and rib-avirin. Interestingly, even for patients who exhibited a com-plete hematological remission, no molecular response wasevidenced, as monoclonal immunoglobulin gene rearrange-ment was still detected in peripheral blood after treatment;however clinical relapses did not occur if viremia remainednegative. Overall, these observations may suggest differencesin oncogenic potential between HCV-driven B-cell clones incryoglobulinemia with respect to splenic lymphoma and arein favour of a model in which a continuous viral stimulationleads to cryoglobulinemia and, subsequently, in a subset ofpatients, to indolent lymphoma.

Another study reported first-line AT with interferon andribavirin in 8 HCV-positive patients with different subtypesof marginal zone lymphoma (4 splenic marginal zonelymphomas with or without villous lymphocytes, 1 dissem-inated marginal zone lymphoma, 1 leukemic marginal zonelymphoma, and 2 intestinal MALT lymphomas); 5 out of 8patients (60%) obtained a response, which was correlated tovirological response in most cases [70].

Among most robust experiences, an Italian multicenterstudy [71] reported results of AT in 13 indolent B-cellNHL (4 splenic marginal zone lymphomas, 2 primary nodalmarginal zone lymphomas, 2 extranodal lymphomas ofMALT, 4 lymphoplasmacytic lymphomas, and 1 follicularlymphoma) carrying HCV infection. All patients underwentAT alone with PEG-interferon and ribavirin, 10 as first lineand 3 as second or third line of therapy. Eleven patientscompleted planned treatment, while 2 discontinued itbecause of severe adverse effects. Among 12 assessablepatients, 7 achieved complete response, 2 partial response(overall response rate 75%), 2 had stable disease, and oneprogressed during therapy. Hematologic responses resultedhighly significantly associated to clearance or decrease inserum HCV viral load, as 7 out of 9 achieved SVR, one hadreduction in viremia, and 1 had no change in viral load.Virological response was more frequent in HCV genotype2; however, hematologic response did not correlate with theviral genotype. One of the greatest accomplishment of thisstudy was the demonstration of the efficacy of AT in a widerange of HCV-positive low-grade NHL subtypes other thansplenic marginal zone lymphoma, as complete responseswere actually observed without significant differences in allindolent NHL histologies.

6.2. Recent Studies and Future Perspectives. Recently Mazzaroet al. [75] reported a comparison of PEG-interferon andstandard interferon (plus ribavirin) as first-line treatmentin 18 patients with HCV-positive low-grade B-cell NHL (1follicular lymphoma, 1 splenic lymphoma with villous lym-phocytes, and 16 lymphoplasmacytic lymphomas). Com-plete responses as well as SVR were higher in the grouptreated with PEG-interferon (6/10 patients, 60%) withrespect to the group treated with standard interferon (3/8

patients, 37%). Achievement of hematological responsesignificantly related to the disappearance of HCV-RNA, asall patients who experienced SVR developed hematologicalCR. In the previously cited study on subcutaneous “lipoma-like” extranodal marginal zone B-cell lymphoma of MALT[78], one patient was treated with interferon and ribavirin asfirst- line treatment and obtained a rapid virological responseand a partial lymphoma response. Interestingly, anotherpatient who relapsed 20 months after CHOP therapy plusradiotherapy was treated with interferon alone for 6 monthsand achieved a complete regression of nodules after 1 monthof therapy together with virological response. Eight monthsafter stopping AT HCV-RNA returned positive and 3 monthslater a subcutaneous lymphoma relapse occurred.

In perspective, several lines of future clinical research canbe pursued with the aim to further improve these results.First, it has to be investigated if the combination of rituximaband AT tested in symptomatic cryoglobulinemia by Saadounand colleagues (rituximab weekly for 4 doses followed byPEG-interferon weekly plus ribavirin daily for 48 weeks)[14] is able to obtain a better long-term control of diseasealso in indolent B-cell NHL. Second, it has to be tested ifnew antiviral combinations with new anti-HCV agents (i.e.,PEG-interferon and ribavirin plus boceprevir or telaprevir),that guarantee better rates of SVR in genotypes 1 hepatitis,could allow to increase the rate of hematologic responsealso in patients with more resistant genotypes. Finally, ithas to be explored if future interferon-free regimens withdirect antiviral agents only, could consent the access to ATalso for HCV-positive NHL patients with contraindicationsto interferon use, for example because of advanced age,cytopenias, and/or comorbidities.

6.3. Role of Antiviral Therapy in Aggressive B-Cell Lymphomas.As previously mentioned, AT seems to be less efficacious thanstandard immune-chemotherapy for HCV-positive aggres-sive lymphomas (diffuse large B-cell lymphoma and mantlecell lymphoma) with respect to indolent ones. This isprobably related to the lost of antigen dependence resultingfrom acquisition of additional mutations that are possiblyresponsible of more aggressive behaviour, although anecdo-tal cases of diffuse large B-cell lymphoma [79] and mantlecell lymphoma [80] treated with AT and obtaining remissionhave been reported. Nevertheless, many researchers haveexplored the option to integrate AT in the context ofimmune-chemotherapy programs in HCV-positive diffuselarge B-cell lymphomas. Although some rare cases of concur-rent delivery of AT and immune-chemotherapy with the aimto prevent or to treat hepatitis flares have been reported [81],treatment with interferon or PEG-interferon with or withoutribavirin is usually not feasible because of hematologictoxicity, as showed by a pilot study by Musto and coworkersin 4 patients with diffuse large B-cell lymphoma [82]. Thesame authors explored the option to perform sequentialAT with PEG-interferon and ribavirin for 3 months inresponding HCV-positive patients with diffuse large B-celllymphomas after 6–8 cycles of R-CHOP. They found thatthis strategy was effective, better tolerated and resulted inhigh rate of virus clearance in the first 12 patients treated.


These preliminary data have been indirectly supported bya subsequent study that described the outcome of 69 HCV-positive NHL patients treated with chemotherapy; of them,25 (14 with aggressive NHL and 11 with indolent NHL)subsequently underwent to AT and 8 out of 25 achieved anSVR [83]. None of the HCV-positive patients who obtaineda SVR experienced NHL recurrence, while 29% of thenonresponders relapsed. Moreover AT resulted associatedwith better disease-free survival in multivariate analyses.These preliminary experiences confirmed that AT is notfeasible in concomitance with immune-chemotherapy inHCV-positive DLBCL because of an excess of hematologictoxicity. On the other hand they suggest that a course of AT inpatients in remission after immune-chemotherapy appearsto be an attractive option with the aim to prevent hepatitisreactivation in view of long-term control of the lymphoma.However these data have to be confirmed in larger series andpreferentially in prospective manner.

7. Conclusions

In conclusion, anti-HCV treatment with interferon and rib-avirin seems to be indicated for indolent B-cell NHL sub-types (in particular of marginal zone type) that not needimmediately conventional immunochemotherapy. The lym-phoma regression observed in some patients with interferon-based treatment is strongly in favour of a causative role ofHCV in a subset of patients with indolent NHL. For thisreason, it is likely to foresee that future improvements in ATmay directly result in increase in cure rates of HCV-associated indolent NHL.

Acknowledgment

This work was supported by AIRC, My First AIRC Grant2011 (to L. Arcaini).

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[2] P. G. Isaacson, M. A. Piris, F. Berger et al., “Splenic B-cellmarginal zone lymphoma,” in WHO Classification of Tumoursof Haematopoietic and Lymphoid Tissues, S. Swerdlow, E.Campo, N. L. Harris et al., Eds., pp. 218–219, IARC Press,Lyon, France, 4th edition, 2008.

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Review Article

Rheumatoid Factor, Complement, and Mixed Cryoglobulinemia

Peter D. Gorevic

Mount Sinai School of Medicine, Division of Rheumatalogy, Annenberg Building, Room 21-056,One Gustave L. Levy Place, New York, NY 10029-6574, USA

Correspondence should be addressed to Peter D. Gorevic, [email protected]

Received 21 May 2012; Accepted 26 June 2012


Copyright © 2012 Peter D. Gorevic. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Low serum level of complement component 4 (C4) that occurs in mixed cryoglobulinemia (MC) may be due to in vivo or exvivo activation of complement by the classical pathway. Potential activators include monoclonal IgM rheumatoid factor (RF), IgGantibodies, and the complexing of the two in the cold, perhaps modulated by the rheology and stoichiometry of cryocomplexesin specific microcirculations. There is also the potential for activation of complement by the alternative and lectin pathways,particularly in the setting of chronic infection and immune stimulation caused by hepatitis C virus (HCV). Engagement ofC1q and interaction with specific cell surface receptors serve to localize immune complexes (ICs) to the sites of pathology,notably the cutaneous and glomerular microcirculations. Defective or saturated clearance of ICs by CR1and/or Fc receptors mayexplain persistence in the circulation. The phlogistic potential of cryoprecipitable ICs depends upon the cleavage of complementcomponents to generate fragments with anaphylatoxin or leukocyte mobilizing activity, and the assembly of the membrane attackcomplex (C5b-9) on cell surfaces. A research agenda would include further characterization of the effector arm of complementactivation in MC, and elucidation of activation mechanisms due to virus and viral antigens in HCV infection.

1. Introduction

Mixed cryoglobulins (MCs) are cold-precipitable rheuma-toid factors (RFs) that are easily identifiable and char-acterized by immunofixation of cryoprecipitate obtainedfrom serum carefully collected from blood kept at andallowed to clot at core body temperature, and then cooledto 4◦C [1]. Type 2 MCs are almost invariably composedof monoclonal IgM kappa RF and polyclonal IgG, and itis the complexing of the two that is a requisite for theformation of cold-precipitable immune complexes (ICs);both the IgM heavy-and light-chain variable regions dis-play a striking clonality that is mirrored in cross reactiveidiotypes (CRIs) as well as mu heavy chain and kappalight-chain V-region gene usage. Type 2 MCs are heavilyrepresented among cryoglobulins associated with chronichepatitis C virus (HCV) infection, and those found inpatients with primary Sjogrens syndrome, both of whichmay be complicated by clonal B-cell proliferations andspecific types (e.g., mucosa-associated (MALT); Splenic) of

non-Hodgkin’s lymphoma [2]. Among patients with type2 MCs associated with HCV, prominent associations withextrahepatic disease manifestations such as leukocytoclasticvasculitis, arthropathy, neuropathy, and membranoprolifer-ative glomerulonephritis have been found in multiple series.Complement abnormalities were described in early seriesof “essential mixed cryoglobulinemia” [3], and type 2 MCsare likely to have a striking complement profile notablefor normal or low levels of component 3 (C3) and oftenundetectable levels of component 4 (C4); the latter (Figure 1)provides a “signature” which may in fact be used to anticipatethe presence of significant (>1 mg/mL) amounts of type2 cryoglobulin in blood [4]. The purpose of this paper isto update older information with regard to complementmeasurements in type 2 MC, with particular attention to thevarious effects of HCV infection and the central role of RF.

2. The Complement System

The complement system comprises 30 serum and cell-surface proteins tightly regulated to respond to activators


C4 C3 Factor BHemolytic units

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Figure 1: C3, C4, and factor Bb levels determined by hemolyticassay in patients with type 2 MC (4).

by three independent pathways (classical: CP, alternative,AP, and Mannan-binding lectin: MBL), evolved primarilyto recognize and destroy pathogenic microorganisms [5].Temperature-dependent activation of both CP and AP invitro has been reported among mixed (IgM-IgG, IgM-lipoprotein) and monoclonal (IgG) cryoglobulins. Activa-tion of AP has been correlated with the presence of IgA inmixed cryoglobulins and with an IgG3 monoclonal cryo-globulin occurring in a patient with membranoproliferativeglomerulonephritis [6]. The selective depression of C4 notedin type 2 MC implicates the CP and is reflected in anextended serum profile, which includes variably low levelsof C1q and C2, normal levels of factor Bb (Factor 1), andelevated levels of MBL; C3 levels may be normal, exceptin patients with severe disease manifestations (glomeru-lonephritis, neuropathy) [7]. In HCV-associated MC, thisprofile (a) correlates inexactly with the level of cryoglobulinand titer of RF, (b) may occasionally be found in the absenceof a detectable cryoglobulin, (c) may occur in the absenceof RF in the serum supernatant after cryoprecipitation, (d)correlates only poorly with symptomatology in serial studies,and (e) may persist with cryoglobulinemia after apparentclearance of the virus [8–10]; these observations suggest acomplexity of pathways to C4 depletion extending beyond ICactivation and HCV infection. The mechanism responsiblefor the selective depression of C4 remains unclear; whereas anovel control mechanism involving Cb-binding protein (C4-bp) and C3b inactivator was implicated in one early study[11], this was not reflected in the levels of C4-bp in seraof patients with MC [12]. Whether cryo-RF might interferewith complement activation at the level of C3 has not beenaddressed.

3. Rheumatoid Factors

Rheumatoid factors are IgM antibodies with specificitylargely for the Fc portion of IgG; potential triggers tomRF production include (a) direct infection by virus, (b)chronic antigenic stimulation by ICs, (c) stimulation in theform of repetitively arranged epitopes on viral particles, or(d) molecular mimicry. Early studies suggested additionalreactivities of MC IgM with idiotypic determinants in theF(ab’)2 of MC IgG, possibly reflecting the fact that in MCthe IgG is reactive with viral antigens [13], some of whichcan also be demonstrated within the cryoprecipitates [6].

ICs containing IgG and IgM isotypes are well known to beactivators of the CP, providing several mechanisms by whichC1q binding to the CH2 domain of IgG, and/or to the CH3 orCH4 domains of IgM may occur in MC [5]. RFs in type 2 MCare distinct from those found in rheumatoid arthritis (RA)with regard to virtually universal cold perceptibility, clonality(CRIs and skewing of V-region gene usage), and in that theantiglobulin activity is unique to the IgM isotype [14]. Directactivation of complement by locally produced RF-containingICs has been well demonstrated in the joint space of patientswith RA, resulting in depression of C4 [15], but has notbeen found in serum, except for a small number of patientswith very severe (and likely overlap) disease, characterized byrecurrent infections [16]. By contrast, serum levels of C4, andmore specifically C3, may be elevated in serum of patientswith RA by virtue of their being acute-phase reactants(APRs), a phenomenon that has also been described in HCVinfection, and used to monitor efficacy of treatment [17];elevation of complement component APRs might mask moresubtle activation due to ICs in the circulation unless specificcleavage products (e.g, C3b and C5a) are also assayed.

MC formation provides a fertile substrate for in vitroand, by extension, potential for in vivo, complement acti-vation. Both IgG and IgM may be recognized by theglobular heads of C1q, which has been identified as aconstituent of cryoprecipitates in some studies. Althoughbinding of C1q to monomeric IgG in cryocomplexes mightbe anticipated to be offset by more effective binding toIgM, this could be mitigated by clustering of IgG in theICs, complex formation of IgG with viral antigens, andincreased representation of the IgG3 subclass, which isknown to be most effective for CP activation [5, 6]. Anadditional factor might be the stoichiometry of IgG andIgMRF in the ICs, which has been reported to influencein vitro cryocomplex formation. Among IgM-IgG mixedcryoglobulins, fractionation experiments have establishedthat the majority of anticomplementary activity is associ-ated with IgM RF; in mixing experiments of dissociatedand purified IgG and IgM from two patients with HCV-associated type 2 MC, the bulk of complement activationappeared to localize to the IgMRF constituent (Figure 2).Nevertheless, in vitro studies indicated that IgM RF/IgGimmune complexes may not fix C3 and C4 efficiently in spiteof fluid-phase activation [18], and appear to bind poorly toerythrocyte complement receptor 1 (CR1; CD35), the mostabundant C3b/C4b receptor in blood [19]. A mechanism foractivation due to MBL-MASP2 binding to underglycosylatedRF, or direct activation by HCV RNA, has not been formallytested. Since the valence of IgM is five, in spite of thepotential for 10 antigen binding sites, it seems possible thatthe stoichiometry of cryocomplexes might be influencedby the binding affinity for determinants on the IgG, theaggregation state of IgG, specificity for other components ofthe cryocomplex, cold-related aggregation, and the rheologyof specific microcirculations such as exists in the skin andthe afferent arterioles of the glomeruli, where increasedprotein concentration and skin temperatures significantlybelow core levels might facilitate the occurrence of such


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IgMIgG

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ent

Inh

ibit

ion

(b)

Figure 2: Total hemolytic complement activity was determined after reconstitution of varied molar ratios of separated IgM and IgG fromtwo type 2 cryoglobulins (Vi; Bu). Separated IgM was 100% monoclonal IgMk with RF activity; the IgG fraction had immunoblot reactivityto multiple HCV linear antigens. Isolated cryoglobulin was significantly (40 and 60% total serum RNA by RT-PCR) enriched in HCV RNA.Separated IgM and IgG fractions were mixed at 37 and cooled O/N to 4 C. Results are compared to control IgG and IgM, the latter froma patient with Waldenstroms macroglobulinemia without RF activity. Both cryoglobulins were obtained from patients chronically infectedwith HCV, with active cutaneous vasculitis and membranoproliferative glomerulonephritis. (courtesy of Dr. B. Ghebrehiwet).

molecular events and cell interactions, thus allowing morerobust complement activation at the sites of pathology [20].

4. Cold-Dependent Activation ofComplement (CDAC)

In sera manifesting CDAC, a similar profile of low CH50

and hemolytic C4 with normal hemolytic C5-C9 is seen at4◦C, with normal values being obtained in EDTA-treatedplasma and in serum kept at 37◦C. First described as an invitro phenomena in occasional sera [21], it was subsequentlyshown to be prominently associated with HCV infection,and not with cold-associated symptomatology (raynaud oracrocyanosis). It does, however, correlate with the degree ofliver damage and inversely with a response to treatment withinterferon-alpha. In contradistinction to cryoglobulinemia,C1q and C4 antigenic levels are normal in CDAC. Cryopre-cipitates (cryoglobulins or cryofibrinogens) are usually notfound. CDAC may be consequent to HCV-antibody-mRFcomplexes with differing stoichiometry [22, 23]. CDAC,RF, cryoglobulinemia, and elevated levels of IgM-containingICs are all prevalent in HCV infection. Dissociation of thethermal properties of cryoprecipitation and complementactivation has been noted in specific instances in which it hasbeen studied.

5. Constituents of Mixed Cryoglobulins

Early studies of mixed cryoglobulins associated with severeRA provided the first indication that as much as onequarter to one-third of the cryoprecipitable material wasnonimmunoglobulin [24]. In HCV-associated MC, otherconstituents include C1q, C-reactive protein (CRP), HCVantigens, and molecules of the lectin complement pathway(MBL and MBL-associated serine protease-1), the latterassociated with membranoproliferative glomerulonephritis[7, 25]. Though the quantitative contribution of theseother components has not been determined, they providealternative routes for activation of complement via the CP

or MBL, for example, activation of C1q by CRP in thepresence of phosphocholine produced by apoptotic cells.Although most of the demonstrable reactivity to HCVantigens in MC appears to reside in the IgG fraction [26], thepossibility remains that significant antibody activity—and byextension anticomplementary activity—to conformationalantigens (i.e, envelope proteins; encapsulated or unencapsu-lated virus) might be retained in the IgM RF fraction [27].

6. Phlogistic Potential of Mixed Cryoglobulins

The ability of cryoaggregates to generate vasoactive sub-stances and proinflammatory mediators to produce tissuelesions is suggested by elevated levels of complement frag-ments with anaphylatoxin activity (C3a, C5a) in serum,as well as the ability of isolated cryoproteins to activatebasophils, cause platelet aggregation, and interact withkallikrein-kinin in vitro [6]. In addition, C5a is a potentchemotactic factor that might be responsible for the influxof inflammatory cells to the site of complement activation.Direct activation of C1 by kallikrein provides an independentmechanism for activation of both the CP and AP separatefrom a role for ICs in MC [5].

7. Defective Clearance of Cryoglobulins

C1q binding has been used as an assay for the identificationof both cryoprecipitable and noncryoprecipitable ICs inthe sera of patients with MC [28]. Defective clearanceof cryoglobulinemic ICs by Fc receptor-mediated mecha-nisms correlates with persistence in the circulation, andthe clinical manifestations of nephritis and neuropathy [4];cryoglobulin-induced cytokine production via Fc gamma IIaligation in MC has been proposed as a mechanism for thegeneration of TNF-alpha and IL-10, and for the growth ofmalignant B-cells in this disorder [29]. Similarly, decreasederythrocyte CR1/CD35 (receptor for C3b and membranecofactor protein) in both MC and chronic liver disease hasbeen linked to deficient reticuloendothelial system (RES)


clearance, defective cleavage of C3b and C4b, and discor-dant complement activation at different concentrations andtemperatures of RF [19, 30]. CR1 numbers were decreasedwhen peripheral blood erythrocytes were analyzed with amonoclonal antibody to this receptor. Defective immuneadherence and elimination of other ICs, such as hepatitisB surface antigen/antibody, may lead to trapping in tissues,resulting in clinical vasculitis or nephritis. Inhibition ofneutrophil and monocyte chemotaxis, as well as bactericidalfunction, has been attributed directly to the effects onthese cells of cryoprotein constituents and may relate to anincreased incidence of infections in some patients [31].

8. Hepatitis C Virus and Complement

The detection of HCV core antigen in cryoprecipitates hasbeen linked to the presence of unencapsulated nucleocapsidparticles as a constituent of MCs [27]. A receptor for theglobular head of the C1q molecule (gC1q-R) appears to bea natural ligand for HCV core antigen [32]. The structuralsimilarity between HCV core antigen and C1q may explainthe presence of cross-reactive anti-C1q in HCV-associatedMC [33]. MCs provide a platform for the formation ofqC1q-R-HCV core complexes, and their localization to sitesof vasculopathy, where they might bind to receptors onendothelial cells, as demonstrated by immunohistology [34].Anti-C1q and anti-CRP antibodies have been linked todisease severity, and to autoimmunity in HCV infection[33, 35]. In addition to CP activation, the binding of MBLto HCV envelope glycoproteins E1 and E2 [36] might leadto activation of the lectin pathway due to homology ofMBL to C1q, resulting in cleavage of C4 and C2 followinginteractions with MBL-associated proteases [5]. This hasbeen corroborated in immunohistological studies of HCV-associated membranoproliferative glomerulonephritis [7].An alternative mechanism for the depression of C4 andperhaps C3 has been provided by the demonstration of theability of HCV viral proteins to regulate synthesis of C3 andC4 via specific transcriptional repression [37, 38].

9. Membrane Attack Complex (MAC)

The MAC is dependent on the cleavage of C5 into C5aand C5b leading to the assembly of C6–9 and lytic activitytargeting membranes at the site of tissue pathology. Rela-tively little information has been accumulated to implicateterminal complement activation and the generation of MACin tissue lesions associated with extrahepatic HCV infection.

10. Discussion

The low level of C4 characteristic of some sera from patientswith type 2 MC may be due to activation and cleavage,clearance abnormalities, or reduced synthesis. Activationmay proceed via the CP, AP, or MBL pathways, maskedby the increased synthesis of this APR due to the inflam-mation of liver damage and/or IC disease. Activation maybe consequent to the IgM RF and/or IgG fractions of

type 2 MC, complex formation, and other constituents ofcryoprecipitates, including HCV viral antigens, viral RNA,and CRP. Cryoglobulin formation provides a marker for thepersistence of ICs in the circulation of affected individuals, aswell as for the development of occlusive and/or inflammatoryvasculopathy, particularly in the skin. Persistence of ICsmay result from defective or saturated clearance mechanismsinvolving complement (CR1 and other), immunoglobulin Fc(RIIa and other), and/or C1q (gC1qR and other) receptors.Tissue damage due to complement activation requires thegeneration of fragments with anaphylatoxin (C3a, C4a, andC5a) and leukocytosis mobilizing (C3d and C3e) activity,and the engagement of their specific receptors; it is reflectedin footprints for the assembly of the MAC complex at sites oftissue injury. Lastly, polymorphisms of proteins that tightlyregulate the complement system might be interrogated todetermine the environmental and genetic determinants ofcomplement abnormalities characteristic for type 2 MC[39, 40].

Acknowledgment

This work was supported by the Seaver Foundation.

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Review Article

Molecular Signature in HCV-Positive Lymphomas

Valli De Re,1 Laura Caggiari,1, 2 Marica Garziera,1

Mariangela De Zorzi,1 and Ombretta Repetto1

1 Unit of Clinical and Experimental Pharmacology, IRCCS, Centro di Riferimento Oncologico, National Cancer Institute,33081 Aviano, Pordenone, Italy

2 Clinical and Experimental Pharmacology, Centro di Riferimento Oncologico, IRCCS, 33081 Aviano, Pordenone, Italy

Correspondence should be addressed to Valli De Re, [email protected]

Received 24 April 2012; Revised 29 June 2012; Accepted 3 July 2012


Copyright © 2012 Valli De Re et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hepatitis C virus (HCV) is a positive, single-stranded RNA virus, which has been associated to different subtypes of B-cell non-Hodgkin lymphoma (B-NHL). Cumulative evidence suggests an HCV-related antigen driven process in the B-NHL development.The underlying molecular signature associated to HCV-related B-NHL has to date remained obscure. In this review, we discussthe recent developments in this field with a special mention to different sets of genes whose expression is associated with BCRcoupled to Blys signaling which in turn was found to be linked to B-cell maturation stages and NF-κb transcription factor. Evenif recent progress on HCV-B-NHL signature has been made, the precise relationship between HCV and lymphoma developmentand phenotype signature remain to be clarified.

1. Introduction

In the early 2000s, a large body of experimental and epi-demiological evidence established an association betweenB-cell non-Hodgkin lymphoma (B-NHL) and hepatitis Cvirus (HCV). Epidemiological studies demonstrated thatHCV-related type-II mixed cryoglobulinemia herein named(MC), a B-cell lymphoproliferative autoimmune disease,favor lymphoma progression [1]. Approximately 1 of 20instances out of B-NHLs in Italy may be attributable to HCV[2, 3]. HCV incidence was found to be higher in the southand on the islands [3]. The burden of clinically relevantHCV-positive cases in Italy is on the decline [4].

As of today, the precise mechanism of lymphoma onsetremains unclear. HCV has been demonstrated to infect B-cells but the level of replication is low and is only demon-strated in a few cases. The mechanism of B-cell tropismremains elusive, and cell cultures producing HCV are limited[5–7]. Alternatively, though not necessarily in opposition,cumulative evidence supports a role of HCV as an etiologicalagent for indirect stimulation of specific B-cells, resulting inprogressive clonal expansion of B-cells [8–10].

The incidence of cryoglobulinemia and indolent HCV-related B-NHL decreases after HCV eradication, data rein-forcing the suggestion of a contribution of chronic antigenicstimulation to the physiopathologic process of HCV B-NHLs[11–13].

Clinically, HCV has been associated with different his-totypes of B-cell B-NHLs which are indistinguishable fromtypical B-NHL, except for the presence of HCV, the coexis-tence of liver disease, and the presence of cryoglobulinemia.Because indolent HCV B-NHLs are currently considered aprogression of MC related to HCV infection, they are treatedin the same way as MC with antiviral therapy (such as pegy-lated interferon and Ribavirin) [14, 15]. New approaches,such as anti-CD20 monoclonal antibody, have also beenproposed alone or in addition to antiviral treatment [11, 16].HCV-B-NHL has been treated like other lymphomas whensymptomatic.

The strong association between HCV infection and B-NHLs has lead to search for molecular signatures that canpredict patients’ characteristics, enhance understanding ofbiological mechanisms of lymphomagenesis, and could havediagnostic/clinical usefulness.


This paper takes into account gene expression profiling,characterization of B-cell maturation stages, experimentalantigen-induced B-cell growth, and immunoglobulin secre-tion as well as immune-regulatory molecules involved inthese processes, which, taken together, provide powerfulmeans to better define HCV-lymphoma entities. Despitethese studies, the identification of the molecular signatureof HCV-B-NHLs is not completely defined yet and weunderscore the need for further studies.

2. HCV + B-NHL Histotypes

HCV infection has been associated with different histotypes.Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoma (less than 1% of all B-NHLs) but is acommonly found characteristic of HCV infected population,they develop it in about one-third of cases [17, 18]. SMZLdisplays a strongly homogeneous signature implying theexistence of a single molecular entity [19]. Phenotypically,SMZL is usually negative for CD10, CD23, and CD123.They coexpress IgM and IgD, with surface immunoglobulinlight chain restriction. Of the genes deregulated in SMZLs,special mention should be made for the genes involved inBCR signaling, tumor necrosis factor signaling, and NF-κBactivation [20–22].

A higher prevalence of HCV positivity was also observedamong lymphoplasmacytoid/lymphoplasmacytic/immuno-cytoma and diffuse large cell histotypes than among HCV-negative counterparts [18]. In primary hepatic lymphomas,mainly of DLBCL type, the prevalence of HCV infection isagain higher than that in the HCV-negative population [23].

3. B-Cell Receptor

It has been previously demonstrated that the B-cell receptor(BCR) repertoire expressed by clonal B-cells involved withHCV-associated MC as well as with B-NHL is not random,with VH1-69 and VH3 heavy chain and VK3-20 and VK3-15 light chain genes being the most represented [9]. Thesedata suggest a model of antigen-driven origin for theselymphoproliferative disorders with the recognition of alimited number of HCV antigens, that is, NS3 [24], E2[9, 25], and indirectly core-antibody complexes [26, 27].Moreover, core antigens are proposed as responsible forvascular damage [28] and NS3 antigen as responsible formembranoproliferative glomerulonephritis [29, 30].

4. Pauciclonality of PeripheralB-cells in Both Resolved andChronic HCV-Infected Patients

Pauciclonality of the peripheral B-cell population is acharacteristic of HCV-infected patients with MC and/orB-B-NHL [31, 32] and is also a distinguishing feature ofsubjects who spontaneously resolved HCV infection eventhough they did not present any clinical manifestationof lymphoproliferative disease [33]. The most important

difference between expanded B-cells of resolved and chron-ically infected patients has been shown in the B-cellCD27− subpopulation. B-cell clones from patients whoare spontaneously resolvers preferentially used similar VH,DH, and JH gene segments compared to blood samplesfrom healthy donors, but with a different frequency ofthe usage of some gene segments with respect to patientswith chronically evolving HCV infection (mainly the VH1-69 gene) and higher antigen selection, as shown by thenumber and characteristics of somatic mutations [33]. CD27expression has been used to distinguish between memoryand naive B-cells in humans; however, low levels of mutatedand isotype-switched CD27− cells, typical of a mature B-cell, are also seen in healthy individuals [34]. The atypicalenrichment of VH1-69-positive cells in the CD27− B-cell compartment in resolved individuals with respect tochronic HCV-infected patients suggested an accumulationof these “VH-designated” B-cells in these patients [27].Reported data did not discriminate between the CD27− B-cell subtypes; therefore, it is impossible to distinguish specificB-cell subtype(s) associated with HCV resolvers as of today.

On the other hand, VH1-69+ cells are associated withmature CD27+ clonal B-cell in HCV-infected patients withMC or B-NHL [31, 35]. Data suggests that MC or B-NHLmalignant B-cell clones may develop from VH+ CD27− B-cell subtypes involved in HCV clearance [33].

5. Cluster of Differentiation for HCV-B-NHLB-Lymphoid Cells

In recent years, it was clearly demonstrated that CD27 isnot a universal marker of memory B-cells in human; infact, its expression distinguishes between different subsets ofmemory B-cells, and CD27 expression occurs in a distinctdevelopmental fashion of CD27 negative B-cells [34, 36].Naive and resting B-cells usually do not express CD27, but itsexpression can be induced by activation of B-cells, resultingin sustained expression over long periods of time. The CDR-H3 repertoire of the CD27− cells is significantly differentfrom the CD27+ cells, indicating that perhaps the lack of aCD27 molecule might be related to binding properties of theimmunoglobulin CDR-H3 region [34].

An accumulation of “atypical memory” CD27− B-cells has been described in chronic diseases such as inHIV-infected viremia [37, 38], in plasmodium falciparum-infected patients [39], in individuals from malaria endemicarea [40], and in patients with autoimmune diseases suchas systemic lupus erythematous (SLE) [39] and rheuma-toid arthritis [41]. Therefore, an accumulation of mem-ory CD27− cells is a characteristic feature occurring inseveral chronically infectious and autoimmune diseases.One hypothesis is that the CD27− memory compartmentcontains B-cells assigned to produce antibodies to counteractsome infections, while at the same time, this compartmentalso contains autoreactive B-cells. Memory CD27− B-cellshamper the development into antibody-secreting plasmacells through decreased levels of stimulatory molecules andan increased levels of inhibitory molecules expression [34,


36, 39, 42]. Thus, it is suggested that these B-cells become“exhausted” after extensive proliferation driven by microbialantigens. This action should reduce the negative effect ofautoreactive antibodies, but by the same mechanism shouldfavor the achievement of a chronic infection due to areduction in the titer of efficient antibody production.

With regard to HCV-infection, while HCV resolversshowed an accumulation of VH1-69 CD27− B-cells com-pared to HCV-chronic patients [33], in MC condition areduced number of naıve B-cells (CD27−, CD21+, andCD10−) due to an increasing sensitivity to undergo apopto-sis was found [43]. Conversely, in MC, Holz et al. evidencedan expansion of both the T2 immature transitional B-cell subset (CD27−, CD21+, and CD10+) and an increaseof activated B-cells (CD27+, CD21−, and CD10−) whichshowed apoptotic resistance [43]. The activated B-cell subsetpredominantly expresses the VH1-69 segment, suggestingthat this B-cell population has to induce the MC disorder[31, 35, 44]. Eradication of HCV with peg-interferontherapy, is associated with a decrease in the number ofthese last B-cells, but the authors showed that marginalzone-like counterpart (VH1-69, IgM+, CD27+, CD21+,CD11c−), they hypothesized that is was an ancestor ofactivated B-cells (CD27+, CD21−), may persist after viraleradication and thus should maintain the autoimmunity[45]. Moreover, B-cell depletion using Rituximab treatmentfor patients who failed antiviral therapy [11] highlightsthe relationship between clinical MC response and therestoration of T1/T2 transitional immature B-cell ratios bya reduction of the T2 B-cell proliferation, underscoring animportant role of this B-cell population in MC pathogenesis[43].

Clonal B-cells from patients with HCV-related B-NHLshad similar mature CD27+ immunophenotype to MC, butin these cases the mechanism of B-cell homeostatic is brokenand B-NHLs histotype was heterogeneous.

Lymphoplasmacytoid. Lymphoma is a low-grade B-NHL,involving the spleen, bone marrow, and lymph nodes. Itproduces cryoglobulins and may originate from B-cells thathave bypassed the germinal center. Among these B-NHLs,the most typical form associated with HCV infection isthe splenic lymphoma. Lymphoplasmacytoid can transformto diffuse large B-cell lymphoma (DLCBL) and originatefrom memory marginal zone-like B-cells, predominantlyIgM+ IgD−, mutated IgVH, CD10−, CD5−, and cyclinD1−. When the neoplastic cells circulate in the peripheralblood they are termed villous lymphocytes due to theircharacteristic appearance [17].

DLCBL. DLCBL is the most common type of aggressivelymphoma with frequent extranodal involvement, and itsimmunophenotype and genetic features are variable andoften aberrant. Primary hepatic DLCBL also shows a highprevalence of HCV infection. Since HCV primarily affectsthe liver, these data underscore the increase risk of lym-phomagenesis following HCV infection in [23].

6. Genetic Alterations and BLyS/BAFFExpression in HCV-B-NHLs

The exact process for altered B-cell homeostasis in HCV-B-NHLs is not yet understood, although accumulation ofseveral genetic anomalies has been highlighted. In particular,trisomy 3q [46] and possibily a higher human telomerasegene (TERC) at 3q23.3 copy numbers were observed inHCV-associated NHL than in HCV-positive patients [47].Moreover, polymorphisms in oxidative stress genes [48];deregulation of NF-κB, BCR and TLR pathways in splenicmarginal zone lymphomas have also been reported [22, 49].T(4; 18) translocation involving the Bcl2 gene has also beenhypothesized, but this translocation has been found only insome cases, mainly of MALT histotype (lymphoma involvingthe mucosa-associated lymphoid tissue) [50].

Several pieces of evidences suggest an important roleof B-lymphocyte stimulator factor (BLyS), a TNF familymember also known as B-cell activating factor (BAFF)which is expressed in B-NHL and MC [51–53]. The pro-tein is a potent coactivator of immunoglobulin produc-tion. Transgenic mice overexpressing BLyS develop B-cellhyperproliferation together with the production of highlevels of immunoglobulins like IgM, rheumatoid factor, andcryoglobulins [54]. BLyS typically activates NF-κB, JNK,and ERK pathways that in turn lead to B-cell survival,proliferation, and differentiation (Figure 1). BCR signal gen-erates the canonical NF-κB signal and in some B-cell stagesupregulates expression of NF-κB2 genes, resulting in theproduction of p100 protein [55]. BLys-receptor activationprovides an accumulation of p52 protein deriving from p100,which activates NF-κB by the noncanonical pathway [56](Figure 1). BLyS also plays a role in Ig class switching inmature B-cell differentiation [57].

7. Maturation of B-cells IsControlled by BCR/BLyS Interaction

A strong association between high level of BLyS and cryo-globulinemic syndrome is now clearly confirmed.

High expression levels of BLyS are known to increasethe survival of positive selected immature bone marrowB-cells (IgM+, CD23−), regulate peripheral/spleen T2/T3transitional stages, and improve survival of follicular/splenicmature B-cells [55, 58]. BLyS may induce B-cell proliferationin bone marrow only when acting together with anti-IgM(Figure 2). Several studies also found that BCR ligationupregulated BLyS-receptor expression in transitional T2 andmature B-cells. Conversely, the FcγIIB receptor, a receptorfor the Fc fragment of IgG immunoglobulin, present on B-cells, abolishes the effect by reducing the expression of BLyS-receptor levels [27, 59]. It is hypothesized that alteration inthe homeostasis of B-cells development, including autoreac-tive B-cells, may be caused by excessive levels of BlyS [51, 60].

Recently, it has been suggested that physiologically BLySsteady-state concentrations may be under the feedbackcontrol of a number of B-cells present in the individualand BLyS-receptors on various amounts on B-cell surfaces


RelBRelA

Canonical pathway Non canonical pathway

TCR, IL-R, BCR, etc… Blys-R, CD40, etc…

Receptor activation Receptor activation

Nemo Nemo

Phosphorilation

p105

p50

Proteasomal

degradation

C-Rel p50

Transcription

Nuclear translocation

NIK

P

P

P

p100

p52

IKK (αβγ) complex IKK (α) complex

NF-κB target genes

α α αβ

β

Figure 1: NF-κB members and NF-κB signaling. The NF-κB family is composed of five related transcription factors: p50, p52, RelA (p65),c-Rel, and Rel-B. These transcription factors are related through homology domains in which they form homodimers and heterodimersthat bind NF-κB DNA sites, thus modulating gene expression. P50 and p52 are derived from p105 and p100 precursors, respectively. NF-κB is silenced by interactions with inhibitory IkB family members in the cytoplasm. There are two NF-κB signaling pathways known asthe canonical pathway (or classical) and the noncanonical (or alternative) pathway. In both pathways, IkB kinase is activated and inducesproteasomal degradation of the IkB inhibitor, thus allowing the translocation of the transcription factor subunits into the nucleus andinduce transcription of target genes. BCR crosslinking provides the canonical NF-κB signal and p100 production, while BLys receptor induceaccumulation of p52, a protein deriving from p100 that activates NF-κB 2 via the noncanonical pathway.

that both bind and then subtract BLyS molecules fromthe serum [61]. BLys is necessary for B-cell survival andproliferation; therefore, when the number of B-cell decreases,BLys concentration increases and vice versa, thus preservingthe stead-stay level of B-cells in the peripheral blood.Moreover, it was found that transitional and naive IgD+CD27− B-cells require more BLys-induced survival signalsthan the CD27+ switched memory or marginal zone-like B-cells [62]. This effect has been ascribed to a difference inBLyS receptor family expression in immature, transitional,and antigen-experienced mature B-subsets. BLyS binds threereceptors: transmembrane activator and calcium-modulator(BCMA), cyclophilin ligand interactor (TACI), and BR3(BLyS receptor 3), with decreasing affinity for BLyS in thefollowing order: BR3 > TACI > BCMA [63, 64]. BR3 wasfirst observed in immature B-cell; after antigen-encounters,a shift from BR3 to BR3 plus TACI expression occurs, whileBMCA is present only during plasma cell differentiation [64,65]. BLyS receptor should then regulate B-cell maturationacting on different NF-κB pathways (TACI-receptor activatescanonical NF-κB pathway, while BR3 receptor activates NF-κB only through noncanonical signaling (Figure 1)). As a

consequence, different subset of B-cell competes for BLySmolecules regulating the quantity of B-cells in each subset.This model of BLyS/B-cell interaction is strongly supportedin X-linked agammaglobulinemia patients and in severalanimal models where mature Blys-receptor+ B-cells were notgenerated, and where soluble BLys levels were then foundhigher than that in healthy controls while in other geneticallydefined primary immunodeficiencies, patients who showswitched BlyS receptor positive memory B-cells, have normalBlyS levels [61].

HCV B-NHLs are almost always transitional/activatedIgM+ K+ B-cells [9], a characteristic shared betweensome immature and mature B-cells which have not yetaccomplished immunoglobulin switching. In bone marrow,immature B-cells with BCR receptor reactivity (includingself-molecules reactivity) below the threshold induce a neg-ative selection survive and proliferate when BLyS-receptorsexpression is associated with high IgM expression (Figure 2).Immature transitional B-cells require both a tonic BCR sig-naling for survival and proliferation [66] and BLyS receptorsexpression [67], whose expression is in turn regulated bythe BCR signal itself [64, 68]. Thus, in immature B-cells,


High BCR/antigen affinity

Blys-R(BR3)

Weak BCR/antigen affinity

BCR-crosslinking

Rag 2

Rag 2

No BCR/antigen interaction

Bone marrow (CD23−)Immature B-cell

+

+

−IgL-k+

IgL-k+IgL-k+

IgL-k+

Positive selectionNegative selection

Functional BCR editing

Nonfunctional

BCR editing

SurvivalDeath

Transitional stage

T1NF-κB p100 ↑

p52 ↑

Figure 2: A model of BCR/BLyS interaction of immature B-cells to transitional B-cells. In bone marrow, weak BCR linkage of immatureB-cells induces BLyS-receptor expression and a down-expression of RAG-2, an enzyme involved in BCR editing. In addition, immatureB-cells can be rescued from the negative selection of the BCR signaling apoptotic pathway. After a functional (but not strong self-reactive)BCR editing is accomplished. In case of high BLyS level, some weak self-reactive B-cells (weak BCR/antigen affinity) can be developed intoT-transitional immature B-cell stage.

the apoptosis was induced by a strong BCR-crosslinking thatgenerated a BLyS-receptor downregulation, while in tran-sitional mature B-cells, BCR-crosslinking was accompaniedby BLyS-receptor upregulation and B-cell proliferation. It isproposed that high levels of serum BLyS may rescue weakself-reactive B-cell clones that usually die at the transitionalstage from apoptosis [58].

It has been well documented that in the general pop-ulation BLys mediated signaling is involved in the survivaland proliferation of some B-NHLs [69], with a differenteffect on BLyS response related to different stage on matureB-cell malignancies [70]. Unlike normal B-cell counterpart,most B-NHLs express BLyS in an autocrine fashion and/orpresent mutation in the cytoplasmatic region of the BLySreceptor [71]. With regard to HCV-B-NHL, as well as MC,an increase in BLyS levels was found. Although a long-term production of BLyS molecules in response to chronicinfections and inflammation cannot be excluded, a BLysautocrine production and/or BLyS receptor mutation shouldbe possible. Studies are required to elucidate this question.

8. Immune Response against the VK3-20Protein and Shift in TH2 Immune Response

Dendritic cells (DC) are the most powerful cells that processantigen material and present it on the surface of othercells of the immune system; they also play an importantrole as part of the immune regulatory network. Dependingon their lineage (myeloid or plasmacytoid: CD11, CD83,

CD86, and HLA-DR class-II markers) and stage of differen-tiation and activation (CD40 and CD80 markers), dendriticcells may either promote a strong T-lymphocytes-mediatedimmune response or an anergic state. The frequency ofmDC and pDC, along with the expression of CD40 andCD80 markers, indicate the capacity of recombinant VK3light immunoglobulin to specifically induce DC activationand maturation in healthy subjects as well as in HCV-positive patients [72]. Buonaguro et al. also observed apoor monocyte activation and maturation [72]. In vitro,BCR stimulus of mature peripheral B-cells only (CD19+and CD27+) yielded a significant increase in expressionof activation markers CD80 and CD86, even though thetrend for CD86 is not significant on B-cells of HCV-infectedpatients as compared to healthy control subjects [73].Moreover, after VK3 stimulation, a higher production ofTH2 cytokines (IL-6, IL-4, IL-10, and TNF-α) was observedin HCV-positive patients. A shift in TH2 cytokine expression,characterized by an elevated production of some cytokines,had already been reported in other chronic infections suchas HIV, Helicobacter pylori and other virus-related diseases[74, 75]. On the other hand, patients with MC have increasedlevels of TH2 cell-derived cytokines, that is, IL-2 and IL-5 [76]. Therefore, it is suggested that the TH2 immuneresponse, by means of T-cell-dependent B-cell stimulation,may promote autoantibody production, MC disease, and B-NHL.

Recent studies propose a crosstalk between TH2 cytoki-nes production and abnormal B-cell activation. The resultsof these studies indicated an association between HCV and


suppressors of cytokine signaling (SOCS), which negativelyregulates the cytosol-to-nuclear JAK-STAT signaling whichultimately induces interferon signaling and apoptosis of thecell [77–79]. However, how cytokines might be involved inthe development of B-cell clonal expansion, mixed cryo-globulinemia, and B-NHL in patients who are chronicallyinfected with HCV still remains unknown.

9. HCV-Positive B-NHL Showed RestrictedCombination of HLA Class-II Genes

Immune complexes may be internalized in B-cell throughBCR-ligation, then processed and presented in the contextof HLA class-II molecules to T-cell costimulation in thegerminal center of follicles [80]. This T/B-cell interactionis necessary for immunoglobulin class switch recombina-tion, somatic hypermutation, and specificity-based selectionunderlying immunoglobulin affinity maturation. RestrictedIg genes may be presented by means of a limited numberof HLA molecules. Possibly because of this, it is evidencedthat the DR5-DQ3 HLA combination was strongly associatedwith the HCV (+) MC (+) B-NHL group of patientscompared with bone marrow donor population, while thecontribution of DR1-DQ1 was higher in cases of HCV(+) B-NHL without MC [81]. The most common DR-DQcombination class used in HCV-infected patients withoutlymphoproliferative diseases and in subjects with HCV-related hepatocellular carcinoma was also different [82, 83].

10. Decreased miRNA26b ExpressionAssociated with HCV-Related MarginalZone Lymphomas

MicroRNAs (miRNAs) are a class of small noncoding RNAsthat bind to partially complementary sites in the 3′ untrans-lated regions (UTR) of target mRNAs and modulate geneexpression by facilitating translational repression or mRNAdegradation. Liver-specific miRNA miR-122 was found tobe associated with inhibition of HCV replication. MiR-122regulates virus production by directly interacting with the 5′

end of the HCV RNA genome [84].Only one study, to date, explores miRNA patterns and

HCV-related B-NHL [85]. A reduced expression of miRNA-26b has been found in HCV-positive versus HCV-negativepatients with SMZL. This latter miRNA seems to be moststrongly associated with specific HCV-related MZSLs, sincethe miRNA pattern is different in chronic HCV-relatedsubjects and in those with HCV-related hepatocarcinoma.One predicted target of miRNA26b is the NIMA-relatedkinase NEK6, which has a critical role in mitotic cell cycleprogression and is upregulated in various human cancers.In the same study, no statistically differential expressionbetween SMZL and nonneoplastic splenic tissue was found.However, a trend towards a significant difference in theexpression of 7 out of 381 miRNAs was tested. Notably,from the list of genes reported, the upregulation of miR-21 and miR-155 was associated with several genes involvedin NF-κB signaling. Genetic alterations involving the NF-κB

pathway were also found in the SMZL subtype of the generalpopulation [21, 86].

The overall data reported indicated the importanceof NF-κB signaling in lymphoma pathogenesis and theinvolvement of specific pathways in HCV-infected patients.

11. Toll-Like Receptors 2 and 4 andIL-6 Expression and Associationwith HCV-B-NHL

The innate immune response involving toll-like receptors(TLRs) has been shown to play an important role in thepathogenesis of many viruses. The continuous interactionbetween viruses with TLRs may induce a chronic activationof inflammatory cytokine responses that are a risk factor fortumor development. Compared with peripheral blood cellsfrom healthy individuals, TLR4 was found to be upregulatedin B-cells after HCV infection [87]. TLR4 expression wasfound to be mediated by the NS5A HCV protein and mayprovoke B-cell activation through interleukine 6 produc-tion. Moreover, the core HCV-protein may trigger B-cellactivation through TLR2 interaction [88]. The productionof proinflammatory interleukine 6, which stimulates B-cell activation, has been suggested to contribute to thedevelopment of cryoglobulinemia and B-NHL [88]. BCRand TLR signalling pathways were also found to be targetedby genetic changes in SMZL [22]. Moreover, in the generalpopulation, genetic variation in TLR1-TLR6 region geneswas associated with B-NHL risk, with a specific associationfor TLR2 variant and MZL [89]. Although all these data areindicative of a functional relation between TLR and HCV-B-NHL, the real relationship between these molecules remainsinconclusive.

12. Conclusions

In conclusion, lymphomas that develop in HCV-infectedpatients seems to combine disease-specific signatures anddifferent sets of genes whose expression is associated withBCR coupled to Blys signaling, which in turn has beenlinked to B-cell maturation stages and to specific NF-κB transcription factors. This paper highlights the closelink between the specific contribution of these genes incomparison to normal and to chronic HCV-infected B-cells. This paper underscores that HCV-related lymphomasare subject to specific deregulation induced by the virusinfection, although the precise relationship between HCVand lymphoma development and phenotype signature needsto be clarified. Identification of molecular signatures inlymphomas occurring in the HCV-infected population couldfacilitate a more rational approach to the diagnosis as well asmore tailored treatments and/or prevention.

Acknowledgments

The authors thank Mrs. Anna Vallerugo, M.A., for her writ-ing assistance. This study was supported by an AIRC Grant


number 1026, assigned to Valli. De Re. Authors had noconflicts of interests in any part of this submitted paper.

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Review Article

The Place of Immunotherapy in the Management ofHCV-Induced Vasculitis: An Update

Laurent Chiche,1 Stanislas Bataille,2 Gilles Kaplanski,1 and Noemie Jourde2

1 Department of Internal Medicine, Centre de Competence pour les Maladies Autoimmunes Systemiques PACA Ouest,Hopital de la Conception, Marseille, France

2 Department of Nephrology, Hopital de la Conception, Universite Aix-Marseille, Marseille, France

Correspondence should be addressed to Laurent Chiche, [email protected]

Received 26 April 2012; Accepted 3 July 2012


Copyright © 2012 Laurent Chiche et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Patients with chronic hepatitis C virus (HCV) can develop systemic cryoglobulinemic vasculitis. Combination of pegylated-interferon α and ribavirin is the first-line treatment of this condition. However, in case of severe or life-threatening manifestations,absence of a virological response, or autonomized vasculitis, immunotherapy (alone or in addition to the antiviral regimen) isnecessary. Rituximab is to date the only biologic with a sufficient level of evidence to support its use in this indication. Severalstudies have demonstrated that rituximab is highly effective when cryoglobulinaemic vasculitis is refractory to antiviral regimen,that association of rituximab with antiviral regimen may induce a better and faster clinical remission, and, recently, that rituximabis more efficient than traditional immunosuppressive treatments. Some issues with regard to the optimal dose of rituximab or itsuse as maintenance treatment remain unsolved. Interestingly, in balance with this anti-inflammatory strategy, a recent pilot studyreported the significant expansion of circulating regulatory T lymphocytes with concomitant clinical improvement in patientswith refractory HCV-induced cryoglobulinaemic vasculitis using low dose of subcutaneous interleukin-2. This paper provides anupdated overview on the place of immunotherapy, especially biologics, in the management of HCV-induced cryoglobulinaemicvasculitis.

1. Introduction

Chronic hepatitis C virus (HCV) infection is associated withnumerous and mostly autoimmune extrahepatic complica-tions. One of the most serious is cryoglobulinaemic vasculitis(CV), which develops in 5–10% of infected patients. CVis a systemic small-vessel vasculitis that affects mostly skin,joints, nerves, and kidneys and can sometimes have a life-threatening presentation [1]. The identification of HCVas the main causal agent for CV has completely modifiedthe management of this virally induced vasculitis. Indeed,circulating immune complexes responsible for organ damageare the result of B-cell expansion and the production ofpathogenic IgMs with rheumatoid-factor activity, which isdriven by the underlying chronic viral infection. Thus,

obtaining a sustained virological response (SVR) has becomethe main treatment for HCV-induced CV. Fortunately, thecombination of pegylated-interferon α (peg-IFN-α) plusribavirin has resulted in an SVR in up to two-thirds ofpatients, depending on the genotype of HCV [2–4].

However, in some situations, immunotherapy alone orin addition to antiviral treatment is necessary to treatHCV-induced CV (Figure 1). For a long time, immunother-apy for CV has been largely empirical, relying on tradi-tional immunosuppressive options. However, recent studies,including some with a prospectively controlled design, haveaddressed the place of biologics in this setting. Herein,we aim to provide an updated overview of the place ofimmunotherapy, especially biologics, for the management ofHCV-induced cryoglobulinaemic vasculitis.


Cryoglobulinemia

An

tivirals

Imm

un

oth

erap

y

(A)

(B)

(C)

(D)

Clinical manifestationsB-cell activationHCV load

Figure 1: Immunotherapy to manage HCV-induced vasculitis. (A) Antiviral regimen, ideally a combination of interferon plus ribavirin,is the first-line treatment for HCV-induced cryoglobulinaemic vasculitis (CV) when the severity of its manifestations is mild-to-moderate.In addition, short-term low-dose corticotherapy may sometimes be used initially. (B) In cases of severe or life-threatening manifestations(i.e., severe renal involvement), immunotherapy must be initiated immediately. Rituximab has become a preferred choice but, as with otherimmunosuppressive drugs, careful monitoring of viral load and hepatic functions is necessary. Worsening of vasculitis has been reported inpatients just after administration of rituximab, especially in those with serious cryocrit levels, thus, in these patients, corticosteroids and/orplasmapheresis may be initiated before B-cell depletion. An antiviral regimen is initiated either simultaneously or secondarily/sequentiallyin these patients. (C) When an antiviral regimen is contraindicated, poorly tolerated, or fails to induce a sustained viral remission,immunotherapy is also initiated. Corticosteroids should be avoided when possible. Careful monitoring of viral load/hepatic function isnecessary. A prolonged antiviral regimen may be considered when clinical and biological manifestations of MC show an improvement underthis regimen in spite of the absence of viral remission. (D) In cases where CV is still active in spite of obtaining a sustained viral response,B-cell malignancy and low-level viremia should be ruled out before considering that the vasculitis is autonomous and before initiatingimmunotherapy.

2. Immunotherapy in HCV-Induced Vasculitis:For Whom and When?

Eradication of HCV with peg-IFN-α plus ribavirin is thefirst-line treatment for CV (Figure 1(a)). Indeed, whenthis treatment is not contraindicated and sufficiently welltolerated, it allows an SVR in 50% (genotypes 1, 4, 5, 6)to 80% (genotypes 2 and 3) of patients after 48 and 24weeks of treatment, respectively [2–4]. In these cases, noimmunotherapy is needed. However, immunotherapy needsto be considered, alone or in addition to antiviral treatments,in the following situations.

2.1. Severe or Life-Threatening Manifestations. Because ofthe delayed and uncertain response to antiviral therapy,severe and rapidly progressive CV manifestations (i.e., acutenephrotic or nephritic syndrome, extensive cutaneous ulcers,central nervous system or gastrointestinal manifestations,and hyperviscosity syndrome) require prompt and aggres-sive treatment (Figure 1(b)). Indeed, the use of aggressiveimmunotherapy in these settings is indirectly supported

by the results of a recent study that identified a strongassociation between increased mortality and cutaneousulcers (hazard ratio (HR) 5.37) or renal insufficiency (HR3.25) [1]. Concerning peripheral neuropathy, even if notconsidered a life-threatening manifestation, it is a majorcause of morbidity in HCV-associated CV and is oftenrefractory to all treatments. In addition, as any improvementis often delayed, later reevaluation prevents a rapid switchto a different therapeutic option if needed, which increasesthe risk of definitive sequelae. Thus, in the most severe cases,immunotherapy can be a part of first-line treatments [5].

In patients with severe or rapidly progressive mani-festations, antiviral therapy is still an important part oftreatment and can be initiated either concomitantly orsequentially. Concomitant administration, ideally, may pre-vent an increase in HCV viral load and hepatic consequencessecondary to an immunosuppressive strategy. However,some data support the short-term safety of a sequentialstrategy (i.e., starting with an immunosuppressive regimenalone), even in patients with advanced liver disease [6]. Also,sequential administration has some practical advantages.


First, it avoids situations where the physician faces theoccurrence of a side effect within a combined antiviral andimmunosuppressive regimen (e.g., cytopenia), a situationthat complicates the imputability of this side effect to aspecific drug. Also, when renal function is altered, the useof ribavirin is very limited due to increased toxicity. Finally,some authors have reported a paradoxical exacerbation ofCV after the initiation of antiviral regimens [7, 8], which maybe prevented when immunotherapy is started first.

2.2. Absence of a Virological Response. The use of peg-IFN-αcombined with ribavirin as the standard-of-care for HCV-induced CV is supported by several studies in which thistreatment has been found to be safe and well tolerated andhas led to an SVR rate similar to that for HCV-infectedpatients without CV [9, 10]. But, importantly, only patientswith complete clearance of HCV achieve a complete andsustained clinical response, and SVR is not always obtainedfor various reasons. In about one-third of patients, andparticularly those with genotype 1 HCV, a well-conductedantiviral regimen fails [2–4]. In addition, peg-IFN-α plusribavirin is poorly tolerated in 10–20% of patients, leadingto early termination of antiviral regimens. Also, somepatients have major contraindications to IFN and/or rib-avirin, such as advanced age, uncompensated cirrhosis,uncontrolled depressive illness, or untreated thyroid disease.In these patients with CV and no virological response,anti-inflammatory drugs may be warranted to avoid orcontrol severe or debilitating complications (Figure 1(c)).However, a major concern is the potential adverse effects thatimmunosuppressive therapy could have on the underlyinguncontrolled chronic viral infection. Except for severe mani-festations (see above), immunotherapy is administered afterother therapies have been optimized to obtain an SVR.

A failed standard-of-care, especially in genotype 1 HCV,may benefit from the recent development of two direct-acting antiviral agents, boceprevir and telaprevir [11]. Thecombination of one of them to the standard-of-care increasesSVR rates in genotype 1 HCV infection to >70%. Alter-natively, in virological nonresponders, when a clinical andbiological improvement has been observed under an antiviralregimen, some physicians may propose longer treatment forup to 48 or 72 weeks, respectively, for genotypes 2 and 3, andfor genotypes 1 and 4 [12]. Also, because of its immunomod-ulatory properties, interferon may precipitate or exacerbatesome preexisting and often subclinical disorders, especiallythose involving the thyroid, but screening before as wellas close monitoring during treatment improves detectionand early management of these potential complications [13].Finally, the contraindications listed above may be judgedas relative in some patients, when the benefit of treatmentmay overcome the theoretical risks. This is especially true foradvanced age, but also, in some cases, for depressive status,when antidepressant prophylaxis initiated 2 weeks beforeinterferon therapy may be useful for at-risk patients [14].

2.3. “Autonomized” Vasculitis. A few patients may experiencebiological and/or clinical persistence or relapse of CV despite

clearance of their HCV infection. This is probably becauseB-cell expansion has become, at least in part, independentof HCV stimulation (Figure 1(d)). In this setting, underlyingB-cell malignancy must be ruled out first. Indeed, HCV-associated CV has been associated with an increased riskof B-cell lymphoma [15]. Landau et al. reported on eightpatients who presented with a relapse in HCV-induced CV,despite having achieved SVRs. In two out of three patientswhose symptoms of CV persisted and were associated withelevated cryoglobulin levels, B-cell lymphoma was diagnosed[16].

There is also controversy about the possible role of occultHCV infection, that is, detectable HCV-RNA in the liver orperipheral blood mononuclear cells in the absence of serumHCV-RNA [17, 18]. Indeed, it is conceivable that the virus, orpart of it, may still be triggering B-cell proliferation, althoughit is not detected in the serum. However, a recent exhaustivereview on this topic did not reach any firm conclusions[19]. Recently, we reported, for the first time, the presenceof HCV-NS3 viral protein in the kidney of a patient witha similar presentation, but we were unable to conclude onthe significance of this finding [20]. What is certain fornow, is that an ultrasensitive real-time PCR assay should beconducted on the serum and/or cryoprecipitate to rule outlow-level infection, which may have been misdiagnosed asoccult infection in previous studies [21]. Thus, in patientswith an SVR but persistent clinical manifestations of CV,after exclusion of underlying hemopathies and/or low-levelHCV-persistent infections, the autoimmune component ofthe disease may be considered as autonomized and treatedsimilarly to nonvirally related CV [22].

3. Immunotherapy in HCV-InducedVasculitis: Which One?

Various anti-inflammatory drugs that are used success-fully to treat other types of vasculitis are also used totreat HCV-induced vasculitis. However, during the lastdecade, conventional immunosuppressive treatments (i.e.,cyclophosphamide and plasmapheresis) have been progres-sively challenged by biologics. Indeed, the most commoncause of death in patients with CV is infection and, in thestudy of Landau et al. [1], immunosuppressive treatment wasassociated with an increased risk of death, independently ofdisease severity (HR 6.51), suggesting that a more targetedimmune-based strategy would be beneficial. Apart fromthe poor effectiveness of TNF-blockade by infliximab oretanercept, reported by us and others [23–25], or therecent anecdotal report of the successful use of an anti-interleukin(IL)-6 strategy [26], rituximab (RTX) is, to date,the only biologic that has sufficient evidence to support itsuse for this indication. Interestingly, to balance this anti-inflammatory strategy, a recent pilot study reported thesuccess of a proregulatory strategy with low-dose IL-2 [27].

3.1. Anti-Inflammatory Strategy: Rituximab. RTX is a mono-clonal antibody against the CD20 antigen, which is selectivelyexpressed on B cells. The rationale underlying RTX treatment


is that in CV, CD20-positive cells are expanded, activated,and play a pivotal role in cryoglobulin production [28].Several studies have demonstrated that RTX is highly effec-tive when CV is refractory to antiviral regimens [5, 6, 29–31], that the association of RTX with an antiviral regimenmay induce a better and faster clinical remission [32, 33]and, recently, that RTX is more efficient than traditionalimmunosuppressive treatments [34, 35].

With some variations according to the different mani-festations of CV, the overall response rate to rituximab inpatients refractory to antivirals has been reported in recentmeta-analyses to be ≥80% [36, 37]. The delay in response isvariable, but improvement occurs within 1–6 months. Recentstudies that have compared a combined therapy with RTX toantiviral therapy alone show that a combined therapy maybe the best choice for patients with severe manifestationsof CV. Indeed, in a prospective cohort study of 93 patients,combined therapy reduced the time to clinical remissionand improved renal-response rates compared to peg-IFN-α + ribavirin alone [33]. In another prospective study thatincluded 37 patients, those in the RTX group achieved acomplete response more often than patients not receivingRTX (54.5% versus 33.3%) [32].

The rationale for choosing a targeted therapy with RTXinstead of conventional immunosuppressive agents has beenonly poorly supported by evidence, though two recentlypublished studies have filled this gap [34, 35] (Table 1). Thefirst study [34], an open-label randomized controlled trial(RCT) conducted in Italy, compared RTX to conventionaltherapies (i.e., corticosteroids, plasmapheresis, azathioprine,or cyclophosphamide) in 57 patients with severe manifesta-tions of CV. Of note, patients in the conventional-therapygroup, whose treatment failed, had the opportunity tocrossover and receive RTX. At 12 months, the proportion ofpatients who continued their initial therapy was significantlyhigher in the RTX group, and only 13.8% of patients inthe conventional-therapy group continued their initiallyassigned therapy beyond 3 months. The second study [35],conducted in the US, was also an open-label RCT, whichcompared RTX and standard therapy in 24 patients withHCV-related CV. Standard therapy was considered to bemaintenance or intensification of conventional immuno-suppressive therapy, but the patients receiving RTX wereallowed to continue their background immunosuppressivetherapy. At 6 months, clinical efficacy was clearly greater forRTX compared to conventional immunosuppressive therapy.Thus, even though the design of these studies may haveadvantaged RTX (Table 1), the data support a preference fortargeted B-cell depletion with RTX as the agent of choicefor CV. They also provide additional information on themodalities of administration of RTX and its safety.

Indeed, as in other autoimmune conditions [38], thereis no consensus on the choice of the modality of administra-tion, that is, a “rheumatological” regimen: 4 weekly infusionsof 375 mg/m2 versus a “hematologic” regimen: 2 biweeklyinfusions of 1000 mg, which are equally used in practice aswell as in RCT (Table 1). However, Sene et al. have raisedthe issue of serum sickness following the use of RTX therapyfor CV, especially in patients with the highest cryoglobulin

levels and the lowest C4 levels [39]. RTX may form a complexwith cryoglobulin, which could increase cryoprecipitationand induce severe systemic reactions, including serumsickness. Consequently, these authors propose the use of alower starting dose of RTX (i.e., rheumatological regimen),possibly preceded by corticosteroids and/or plasmapheresisto avoid side effects. Overall, short-term reactions to RTXinfusions do not seem to be more frequent in CV thanin other autoimmune conditions that are treated with aclassical premedication of 100 mg of methylprednisolone,antihistamine drugs, and paracetamol.

The safety of RTX, especially when RTX is used withoutthe cover of antiviral agents, was supported in both RCTs,even though HCV load was not monitored in the Italianstudy [34]. RTX was not associated with significant liverimpairment despite transient increases in HCV viral load,as already reported when RTX was given to patients withliver cirrhosis [6]. Nevertheless, data on HCV load andliver enzymes come from small sample-sized studies [40]with short-term followups, thus, this needs further evidence.RTX is also associated with a significant risk of infection,especially in patients with renal failure and advanced ageand in those receiving high doses of corticosteroids [41].This warrants the same precautions recommended for otherautoimmune conditions with regards to vaccination andspecific followup [42], including also early identification ofrare but potentially severe complications related to RTX (i.e.,anti-Pr cold agglutinins syndrome or progressive multifocalleukoencephalopathy).

3.2. Proregulatory Strategy: IL-2. Recently, Saadoun et al.obtained significant expansion of circulating regulatory Tlymphocytes (Treg) with concomitant clinical improvementin 8/10 patients with refractory HCV-induced CV using a lowdose of subcutaneous IL-2 (Proleukin, 1.5 million IU per dayfor 5 days, then 3 million IU per day for weeks 3, 6, and9) [27]. Their patients were refractory to previous antiviralregimens, but only 1/10 patients had previously receivedrituximab, and only 1/10 had mild renal involvement. Inter-estingly, these patients did not receive any corticosteroidsduring the study period. The limitations of this pilot studyare the absence of a control group, the short follow-up time(a few months), and some potential confounding factors(i.e., there was also a significant increase of CD56 brightNK cells), which prevent definitively concluding that theclinical benefits were solely due to the observed increase inTreg cells. Indeed, in the study by Koreth et al. (publishedat the same time), and also using low-dose IL-2 in patientssuffering from graft-versus-host-disease, Treg-cell countsincreased in all patients but were not statistically differentbetween patients who had and those who did not have aresponse [43]. Nevertheless, these two studies constitute aproof of principle that low-dose IL-2 can be used safelyto promote tolerance, probably through Treg expansion[27, 43].

IL-2 is produced by naive and memory T cells after anti-gen stimulation and binds to a high-affinity receptor consist-ing of three subunits: IL-2Rα (CD25), IL-2Rβ (CD122), and


Table 1: Prospective randomized controlled trials comparing rituximab (R) with a classical immunosuppressive regimen (C).

Studies Sneller et al. (USA) De Vita et al. (Italy)

Methodology

Sample size (R/C) 24 (12/12) 57 (29/28)

Design Prospective RCTOpen-label, monocentric

Prospective RCTOpen-label, multicentric

Followup duration M12 M24

Rituximab 375 mg/m2 × 4No GC premedication

1000 mg × 2100 mg MP iv before each

Other treatments allowed for group R IS/GC already initiated Low dosage of GC

Effective regimen for group C IS/GC already initiated ± increase(only PL = 1 at M5)

GC = 17 or IS = 7 (AZA/CYC)or PL = 5 ± GC

Planned sample size 30 124

Limitations 8-year enrolmentEarly stop after interim analysis

86% switch before M2∗

Early stop after interim analysis

Patients

Underlying VHC infection 24/24 53/57

Previous treatments (R versus C)Unbalanced at randomization

GC = 6 versus 3CYC = 1 versus 0, PL = 2 versus 0

Not provided

Efficacy

Primary endpoint Clinical remission at M6 Survival of initial treatment at M12

Result (R versus C) 10/12 (83%) versus 1/12 (8%) 64% versus 3.5%

Response to retreatment R: 3/3 R: 5/7 C: 6/8

Time of switch of C to R After M6 As soon as failure∗

Number of switches of C to R 9/12 23/28

Response to switch to R 4/7 (2 lost to followup) 14/23

Safety

Infusion-related severe events 1 serum-infusion reaction 1 hypotension with angina

Viral load of VHC No difference Not monitored

Abbreviations: AZA: azathioprine; CYC: cyclophosphamide; GC: glucocorticoids; IS: immunosuppressive; MP: methylprednisolone; PL: plasmapheresis.

γc (CD132). Until recently, almost all clinical trials using IL-2 aimed at boosting effector T lymphocyte (Teff) functionand have taken advantage of the immune-stimulating activityof IL-2. Indeed, this approach was successful in a subset ofpatients suffering from renal cell carcinoma and melanoma[44] and was also tested to boost the immunity of patientswith AIDS [45]. The main limitations to the broader useof IL-2 are its very short half-life in the circulation afterinfusion, which necessitates using IL-2 at levels as high aspossible, and its life-threatening nonspecific toxicities, suchas vascular-leakage syndrome. Recent studies have shownthat the primary function of IL-2 is, actually, the generationand survival of Treg [46], which explains in part why thisapproach failed in its anticancer indication and supports thepossibility that IL-2 may, instead, promote T-cell tolerance inautoimmune conditions, such as CV, where a deficit of Treghas been documented [47].

There are several ways to use IL-2 to boost Treg. IL-2,together with other stimuli, can be used to expand the Treg-cell population ex vivo (Figure 2(a)), in tissue culture, beforetransferring these expanded cells to patients [48]. But thisstrategy is probably too complex to broadly translate to the

bedside. Conversely, in-vivo expansion using subcutaneousinfusion of IL-2 has been already used with variable results inmice and humans. IL-2 can be used at a high dose with coad-ministration of rapamycin to prevent the activation of Teffcells without affecting the Treg-cell response (Figure 2(b)).This protocol has proved to be beneficial in the treatment ofdiabetes in NOD mice [49] but, unfortunately, a clinical trialin new-onset type-1 diabetes patients showed that treatmentwith rapamycin plus a relatively high-dose of IL-2 (4.5 ×106 IU/day subcutaneously, three times a week for 4 weeks)resulted in greater loss of insulin secretion at 3 months and,overall, was considered to worsen pancreatic β-cell function[50]. A low dose of IL-2 alone may also be used, whichfavours the expansion of Treg and has only a minor effecton Teff (Figure 2(c)). This strategy was successful in thetwo clinical studies already mentioned [27, 43] but warrantsconfirmation on a larger scale and additional work is neededto fully understand the role of IL-2 on cells other thanTreg. Finally, an alternative approach (Figure 2(d)) couldbe the use of improved IL-2 formulations or IL-2-specificmonoclonal antibodies, which allow IL-2 to selectively targetTreg cells [51].


(A)

(B) (C) (D)

Rapamycin

Treg

Teff

Complexed

Treg

Treg

Treg Treg Treg

Teff

Teff Teff Teff

IL-2

Ex vivo

In vivo

−++ + +

High-dose

IL-2 IL-2 IL-2

Low-dose

Figure 2: Different IL-2 based approaches to promote the expansion of regulatory T cells (Treg). A first approach consists of using IL-2,together with other stimuli, to expand ex vivo the Treg cells collected from a patient’s tissue culture before transferring these cells to thepatient (A). In vivo, IL-2 can be administered subcutaneously at high doses but can be associated with rapamycin to prevent activation ofeffector T cells (Teff) (B) or given at a low dose for the same reason (C). IL-2-specific monoclonal antibodies can be used to target IL-2selectively to Treg cells (D).

4. Biologics in HCV-InducedVasculitis: Next Steps

In just a few years, biologics have modified the managementof HCV-related CV. Their use has also raised many unsolvedissues. The first concerns maintenance treatment. In patientsrefractory to antiviral regimens and who are successfullytreated with RTX, more than a third will relapse during B-cell recovery, usually between 6 and 12 months [36, 37].However, retreatment with RTX after a relapse seems to beeffective in most cases [34, 35]. Systematic maintenance ofRTX therapy has rarely been reported in CV but may beconsidered in severe forms [52], though the best modalityremains to be determined. Other biologics targeting B cells,such as other anti-CD20 monoclonal (i.e., ocrelizumaband ofatumumab), anti-CD22 (epratuzumab), or anti-BAFF(belimumab) might also prove useful in the management ofthese conditions.

The second concern is the dosage used in RTX regimens.As already stated, both “haematological” and “rheumato-logical” regimens are both used in practice. Visentini etal. recently reported preliminary results from 27 patientsreceiving low-dose rituximab (2 weekly doses of 250 mg/m2):they had a response rate similar to that reported for patientstreated with standard doses [53]. If confirmed, this regimencould reduce costs, improve safety profiles, and be preferred

by patients with nonsevere manifestations. Finally, oneadditional advantage of using RTX to treat CV may bethe reduced exposure to corticosteroids. In the two RCTs[34, 35], responders to RTX therapy received lower totaldoses of prednisone than those allocated to a conventionalimmunosuppressive therapy. The possibility to propose asteroid-free regimen in selected patients with CV and to beonly treated with RTX warrants additional trials.

In conclusion, patients suffering from HCV-inducedvasculitis have and will largely benefit from the progressmade in both antiviral and immunologic research. It seemsthat the place of biologics in the management of this complexcondition is likely to increase in a near future.


The authors declare that they have no conflict of interests.

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Review Article

Rituximab-Based Treatment, HCV Replication, andHepatic Flares

Evangelista Sagnelli,1, 2 Mariantonietta Pisaturo,1, 2

Caterina Sagnelli,3 and Nicola Coppola1

1 Section of Infectious Diseases, Department of Public Medicine, Second University of Naples, 80131 Naples, Italy2 Division of Infectious Diseases, AORN Sant’Anna e San Sebastiano di Caserta, 81100 Caserta, Italy3 Department of Clinical and Experimental Medicine and Surgery “F. Magrassi e A. Lanzara”,Second University of Naples, 80131 Naples, Italy

Correspondence should be addressed to Evangelista Sagnelli, [email protected]

Received 26 April 2012; Accepted 20 June 2012


Copyright © 2012 Evangelista Sagnelli et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Rituximab, a chimeric mouse-human monoclonal antibody directed to the CD20 antigen expressed on pre-B lymphocytes andmature lymphocytes, causes a profound B-cell depletion. Due to its peculiar characteristics, this drug has been used to treatoncohaematological diseases, B cell-related autoimmune diseases, rheumatoid arthritis, and, more recently, HCV-associated mixedcryoglobulinaemic vasculitis. Rituximab-based treatment, however, may induce an increased replication of several viruses such ashepatitis B virus, cytomegalovirus, varicella-zoster virus, echovirus, and parvovirus B19. Recent data suggest that rituximab-basedchemotherapy induces an increase in HCV expression in hepatic cells, which may become a target for a cell-mediated immunereaction after the withdrawal of treatment and the restoration of the immune control. Only a few small studies have investigatedthe occurrence of HCV reactivation and an associated hepatic flare in patients with oncohaematological diseases receiving R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). These studies suggest that the hepatic flares arefrequently asymptomatic, but life-threatening liver failure occurs in nearly 10% of cases.

1. Introduction

Hepatitis C virus (HCV) is responsible worldwide for chron-ic hepatitis, liver cirrhosis, and hepatocellular carcinoma[1]. Besides its hepatotropic characteristics, HCV is also alymphotropic virus [2, 3] responsible for HCV-related B-cellnon-Hodgkin lymphoma (NHL) [4–6], immune-mediatedextrahepatic manifestations, mixed cryoglobulinaemic vas-culitis [7–12], and the presence in serum of rheumatoidfactor and autoantibodies [13–15].

In the last decade, rituximab, a chimeric mouse-humanmonoclonal antibody directed to the CD20 antigen expressedon pre-B lymphocytes and mature lymphocytes [16], hasbeen used increasingly for treating patients with haemato-logical diseases including CD20-positive B-cell NHL [17].Rituximab causes a profound B-cell depletion, peripheralblood B lymphocytes becoming undetectable after a single

infusion, with a complete B-cell recovery from 6 to 9 monthsafter the discontinuation of treatment [18, 19]. Due to itspeculiar characteristics, this drug has also been used fortreating B cell-related autoimmune diseases [20], rheuma-toid arthritis, and, more recently, HCV-associated mixedcryoglobulinaemic vasculitis [21, 22].

It is well known that rituximab-based chemotherapy isfrequently followed by a reactivation of viral infections andcorrelated diseases. A frequent increase in viral replicationunder R-CHOP (rituximab, cyclophosphamide, doxoru-bicin, vincristine, and prednisone) was demonstrated byAksoy et al. for several viruses including hepatitis B virus(HBV), cytomegalovirus, varicella-zoster virus, echovirus,and parvovirus B19 [23]. Worthy of mention is HBV infec-tion, both overt and occult, which in the absence of a specificprophylaxis or treatment frequently reactivates during orafter R-CHOP, with a mortality rate close to 20% and death


being due to liver failure or to an unfavourable progressionof the underlying haematological disease once R-CHOP hasbeen discontinued because of a hepatic flare [24–33].

1.1. Studies on HCV Reactivation and Hepatic Flares due toRituximab-Based Chemotherapy. According to the fragmen-tar data available, R-CHOP can induce an increase in HCVreplication in oncohaematological patients [34, 35], a bio-logical event associated to the development of hepatic flaresin some studies [36–41]. A reasonable explanation for thisassociation may be that R-CHOP induces an increase in HCVexpression in hepatic cells, which may become a target for acell-mediated immune reaction after the discontinuation oftreatment and the restoration of the immune control. Thishypothesis, however, requires support from further studieswith a longer follow up to clarify the relationship betweenHCV reactivation and the occurrence of a hepatic flare andto assess the clinical impact of flares.

The uncertainty dominating this topic arises from theinconsistent data available and from the marked differencesin the studies as regards the design, age of the patients, typeand stage of the oncohaematological diseases, type of chem-otherapy used, and stage of liver disease. In addition, thecriteria used to define HCV reactivation and a hepatic flarediffer from study to study.

Some of these papers are case reports. Akosy et al.described HCV reactivation not associated to a hepatic flarein a patient with HCV-related cirrhosis and NHL treated onlywith rituximab [34]. Nooka et al. published a similarobservation in a patient with diffuse large B-cell lymphoma(DLBCL) and HCV infection who experienced an asymp-tomatic HCV reactivation during R-CHOP [35]. Hsieh et al.described an HCV reactivation with a hepatic flare in apatient with DLBCL receiving R-CHOP [41], and Lake-Bakaar reported a case of HCV reactivation in a patientwith HCV-related mixed cryoglobulinaemia who developeda hepatic flare 2 weeks after starting rituximab treatment[42].

Only a few studies evaluated both HCV reactivation andthe development of a hepatic flare in small series ofpatients with oncohaematological diseases receiving R-CHOP (Table 1). In a prospective study on 8 anti-HCV/HCVRNA-positive patients undergoing chemotherapy, HCV rep-lication was determined both in plasma and peripheral bloodmononuclear cells (PBMC). In this study, Coppola et al.[40] found an increase in HCV RNA of at least 1.5 logIU/mL in plasma and of at least 1.1 log IU/mL in PBMC ofthe 7 patients receiving rituximab and corticosteroid-basedchemotherapy, whereas no change was observed in the onepatient treated with rituximab-sparing chemotherapy. In thisstudy, the patients with HCV reactivation showed a hepaticflare 3–5 months after treatment was discontinued; this waslife threatening only in one patient who had compensatedcirrhosis at the baseline and developed a grade-3 hepaticflare, ascites, and portosystemic encephalopathy with aprogression of Child-Pugh score from A6 to B9 (Table 1).

In a retrospective study on 131 patients with HCV infec-tion and NHL treated with rituximab and prednisone-basedchemotherapy, Ennishi et al. [36] described a hepatic flare

in 36 patients (27%), of these 131, 34 with DLBCL showedHCV reactivation during the follow up, but the prevalence ofhepatic flares in these 34 patients was not reported (Table 1).In nearly 10% of cases, the hepatic flares were life threateningand some patients died of liver failure. The retrospectivenature of the study, however, suggests caution in acceptingthese data as conclusive.

In a retrospective study, Marignani et al. [39] described 3patients with HCV infection and NHL treated with R-CHOPtherapy. Two of these patients experienced HCV reactivationand showed a hepatic flare, which was symptomatic in oneof the two, after chemotherapy was discontinued (Table 1).This patient showed an increase in serum total bilirubin upto 7.8 mg/dL and became asymptomatic in 4 weeks. The thirdpatient did not show an HCV reactivation but developed ahepatic flare after chemotherapy was discontinued.

Pitini et al. recently described HCV reactivation and ahepatic flare in 10 patients with HCV infection and NHLtreated with R-CHOP [38]. The hepatic flare was asymp-tomatic in 8 and symptomatic in 2 patients (Table 1): a 68-year-old male with DLBCL who developed HCV reactivationand a hepatic flare two weeks after the third cycle of R-CHOP and died of acute liver failure and a 65-year-oldfemale with DLBCL who developed severe ascitis after fourcycles of R-CHOP and died 1 week later for the uncontrolledprogression of the underlying oncohaematological disease.

Similar results were reported by Tsutsumi et al. [37] in aprospective study of 4 patients with HCV infection and NHLtreated with R-CHOP; all patients showed HCV reactivationand a hepatic flare during chemotherapy, but no severeclinical events occurred (Table 1).

In a retrospective study on 160 patients with Hodgkin’slymphoma and HCV infection, Arcaini et al. described hep-atotoxicity in 5 (17.9%) of 28 patients with B-cell lymphomatreated with R-CHOP [43]. For 25 patients in this study, 15treated with R-CHOP and 10 with CHOP, circulating HCVRNA was quantified at each cycle of chemotherapy; HCV-RNA quantification did not correlate to liver toxicity.

Petrarca et al. treated HCV-associated mixed cryoglob-ulinaemia with rituximab and found this treatment usefuland safe even in patients with a severe liver disease [44]; thepatients in this study, however, had a low degree of immunedepression and did not receive corticosteroids.

Worthy of mention is the literature showing a frequentoccurrence of hepatic flares in patients receiving high-dosecorticosteroids plus rituximab [36–40], probably because ofa cumulative effect of these drugs, which both induce anincrease in HCV replication, rituximab because of theimpairment of antibody production, [18, 19, 45] and corti-costeroids because of the enhancement of HCV entry to thehepatocytes as a consequence of an overexpression of specificreceptors on the surface of these cells [46, 47].

1.2. Other Studies. Visco et al. reported an associationbetween R-CHOP treatment for DLBCL and a mild increasein the liver enzyme level not requiring discontinuation oftreatment in 5 (14%) of 35 patients [48]; this study, however,gave no information on HCV reactivation.


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Coppola et al. described 28 anti-HCV/HCV RNA-nega-tive patients receiving chemotherapy (rituximab based in61% of cases) for oncohaematological diseases. Thesepatients remained HCV RNA-negative both in plasma andPBMC and no hepatic flare was observed over an observationperiod of 6–24 months [49], most probably because nopatient in this study had an occult HCV infection.

2. Conclusions

Fragmentary information from case reports, small studies,and retrospective investigations suggest, on the whole, thatrituximab-based chemotherapy favours viral replication inpatients with HCV infection and oncohaematological dis-eases. Only a few small studies evaluated both variations inthe HCV viral load and the development of a hepatic flareduring or after R-CHOP treatment, but the results areconcordant and strongly suggest a close association betweenthese two events. A possible interpretation of this associationis that R-CHOP induces an enhancement of HCV replicationfollowed by a spontaneous subsequent decrease once treat-ment is reduced or discontinued. The consequent restorationof the immune control may induce a hepatic flare of varyingclinical impact, that is, asymptomatic, symptomatic or lifethreatening, most probably reflecting the extent of cell-mediated hepatocellular necrosis. The clinical effects seemmore evident in patients treated with a combination ofrituximab and high-dose corticosteroids.

The high prevalence worldwide of HCV infection inoncohaematological patients and the progressive increase inthe use of R-CHOP for treating these diseases may create aheavy burden in the future for the health care authorities inseveral countries.

Acknowledgments

This study was supported by a grant from PRIN 2008,M.I.U.R., Rome, Italy “ottimizzazione della diagnosi eziolog-ica dell’epatite acuta c e studio dei fattori viro-immunologicidi guarigione, di cronicizzazione e di risposta alla terapia coninterferone” and in part by a grant from Regione Campania“Progetti per il miglioramento della qualita dell’assistenza,diagnosi e terapia del paziente affetto da AIDS nei set-tori: immunologia, coinfezioni, informazione e prevenzione2008”.

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Review Article

Morphologic Features of Extrahepatic Manifestations ofHepatitis C Virus Infection

Huaibin M. Ko, Juan C. Hernandez-Prera, Hongfa Zhu, Steven H. Dikman,Harleen K. Sidhu, Stephen C. Ward, and Swan N. Thung

The Lillian and Henry M. Stratton-Hans Popper Department of Pathology, Mount Sinai School of Medicine, New York,NY 10029, USA

Correspondence should be addressed to Huaibin M. Ko, [email protected]



Copyright © 2012 Huaibin M. Ko et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cirrhosis and hepatocellular carcinoma are the prototypic complications of chronic hepatitis C virus infection in the liver.However, hepatitis C virus also affects a variety of other organs that may lead to significant morbidity and mortality. Extrahepaticmanifestations of hepatitis C infection include a multitude of disease processes affecting the small vessels, skin, kidneys,salivary gland, eyes, thyroid, and immunologic system. The majority of these conditions are thought to be immune mediated.The most documented of these entities is mixed cryoglobulinemia. Morphologically, immune complex depositions can beidentified in small vessels and glomerular capillary walls, leading to leukoclastic vasculitis in the skin and membranoproliferativeglomerulonephritis in the kidney. Other HCV-associated entities include porphyria cutanea tarda, lichen planus, necrolyticacral erythema, membranous glomerulonephritis, diabetic nephropathy, B-cell non-Hodgkin lymphomas, insulin resistance,sialadenitis, sicca syndrome, and autoimmune thyroiditis. This paper highlights the histomorphologic features of these processes,which are typically characterized by chronic inflammation, immune complex deposition, and immunoproliferative disease in theaffected organ.

1. Introduction

Hepatitis C is a disease that affects approximately 170 millionpeople worldwide, with a prevalence in the United Statesof approximately 2% of the adult population [1]. Chronichepatitis C occurs in 80% of these cases and can leadto cirrhosis and hepatocellular carcinoma [2]. Extrahepaticmanifestations (EHMs) of hepatitis C virus (HCV) infectionwere first reported in the early 1990s [3] and can affecta variety of organ systems with significant morbidity andmortality. Forty to 75% of patients with chronic HCVinfection exhibit at least one clinical EHM [4, 5].

HCV infection is generally characterized by an indolentclinical course that is influenced by a variety of host, viral,and environmental factors [6]. While HCV may infectother cells outside of the liver, most EHMs are thoughtto be secondary to the host immune response to the viralinfection and not a direct viral cytopathic effect [7, 8]. Thenatural history of HCV infection and its association with

EHMs is only partially understood. Some EHMs, such asmixed cryoglobulinemia, have been strongly associated withhepatitis C both clinically and pathologically, while otherEHMs may be linked to HCV based on higher prevalence,response to antiviral treatment, or anecdotal observation.

2. Mechanisms

While direct infection of extrahepatic tissue cells by HCVhas been documented, the majority of EHMs are thoughtto be secondary to immune-mediated mechanisms, eitherlymphoproliferative or autoimmune in nature.

HCV infection results in upregulation of the humoralimmune system in patients with chronic disease, which leadsto increases in monoclonal and polyclonal autoantibodiesvia chronic antigenic stimulation [7]. It has been postulatedthat anti-HCV-IgG and HCV lipoprotein complexes may actas B-cell superantigens inducing the synthesis of non-HCV


reactive IgM with rheumatoid factor-like activity [9]. Theseautoantibodies, in turn, form immune complexes, whichcirculate through the body and are deposited in small tomedium blood vessels, resulting in complement activationand extrahepatic injury [7–9].

3. Mixed Cryoglobulinemia

HCV is associated with essential mixed cryoglobulinemia(MC), also known as type II cryoglobulinemia. MC is themost documented extrahepatic manifestation of chronicHCV infection and is found in more than half the patients[10–13]. Of these 10% are symptomatic [13, 14].

Cryoglobulins are circulating immunoglobulins thatprecipitate with cold temperature and resolubilize whenwarmed. In type II cryoglobulinemia, the cryoglobulins arecomposed of two or more classes of different immunoglob-ulins of which one is a monoclonal IgM component withrheumatoid factor-like activity [15]. Expansion of rheuma-toid factor synthetizing B cells represents the biologicalhallmark of MC [16]. Many organs including the skin,gastrointestinal tract, and kidney may be involved. Theclassic triad of symptoms in patients with HCV-associatedMC is palpable purpura, weakness, and arthralgia.

3.1. Palpable Purpura/Leukoclastic Vasculitis. Cutaneous vas-culitis of HCV-related MC, resulting in palpable purpura,is reported in 24–30% of cryoglobulin positive patients[4, 17]. It is secondary to small and/or medium vesselvasculitis with deposition of immune complexes in thesmall- and medium-sized dermal vessels [17]. It occursintermittently, preferentially during the winter months, andis nonpruritic. It characteristically begins with involvementof the lower limbs and moves cranially toward the abdomen,less frequently involving the trunk and upper limbs. Theface is always spared. The purpura is papular or petechialand persists for 3–10 days with residual brown pigmentation.In addition, Raynaud syndrome and acrocyanosis are foundin 25–34% of patients [18]. Cutaneous biopsy shows anonspecific mixed inflammatory infiltrate (leukocytoclasticvasculitis) involving small vessels (Figure 1). Mononuclearcells may be seen within the walls of the vessels, and, insome cases, endovascular thrombi and fibrinoid necrosis ofthe arteriolar walls may be seen (Figure 2).

3.2. Membranoproliferative Glomerulonephritis. Glomeru-lonephritis (GN), specifically, type I membranoprolifera-tive glomerulonephritis (MPGN) is a common presenta-tion of type II cryoglobulinemia in patients with chronicHCV infection. Patients may present with proteinuria andnephrotic syndrome [19–21]. On biopsy, a type I membra-noproliferative glomerulonephritis is seen, sometimes withpronounced lobulation of the glomeruli [22]. There may bemassive infiltration of the glomeruli by monocytes as wellas diffuse thickening of the glomerular capillary wall [23].Periodic acid-Schiff (PAS) positive “hyaline thrombi” canbe seen within the capillary lumina (Figure 3). The lightmicroscopy appearance may also appear as type III MPGN,

Figure 1: Leukocytoclastic vasculitis: predominantly lymphocyticmixed inflammatory infiltrate involving small vessels in the dermis(hematoxylin-eosin, original magnification ×200).

Figure 2: Leukocytoclastic vasculitis: fibrinoid necrosis of dermalvessels (hematoxylin-eosin, original magnification ×100) (photocourtesy of Dr. Rajendra Singh).

acute exudative and proliferative glomerulonephritis. MPGNmay be indistinguishable from allograft glomerulopathy intransplant patients [24]. As in other organs affected by mixedcryoglobulinemia, leukoclastic vasculitis can be seen in thekidney.

On immunofluorescence (IF) studies, coarsely granulardeposits of IgG, IgM, and C3 are visualized in the capillarywall. On occasion, large intraglomerular deposits of C3and other immunoreactants are seen forming “thrombi”within the glomerular capillaries [22]. Morphologically, thelocation and presence of the deposits is best seen on electronmicroscopy (EM). EM shows dense, immune-type mesangialand subendothelial deposits along the glomerular capillarywalls (Figure 4). At high magnification, the cryoglobulindeposits often appear as organized tubular, cylindrical, orcrystalloid deposits (Figure 5) [25]. Intramembranous andsubepithelial deposits may be seen rarely, in addition to thesubendothelial deposits [23, 25]. EM is useful in determiningthe presence and site of the deposits, which may be difficultto determine by IF [26].

4. Skin (Not Associated withMixed Cryoglobulinemia)

4.1. Porphyria Cutanea Tarda. The prevalence of HCVinfection in patients with porphyria cutanea tarda (PCT)varies according to region [9, 27]. The prevalence is higher insouthern Europe (65%–91%) compared to northern Europe(8–17%). In Australia and New Zealand, the prevalence is


Figure 3: Membranoproliferative glomerulonephritis: PAS-positive“hyaline thrombi” seen within the capillary lumina (PAS, originalmagnification ×400).

Figure 4: Membranoproliferative glomerulonephritis: endocapil-lary proliferation with extensive subendothelial deposits along theglomerular capillary walls. Mesangial deposits are present as well(electron microscopy).

about 20%; while, in the United States, the prevalence isreported to be 50–75% [27]. PCT results from decreasedactivity of the uroporphyrinogen decarboxylase enzyme;however, the mechanism that links this phenomena tochronic HCV infection is unknown. In most cases, HCVexposure and liver dysfunction precede the onset of PCT,suggesting that the HCV infection may uncover an existingporphyrin metabolism defect in susceptible patients. His-tologically, PCT is characterized by cell poor subepidermalbulla with increased hyaline material in the vessel walls andbasement membrane. The hyaline material is reactive in PAS-diastase-resistant staining [9]. The dermal papillae are rigidwith festooning (Figure 6).

4.2. Lichen Planus. Lichen planus (LP) is a relatively com-mon inflammatory skin disease in the general populationand is thought to be related to autoimmunity [28]. Therelationship between HCV infection and LP is controversial;however, literature analysis has found that, in most studies,the proportion of HCV-positive patients is higher in theLP group compared to the general population with theprevalence of HCV ranging from 16% to 55% and 1-2%, respectively [5, 29–31]. HCV-related LP lesions aresimilar to those of classic LP with the exception of oral

Figure 5: Membranoproliferative glomerulonephritis: subendothe-lial electron dense deposits in a patient with mixed essential cryo-globulinemia showing microtubular architecture. Microtubulesmeasure approximately 30 nm in diameter (electron microscopy).

Figure 6: Porphyria cutanea tarda: subepidermal bulla and festoon-ing of the dermal papilla are prominent. There is no significantinflammatory infiltrate (hematoxylin-eosin, original magnification×100).

involvement, which also occurs in the majority of HCV-related LP. Histologically, LP is characterized by band-like,subepidermal, lymphohistiocytic infiltrate with interfacechange, “sawtooth” rete ridges, and pigmentary continence(Figure 7) [9].

4.3. Necrolytic Acral Erythema. Since its initial descriptionin 1996, necrolytic acral erythema has been described asa dermatosis, which is almost exclusively associated withHCV infection [32]. It is characterized by pruritic, sym-metric, well-demarcated, hyperkeratotic, erythematous-to-violaceous, lichenified plaques with a rim of dusky erythemaon the dorsal aspects of the feet and extending to the toes.Disease remission has been described after oral zinc admin-istration. Thus, it is thought that zinc dysregulation, which


Figure 7: Lichen planus: there is a band-like infiltrate of lympho-cytes at the epidermal-dermal junction with damage to the basal celllayer and pigment incontinence. The epidermis has a saw-toothedappearance (hematoxylin-eosin, original magnification ×200).

Figure 8: Necrolytic acral erythema: epidermal pallor in thestratum corneum, hyperkeratosis, dyskeratotic keratinocytes, spon-giosis, and a superficial perivascular mixed inflammatory infiltratein the dermis are seen (hematoxylin-eosin, original magnification×200) (photo courtesy of Dr. Rajendra Singh).

can occur in hepatitis C, is related to the pathogenesis of theselesions [33]. Morphologic features include a nonspecific pso-riasiform pattern, acanthosis, papillomatosis, and hyper- andparakeratosis, with necrotic keratinocytes in the superficialepidermis. There is a superficial, perivascular inflammatoryinfiltrate comprised predominantly of lymphocytes. Somelymphocytes extend to a hyperplastic epidermis where thereis spongiosis and foci of sharply demarcated parakeratosis(Figure 8) [34].

5. Kidney (Not Associated withMixed Cryoglobulinemia)

5.1. Membranous Glomerulonephritis. Membranous GN mayalso occur in the setting of chronic HCV infection [19, 22]. Incontrast to patients with HCV and MPGN, there is little evi-dence linking MGN to cryoglobulinemia. Patients with HCVand MGN do not appear to demonstrate cryoglobuline-mia or rheumatoid factor [35], and hypocomplementemiais rarely found [36, 37]. Typically, early-stage MGN isseen, with no evidence of endocapillary proliferation [22].Focal segmental glomerulosclerosis may be present [36].IF studies show diffuse glomerular basement membrane

granular deposits of IgG [37]. Membranous GN appears assubepithelial electron dense deposits on EM [36, 37].

5.2. Diabetic Nephropathy. Chronic infection with HCV isassociated with insulin resistance (see the following). Thegross and microscopic features of HCV-associated diabeticnephropathy are the same as diabetic nephropathy not asso-ciated with HCV. The kidneys may be enlarged in the earlystages of disease due to hyperfiltration and hypertrophy ofthe glomeruli within the cortex. As the disease progresses, thekidney becomes scarred, with loss of nephrons, and decreasesin size. However, end-stage disease does not commonlyshow gross contraction [38]. The corticomedullary junctionarteries may be prominent due to arteriosclerosis, and themain renal artery may show atherosclerosis [22].

The microscopic features include diffuse mesangial scle-rosis with thickening of glomerular capillary walls andthickening of the tubular basement membranes. Nodularlesions, characterized as eosinophilic material within themesangium first described by Kimmelstiel and Wilson in1936, may be seen (Figure 9) [39]. Microaneurysms ofglomerular capillary loops may precede the developmentof large nodules [22, 40]. On IF, linear staining along theglomerular capillary walls with IgG is seen [40, 41].

Exudative lesions, otherwise known as hyalinosis lesionsor fibrous caps, are seen in 60% of diabetic kidneys and maybe secondary to ischemia due to atherosclerosis [40]. Theselesions consist of PAS-positive eosinophilic material thataccumulates between endothelial cells and the glomerularbasement membrane of the capillary loops, eventually fillingthe lumen of the capillaries [42, 43]. Hyalinosis lesionsstain brightly on IF for IgM and C3 [41]. Hyalinosis lesionsmay also contain fibrinogen, lipoprotein, complement, β-lipoprotein, and small amounts of IgG [41]. “Capsular drop”is a lesion that stains similarly to hyalinosis lesions in thekidney. Capsular drop is identified as a round accumulationof eosinophilic material between the basement membraneand the parietal epithelial cells of Bowman capsule [43].Adhesions between the glomerular lobule and Bowmancapsule may be observed [22].

Other nonspecific findings that are associated withdiabetic renal disease include hyaline arteriolosclerosis, inter-stitial fibrosis, chronic inflammatory infiltrates, and obsoleteglomeruli [22].

6. Hematologic

6.1. B-Cell Non-Hodgkin Lymphoma. HCV is the stimulusnot only for the apparent benign lymphoproliferative processunderlying a wide spectrum of clinical features but also forthe progression to frank lymphoid malignancy in a subgroupof patient [44]. Associations between chronic HCV infectionand lymphoproliferative disorders have been described [45].In patients with B-cell non-Hodgkin lymphoma (NHL),up to 13% have the HCV antibody [46, 47]. In addition,approximately 10% of patients with type II cryoglobulinemiaassociated with HCV developed NHL over a 10-year follow-up period [17]. It is believed that HCV E2 antigen binding


Figure 9: Diabetic Nephropathy: extensive mesangial expansion is seen, with rounded acellular mesangial nodules (Kimmelstiel-Wilsonnodules) (hematoxylin-eosin and PAS, original magnification ×400).

to host CD81 receptors leads to B-cell proliferation, whichmay result in lymphoma [46, 48]. Regression of low-gradeNHL has been observed in association with HCV therapy[49, 50]; however, high-grade HCV-associated lymphomarequires chemotherapy.

The majority of HCV-associated lymphomas are extra-nodal and located in the liver (primary hepatic lymphoma)and salivary glands [49, 50]. The bone marrow and spleenmay also be involved [51, 52].

Morphologically, HCV-associated lymphomas representa variety of histological subtypes. Marginal zone, lympho-plasmacytic, and diffuse large B-cell lymphomas are themost common histotypes associated with HCV [53, 54].HCV has also been associated with follicular lymphoma [55,56] as well as mucosa-associated lymphoid tissue (MALT)lymphoma, and mantle cell lymphoma [54].

Overall, marginal zone lymphoma appears to be themost frequently encountered low-grade B-cell lymphoma inHCV patients [52]. HCV infection is documented in approx-imately 35% of patients with nongastric B-cell marginalzone lymphoma [53]. Splenic marginal zone lymphoma, inparticular has a high prevalence of HCV infection and isoften associated with type II cryoglobulinemia [51, 53, 57].Morphologically, a central zone of small round lymphocytessurrounds the germinal centers, commonly replacing thereactive germinal centers in the splenic white pulp. The redpulp is infiltrated with small lymphocytes and ill-definednodules of larger cells (Figure 10) [58]. The tumor cells stainpositively for CD20, CD79a, and BCL2 in the majority ofcases. They are negative for CD5, CD10, CD23, and annexinA1 [58].

HCV-associated lymphoplasmacytic lymphoma (LPL)has been associated with type II cryoglobulinemia in somestudies and may be related to geographic location [58].Morphologically, LPL presents as a relatively monotonousproliferation of small lymphocytes, plasma cells, and plasma-cytoid lymphocytes. Dutcher bodies (plasma cells with PAS+intranuclear inclusions) and mast cells may be seen [58].

Primary hepatic diffuse large B-cell lymphoma (DLBCL)is also associated with HCV. DLBCL may present onhistology as large lymphoid cells with vacuolated nuclei in a

diffuse infiltrating pattern intermingled with small lymphoidcells. The large cells typically stain positively for CD20,CD10, and CD25 [59]. On occasion, NHL may be seenconcurrently with hepatocellular carcinoma [60].

7. Autoimmune/Inflammatory

7.1. Type 2 Diabetes Mellitus. Chronic infection with HCV isassociated with insulin resistance, metabolic syndrome, andtype 2 diabetes [61]. The prevalence of type 2 diabetes hasbeen reported to be 14–50% in patients with chronic HCVinfection [62]. HCV-associated diabetes is characterized byinsulin resistance and does not appear to be associatedwith antibodies directed towards the beta cells in the isletsof Langerhans [63]. The mechanism for insulin resistanceis unclear, but it is thought to be secondary to viralinduced adipocytokine release or HCV viral proteins directlyinterfering with inflammatory or muscle insulin signalingpathways [64]. HCV-related type 2 diabetes mellitus occursin association with hepatic steatosis, insulin resistance, andhigh levels of both tumor-necrosis factor and CXCL10 [65].

7.2. Sialadenitis/Sicca Syndrome. The association betweensialadenitis and HCV infection was first postulated in 1992,and the reported occurrence of HCV-related sicca syndromeranges from 4 to 57% of chronic HCV patients [4, 61, 66, 67].The large range may be related to differences in diagnosticcriteria [61]. The mechanism by which HCV results in siccasyndrome is not well established. The virus has not beenshown to directly infect salivary gland tissue [68], and itis likely that HCV-related sicca syndrome is the product ofhost immune-mediated mechanism, rather than direct viraleffect [69]. Patients may present with oral or ocular dryness.Histologic examinations of salivary gland biopsies in HCV-infected patients show pericapillary and nonpericanalarylymphocytic infiltration. The glandular canals are typicallyspared (Figure 11) [61].

7.3. Autoimmune Thyroiditis. Autoimmune thyroid diseaseis commonly associated with HCV. Hypothyroidism is


Figure 10: Marginal zone lymphoma of the spleen: (A) there is effacement of splenic architecture by sheets of monotonous small-to-mediumsize lymphocytes (hematoxylin-eosin, original magnification ×200). Immunohistochemical stains show that the lymphocytes are positivefor BCL2 (B) and CD20 (C), and negative for CD10 (D) (immunoperoxidase, original magnifications ×200 ((A) through (D))).

Figure 11: Sialadenitis: there is extensive lymphoid infiltrate withinterstitial fibrosis and acinar atrophy (hematoxylin-eosin, originalmagnification ×100).

seen in 3.5–13% of patients with chronic HCV [70, 71].Patients generally present with the most common casesof autoimmune thyroid disease: Graves’s disease (GD)and Hashimoto’s thyroiditis (HD). The pathogenesis ofHCV-related autoimmune thyroid disease is unknown. Twohypotheses have been proposed: primary viral cytopathiceffect and secondary induced autoimmunity [72]. Interferontherapy may also induce antithyroid antibodies or uncoverunderlying Hashimoto’s thyroiditis or Graves’s disease,which can be refractory to discontinuation of therapy[71, 73].

In the early stages of Hashimoto’s thyroiditis, thethyroid is firm, symmetrically enlarged, and has a tan-yellow appearance corresponding to lymphoid tissue on

gross examination. The gland may become atrophic in end-stage disease. Histological findings include small, atrophicthyroid follicles with lymphoplasmacytic infiltration andwell-developed germinal centers (Figure 12) [26]. The lym-phocytic infiltrate is composed of mixed T and B cells in aneven ratio [26].

In Graves’s disease, the thyroid has marked vascularityand is diffusely enlarged. Histologic examination showsprominent vascular congestion, follicular hyperplasia, andpapillary hyperplasia. The follicular cells appear columnarwith enlarged nuclei that may demonstrate nuclear clearing,mimicking papillary carcinoma (Figure 13) [26]. Reactivelymphocytes are found in the stroma [74].

8. Conclusion

Chronic hepatitis C virus infection is associated with mul-tiple extrahepatic manifestations (EHMs) affecting variousorgans in the body. While there is some evidence thatthe virus may play a direct role in HCV-related B-celllymphomas via direct HCV antigen stimulation of B-cells,most EHMs are generally believed to be secondary to the hostimmune response to the virus.

In some conditions, the histopathologic changes of EHMare related to circulating immune complexes such as typeII cryoglobulinemia, and their subsequent deposition inthe small vessels and glomerular capillary walls, leading toleukoclastic vasculitis in the skin and membranoproliferativeglomerulonephritis in the kidney.

Other HCV-associated entities like sialadenitis, sicca syn-drome, lichen planus, and autoimmune thyroiditis, while notassociated with cryoglobulinemia, appear to be secondary


Figure 12: Hashimoto thyroiditis: there is extensive lymphocyticinfiltrate with germinal center formation. Follicular cells are slightlyenlarged with partial nuclear clearing (hematoxylin-eosin, originalmagnification ×100).

Figure 13: Graves disease: there is follicular hyperplasia with intra-cellular colloid droplets, cell scalloping, a reduction in follicularcolloid, and a multifocal lymphocytic infiltrate (hematoxylin-eosin,original magnification ×200).

to autoimmune processes resulting in chronic inflammatoryinfiltrates.

In porphyria cutanea tarda, the disease process is thoughtnot to be related to host immune response to HCV, but ratherto HCV-associated liver dysfunction.

The role of the virus in insulin resistance in HCV-associated diabetes is unclear, but it is thought to besecondary to either viral induced inflammation or directinterference of the virus on muscle insulin signaling.

In summary, chronic HCV infection may result in a mul-titude of disease processes affecting the small vessels, skin,kidneys, salivary glands, eyes, thyroid, and immunologicsystem. The sequelae of extrahepatic HCV infection are seenhistomorphologically as chronic inflammation, immunecomplex deposition, and immunoproliferative disease in theaffected organs.

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Review Article

HCV-Related Nervous System Disorders

Salvatore Monaco, Sergio Ferrari, Alberto Gajofatto,Gianluigi Zanusso, and Sara Mariotto

Section of Neuropathology, Department of Neurological, Neuropsychological, Morphological and Motor Sciences,University of Verona, Verona 37134, Italy

Correspondence should be addressed to Salvatore Monaco, [email protected]



Copyright © 2012 Salvatore Monaco et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Chronic infection with hepatitis C virus (HCV) is associated with a wide spectrum of extrahepatic manifestations, affectingdifferent organ systems. Neurological complications occur in a large number of patients and range from peripheral neuropathy tocognitive impairment. Pathogenetic mechanisms responsible for nervous system dysfunction are mainly related to the upregulationof the host immune response with production of autoantibodies, immune complexes, and cryoglobulins. Alternative mechanismsinclude possible extrahepatic replication of HCV in neural tissues and the effects of circulating inflammatory cytokines andchemokines.

1. Introduction

Chronic infection with hepatitis C virus (HCV), a hepa-totropic and lymphotropic agent, is a growing global healthissue affecting an estimated 170 million people [1]. Inaddition to be a leading cause of chronic hepatitis, cirrhosis,and hepatocellular carcinoma (HCC), chronic HCV infec-tion has been associated with more than 30 extrahepaticmanifestations (EHMs), affecting a large proportion ofinfected patients [2]. Many EHMs, including a number ofneurological conditions, are immunologic/rheumatologic innature, as a consequence of B-cell proliferation with ensuingproduction of monoclonal and polyclonal autoantibodiesdisplaying rheumatoid factor activity or cryoglobulin prop-erties [3]. In addition, lines of evidence suggest that thebrain, but not peripheral nerves or skeletal muscles, is a per-missive site for viral replication, as evinced from quasispeciesanalysis and the detection of replicative intermediate formsof HCV RNA and viral proteins within the central nervoussystem (CNS) [4]. Additional mechanisms, contributing toneurological dysfunction, are possibly related to the effect ofcirculating inflammatory cytokines and chemokines reach-ing brain tissues across altered sites of the blood-brain bar-rier. Cryoglobulinemia, the most frequent and best-studied

EHM of HCV infection, is detected in up to 50% of HCV-infected patients, inducing symptomatic disease in nearly15% of cases. Cryoglobulins (CGs) are cold-precipitableimmunoglobulins, which, following vascular deposition,elicit inflammation and occlusion of small- and medium-size blood vessels. Three types of CG are recognized, eitheressential or secondary to autoimmune disorders, chronicinfections, and lymphoproliferative diseases [5]. Type I CG(10–15% of cases) is monoclonal Ig; type II CG, a mixtureof monoclonal Ig rheumatoid factor and polyclonal IgG,accounting for 50–60%, is typically found in patients withchronic HCV infection or primary Sjogren syndrome; typeIII CG, polyclonal IgG and IgM rheumatoid factor, is seenin lymphoproliferative disorders, chronic infections, andautoimmune diseases [6]. Up to 95% of type II and III CG,or “mixed CG” (MC), are associated with chronic HCV/HIVinfection. Mechanisms of CG-induced ischemic tissue dam-age are secondary to lymphocytic microvasculitis and/ornecrotizing arteritis, with transmural fibrinoid necrosis,thrombotic lumen occlusion, and polymorphonuclear cellinfiltration. Typical clinical manifestations of symptomaticCG include cutaneous purpura, arthralgias, peripheral neu-ropathy, and membranous proliferative glomerulonephritis[5, 7]. About 17–60% of patients with CG develop peripheral


neuropathy, often at disease onset, while CNS involvementoccurs in approximately 6% of cases. In addition to causingvascular damage, CGs represent an independent risk factorfor carotid plaque formation, hepatic fibrosis, and liversteatosis. Less frequent EHMs of HCV infection, representingpotential threats for neurological involvement, include non-Hodgkin’s lymphoma, diabetes, thyroid abnormalities, andrheumatological diseases. Importantly, most of EHMs mayimprove or even resolve after antiviral treatment, especiallyin subjects attaining a sustained virological response. Thespectrum of CNS and neuromuscular disorders associatedwith chronic HCV infection is reported in Table 1.

2. Neurological Manifestations

HCV-related CNS complications encompass a wide spec-trum of disorders ranging from cerebrovascular eventsto autoimmune syndromes. However, their relatively lowfrequency, in addition to the heterogeneity of neurologicalmanifestations, and the paucity of pathological observations,largely preclude the achievement of reliable information as tothe pathogenesis of different syndromes.

Acute cerebrovascular events, including ischemic stroke,transient ischemic attacks, lacunar syndromes, or rarelyhemorrhages, have been reported in HCV-infected patients[8–10], being the initial manifestation of HCV infection insome cases [11]. The occurrence of occlusive vasculopathyand vasculitis are well-known events [12, 13]. IsolatedCNS vasculitis has been coupled with angiographic evi-dence of multiple focal narrowing of cerebral arteries, andfull recovery has been achieved with corticosteroids andcyclophosphamide [14]. In some patients, CNS ischemicchanges may occur in the setting of an antiphospholipid-associated syndrome [15], or in association with antineu-trophil cytoplasmic antibodies. Recently, HCV has beenconnected with the metabolic syndrome and evidence hasbeen provided that HCV infection represents an independentrisk factor for increased carotid wall thickness and plaqueformation, thus contributing to significant cerebrovascularmortality, especially in patients with elevated HCV-RNAlevels [16].

Acute or subacute encephalopathic syndromes, clinicallycharacterized by cognitive impairment, confusion, alteredconsciousness, dysarthria, dysphagia, and incontinence, havebeen associated with diffuse involvement of the white matterin HCV chronically infected patients with CG and/or circu-lating anticardiolipin antibodies. An ischemic pathogenesisof these rapidly evolving syndromes is supported by MRIfindings showing small lesions in subcortical regions andperiventricular white matter. Moreover, severe and diffuseinfra- and supratentorial white matter alterations, highlysuggestive of vasculitis, are observed in subjects with coin-cidental systemic vasculitis. Pathological evidence of CNSvasculitis-induced ischemic damage was first provided in apatient with MC, peripheral neuropathy, and relapsing mul-tiinfarct encephalopathy [17]; in this case, neuropathologicalexamination showed multiple ischemic lesions, 0.5–3 mm indiameter, in the white matter of cerebral hemispheres and

Figure 1: Axial FLAIR MRI of the brain showing periventricularhyperintense areas in a patient with chronic HCV infection andcognitive changes.

cerebellum, and parenchymal infiltration and accumulationof lymphocytes around small vessels. The occurrence ofpossible vasculitis-induced ischemic changes has been alsoclaimed in a patient with chronic HCV infection, MC,Sjogren syndrome, and sensory neuropathy, who developedskin vasculitis and leukoencephalopathy over a three-yearperiod [18]. Taken together, these reports suggest that activeHCV infection can be responsible for acute/subacute white-matter involvement, when associated with CG, coagulationdisorders, or systemic vasculitis.

In addition to encephalopathic syndromes, slowly evolv-ing cognitive decline, clinically characterized by impair-ment of attention, executive, visual constructive, and spa-tial functions, has been correlated to an increased occur-rence of periventricular white matter high intensity signals(WMHISs) on T2-weighted MRI [19]. In these patients,a relationship between CG level and number of impairedcognitive functions was observed, while no correlationwas found with systemic manifestations of CG, includingperipheral neuropathy. WMHIS changes likely reflect theoccurrence of small vessel disease, which leads to chronichypoperfusion of the white matter and local alteration of theblood-brain barrier in anatomical regions where precapillaryarterioles are widely spaced and present a poor anastomosingnetwork (Figure 1).

The spectrum of CNS syndromes encountered in HCVpatients is not limited to the foregoing vasculitic and vas-culopathic forms, but also includes inflammatory disorders,such as acute encephalitis, encephalomyelitis, and meningo-radiculitis/polyradiculitis. There are reports of patients withrapidly evolving leukoencephalitis with microglial nodulesand perivascular T-cell infiltrates in association with HCV


Table 1: HCV-associated CNS and neuromuscular syndromes.

Disorders Clinical Features

Neurological

Stroke, TIA, lacunar syndromes Focal signs

Acute encephalopathic forms Confusion, altered consciousness, incontinence

Leukoencephalopathy Multifocal signs and symptoms, cognitive dysfunction, tetraparesis, aphasia

Encephalomyelitis Motor, sensory and sphincter deficits, seizures

Myelitis Sensory ataxia, spastic paraplegia

Cognitive/Neuropsychiatric

Fatigue Sensation of physical and mental exhaustion

Psychiatric disorders Depression, anxiety

Cognitive dysfunctionAlterations in verbal recall, working memory, sustained attention, concentration,

learning skills

Peripheral Neuropathies

Sensorimotor axonal polyneuropathies Sensory loss, distal weakness

Large fibres sensory neuropathies Reduced touch and proprioception sensations, sensory ataxia

Small fibres sensory neuropathies Burning feet, pain, restless legs syndrome

Motor axonal polyneuropathies Distal weakness

Mononeuropathies Deep aching pain, truncular deficits

Mononeuropathy multiplex Stocking-glove asymmetric neuropathy

Demyelinating forms Sensory loss, distal weakness, areflexia

Myopathies

Noninflammatory Progressive proximal/generalized weakness, atrophy

Inflammatory Progressive symmetrical proximal weakness, atrophy, dysphagia, interstitial lung disease

genome presence [20] or fatal progressive encephalomyeliticsyndromes, pathologically characterized by neuronal lossand perivascular lymphocyte cuffing in the brainstem andcervical spinal cord [21]. In these cases, available evidencesuggests the occurrence of an immune-mediated processinduced by HCV, rather than a direct effect of the virus.

Sacconi et al. described a patient with acute disseminatedencephalomyelitis (ADEM), an autoimmune postinfectiousCNS disease, developing after HCV infection and responsiveto steroid therapy, further supporting the role of cellularimmunomediated mechanisms in CNS complications ofHCV infection [22]. Chronic HCV infection may also inducehumoral-mediated demyelination, sequentially or simulta-neously involving the CNS and PNS. Recurrent episodesof CNS and PNS demyelination suggestive of antibody-mediated autoimmunity or, in alternative, of a direct cyto-pathic effect of the virus, have been reported in a patient withactive HCV replication [23]. Relapsing forms of central andperipheral demyelination, worsened by interferon treatment,have also been described [24].

Additional examples of HCV-triggered demyelination areobserved in patients with myelitis. Myelitis is considered aninfrequent neurological complication in patients chronicallyinfected with HCV, although available evidence suggests thata high percentage of patients with recurrent inflammatorytransverse myelitis, but not monophasic myelitis, test positiveto anti-HCV antibodies and have serum HCV-RNA [25].HCV-related myelitis occurs acutely [26] or subacutely [27,28], the neurological presentation ranging from transverse

myelitis to acute partial transverse myelopathy, sensoryataxia, or spastic paraplegia; many patients present a recur-rent course and have a multisegmental spinal involvementat MRI, usually at cervical and thoracic levels (Figure 2).Notably, patients with negative imaging have been reported.A common feature of HCV-associated myelitis is the pres-ence of circulating anti-HCV antibodies, but not serumCG or HCV-RNA sequences in the CSF. Neuropathologicalexamination of spinal cord has been performed in a fewsubjects. Biopsy-proven acute demyelination, accompaniedby parenchymal and perivascular infiltration of macrophagesand lymphocytes, but not vasculitis, has been reported ina 46-year-old man with a history of recurrent myelitisand chronic HCV infection [29]. Biopsy specimens of thespinal cord were negative for HCV antigens and HCV-RNA; on the contrary, anti-HCV antibodies, but not HCV-RNA, were found in the CSF. Necrotic changes in asso-ciation with proliferation of hyalinized small vessels andinfiltration by macrophages and T lymphocytes, mimickingneuropathological features of Sjogren syndrome, have beenreported in spinal cord biopsies of a patient with stepwiseprogressive longitudinal myelopathy and chronic HCV infec-tion, suggesting an ongoing immune-mediated and ischemicpathogenesis [30].

3. Cognitive/Neuropsychological Symptoms

More than half of patients with chronic HCV infection com-plain of “brain fog” (fatigue, impaired concentration, and


(a)

(b)

Figure 2: Sagittal (a) and axial (b) T2-weighted MRI sequencesdisclose cervical spinal cord hyperintensity in a patient with HCV-related myelitis.

poor memory) and have a reduced quality of life, regardlessof the severity of liver involvement or virus replication rate.Fatigue, cognitive dysfunction, and mood alterations displaya profound effect on social and physical functioning, thusfurther impacting health-related quality of life (HRQL). Inearlier studies, fatigue was shown to have a major functionalrole in patients with chronic HCV infection [31]. Chronicfatigue is perceived as a sensation of physical and mentalexhaustion, and, when severe, it is accompanied by deficits ofattention tasks, anomia, and word-finding difficulties, in theabsence of verbal memory or cognitive ability impairments.In addition, some HCV patients with severe fatigue alsocomplain of muscle and joint pain, sleep disturbances,restless leg syndrome, headache, and depression. Alterationsin brain metabolism and neurotransmission, responsiblefor dysfunction of the ascending reticular activating sys-tem, putamen, globus pallidus, and the limbic system, areassociated with chronic fatigue. Forton et al. [32] usingmagnetic resonance spectroscopy (MRS) found elevatedcholine/creatine (Cho/Cr) ratio in the basal ganglia andfrontal white matter of HCV-infected patients, which wasnot related to the degree of liver disease, viral genotype, orother factors. These authors first suggested that the abovechanges were secondary to microglial activation, as an effectof HCV brain infection or peripheral cytokines. On theother hand, Weissenborn et al. [33] showed a decrease ofthe N-acetyl-aspartate(NAA)/Cr ratio in the frontal greymatter of HCV-patients, but no changes of the Cho/Crratio. Both findings have been confirmed using a differentapproach for MRS analysis, suggesting the occurrence ofincreased cell membrane turnover and decreased neuronalfunction [34]. More recently, the study of 53 HCV-positivepatients with mild liver involvement and neuropsychiatric

symptoms, disclosed increased Cho and myo-inositol con-centrations in basal ganglia and white matter and increasedCr, NAA, and N-acetyl-aspartyl-glutamate in basal ganglia[35], findings consistent with HCV-induced chronic cellularinflammation. A neurochemical basis for chronic fatiguein HCV-infected patients was suggested by the observationthat treatment with ondansetron, a competitive antagonistof serotonin receptors, was effective in ameliorating fatiguein a patient with HCV infection. A significant improvementof the fatigue and depression scores with ondansetronwas also found in a placebo-controlled randomized studyinvolving 36 patients with chronic HCV infection [36].These findings further support a major role for serotonin-ergic pathway dysfunction in causing fatigue and are inkeeping with data showing decreased serum tryptophanlevels and related reduction in serotonin synthesis [37,38]. Alterations in mesencephalic/hypothalamic serotonin(SERT) and striatal dopamine transporter (DAT) bindingcapacity have been documented by single-photon emissiontomography (SPECT) in HCV patients with pathologicalperformance at psychometric tests [39]. More recently, aninvestigation of 15 HCV patients reporting neuropsychiatricsymptoms was performed by combining neuropsychologicaltests, 18F-fluoro-desoxy-glucose (FDG) positron emissiontomography (PET), and SERT; results showed significantreduction in striatal and midbrain dopamine availabilityand reduced metabolism in limbic, frontal, parietal, andtemporal cortices, confirming a major role for defectivedopaminergic transmission in causing cognitive impairmentin HCV-infected patients [40].

Among EHM affecting HRQL, sexual dysfunction andpsychiatric disturbances play a major role. Most HCVpatients present depression and anxiety, and, using DSM-IV criteria, it has been found that 28% of chronically HCV-infected subjects have depression. In addition, about 15%of patients may suffer of recurrent brief depression [41], acommon subtype of cognition-impairing affective disorder,sharing many indicators with major depressive disorder,except the duration of depressive episodes. The occurrenceof depression has been attributed to psychological factors,or to specific determinants, including immune mechanisms,derangement of the blood-brain-barrier integrity, viralreplication within the CNS, iatrogenic factors, or altereddopaminergic and serotoninergic transmission. Admittedly,mental instability and depression are factors limiting theadherence to specific antiviral treatment, such as conven-tional interferon formulations or pegylated interferons [42].While the emergence of mild depression during interferontreatment can be safely managed with antidepressant at lowdoses, in moderate to severe depression, which affects upto 44% of patients in antiviral therapy, is mandatory toreduce or discontinue interferon treatment, especially in thepresence of active suicidal ideation. Proposed neurobiolog-ical mechanisms of interferon-induced depression remainto be elucidated, although changes in the hypothalamic-pituitary adrenal axis and in the central catecholamine andserotonin systems, as well as downregulation of serotoninsynthesis, have been claimed. However, these mechanismsremain putative and additional studies are warranted [43].


To date, cognitive impairment in chronic HCV infectionhas been reported in most studies, with the exception oftwo reports, showing unimpaired cognitive performancedespite impaired quality of life in patients enrolled afterdiagnosis at blood donation [44], and no deficits in attention,adaptive behavior, and intelligence in young HCV-positivechildren and adolescents with hemophilia [45]. Impairedability in sustained attention and decreased concentrationand psychomotor speed were described earlier [33, 46],although these changes are also found in mildly fatiguedpatients. Fontana et al. [47] showed prevailing alterationsin verbal recall and working memory in about one third ofHCV-infected patients, including subjects with liver cirrhosisand substance abuse history. In this study, depression scoreswere found to be predictors of cognitive impairment. Thestudy on a small cohort of homogeneous state-infectedpopulation with mild liver involvement and similar historyof iatrogenic HCV exposure reported alterations in generalmemory, sustained attention, and delayed auditory recog-nition; importantly, fatigue correlated only with delayedauditory memory recall ability [48]. Investigations employ-ing neurophysiological tests of cognitive processing, such asP300 event-related potentials, have revealed delayed peaklatencies and reduced amplitudes in cognitively impairedHCV-infected patients. The use of P300, as an indepen-dent measure of cerebral information processing, has theadvantage of avoiding the bias of confounding factors suchas fatigue or depression, and, in addition, represents asensitive marker of deranged cortical activation associatedwith conscious attention. Indeed, investigation of a largepopulation of patients with chronic HCV infection hasdisclosed the occurrence of subclinical cognitive dysfunctionin 18% of subjects; in these cases, delayed peak latencies andreduced amplitudes of P300 largely correlated with fatigue[49].

While definitive conclusions regarding the pathogen-esis of cognitive dysfunction, fatigue, and depression inHCV infection require further elucidation, many recentdata support a major role for the virus itself in causingCNS pathology. Accordingly, the detection of negative-strand HCV RNA in brain tissues of infected patients hassuggested that HCV replicates within the CNS; in addition,the diversity of viral quasispecies between the CNS andliver supports an independent life of the virus within thebrain [4]. CNS-specific HCV quasispecies have been foundto share their genomic sequences with those detected inlymphoid tissues and peripheral blood mononuclear cells(PBMC), but not with liver and serum HCV variants. Thesemolecular data have suggested that infected PBMC couldmediate HCV entry in the CNS [50–54] by a “Trojanhorse” mechanism [46]. Furthermore, the detection ofHCV proteins in macrophages/microglia and astrocytes ofpostmortem brain samples from patients coinfected withHCV/HIV or monoinfected with HCV suggests a major rolefor these cells in supporting replication [55]. More recentstudies indicate that the brain microvascular endothelialcells (BMECs) are a preferential site of HCV tropism andreplication. The demonstration that infection of BMECcauses \linebreak apoptosis in vitro has suggested that

alterations of the blood-brain-barrier could be responsiblefor microglia activation, following the entry across the brainof inflammatory cytokines and chemokines [56].

4. Peripheral Neuropathies

The PNS is variably affected in HCV-infected patients,mainly depending on the presence and type of CG,associated comorbidities, and iatrogenic factors. In HCV-associated type I CG, the involvement of PNS is rare, and,therefore, the pathogenesis is not entirely understood; ourexperience is consistent with axonal forms of polyneu-ropathy, pathologically characterized by perivascular infil-trates, endoneurial purpura, and microangiopathy, overallsuggesting an ischemic pathogenesis linked to endoneurialmicrocirculation obstruction [57]. Conversely, in patientswith HCV-associated MC, the involvement of the PNS rangesfrom 26% to 86%, in accordance with the disease stage andthe clinical/electrophysiological protocols for neuropathyascertainment. In most cases, pathological features areindicative of ischemic nerve changes, as a consequence ofsmall vessel vasculitis, or, less frequently, necrotizing arteritisof medium-sized vessels [58]; the presence of circulating CGis predictive of severe PNS involvement and a cryocrit levelhigher than 5% is detected in aggressive vasculitic forms,with recurrent purpura. In patients without CG, immunecomplexes or HCV-induced autoimmune mechanisms mayplay a pathogenetic role in inducing vascular and perivascu-lar inflammation, which may be driven by an intrinsic nervepopulation of immunocompetent and potentially phagocyticcells [59]. The possible role of HCV in inducing vascularinflammation is suggested by pathological/molecular studiesof nerve biopsies showing the presence of nonreplicativeHCV-RNA in epineurial cells, in close spatial relationshipwith mononuclear inflammatory infiltrates, speaking infavor of HCV-mediated cellular inflammation [60, 61].This is in keeping with the detection of positive-strandgenomic HCV RNA in nerve and muscle tissue samplesof patients with peripheral neuropathy, necrotizing arteritisand small-vessel lymphocytic vasculitis, findings suggestiveof an immune-mediated pathogenesis, rather than a directviral damage. Many patients develop a symmetrical sensoryor sensorimotor axonal-type polyneuropathy, with sensoryloss and weakness in distal regions of limbs, a patternsuggestive of a length-dependent process [62], similarlyto the most frequent form of neuropathy encountered inHIV-1 infection [63]. Alternative common presentationsinclude mononeuropathies and mononeuropathy multiplex,the latter producing a stocking-glove asymmetric neuropathyor overlapping syndrome (Figure 3). Cranial nerves areusually spared, although involvement of the abducens, facial,and motor trigeminal nerves has been reported. At variancewith earlier reports, in more recent series of patients withHCV-associated neuropathy, sensory neuropathy representsthe most prevalent form [64, 65]. The asymmetrical sensoryvariants include large-fiber sensory neuropathy (LFSN)and small-fiber sensory polyneuropathy (SFSN). LFSN ischaracterized by sensory loss, paresthesias, numbness, and


Mononeuritis multiplex

Overlappingmononeuritis

multiplex

Distal symmetricpolyneuropathy

Ganglionopathy

Figure 3: Clinical patterns of PNS involvement in HCV infection.

cramps; conversely, SFSN, a painful condition targeting smallmyelinated and unmyelinated sensory axons, is clinicallycharacterized by burning feet, tingling, restless leg syndrome,and, rarely, complex regional pain syndrome type 1 [66];in some patients LFSN and SFSN may coexist. Intriguingly,patients with SFSN may disclose a pattern suggestive ofganglionopathy. Unusual forms of PNS involvement includepure motor polyneuropathies [67] and autonomic neuropa-thy [68].

The spectrum of peripheral neuropathies in HCV infec-tion is not limited to axonal forms, but encompasses a num-ber of demyelinating conditions. A patient with subacutesensory ataxia and IgMk cryoglobulin with demyelinatingand axonal features was reported by Lippa et al. [69]. Inaddition, sensory demyelinating polyneuropathy, responsiveto immunomodulatory treatment, has been also detectedin subjects with polyclonal hypergammaglobulinemia orIgM monoclonal gammopathy, in the absence of CG [70],suggesting the occurrence of humoral immune-mediateddemyelination. Other forms of HCV-related demyelinatingconditions include the Lewis-Sumner syndrome [71] andchronic inflammatory demyelinating polyradiculoneuropa-thy [72].

5. Myopathies

The association between chronic HCV infection and myopa-thy is infrequent, and only few cases of noninflammatoryand inflammatory myopathies have been reported so far.Clinical features of HCV-related myopathies widely rangefrom progressive weakness to relapsing forms, and it isnot unusual to find subjects with only mild elevation ofmuscle enzymes and/or moderate weakness, which leads toconsiderable diagnostic difficulties.

In noninflammatory myopathies, pathological featuresare variegate and include vacuolar changes [73] or necro-tizing myopathy [74], in association with slowly or pro-gressive proximal weakness, and selective atrophy of type 2fibers in relapsing myopathy. The occurrence of oxidativemitochondrial damage has been suggested in a patient with

severe ptosis, diplopia, generalized weakness and respiratoryinvolvement, complex III deficiency and ultrastructural alter-ations of mitochondrial shape and cristae [75]. Moreover, apathogenic role for circulating cytokines and growth factorsin mediating muscle damage has been advanced based onexperimental findings showing that HCV promotes TNF-mediated apoptosis in myocytes [76].

At variance with noninflammatory myopathies, the clin-ical presentation of inflammatory forms is usually subacuteand insidious, and muscle biopsies usually show variousdegrees of focal or diffuse muscle inflammation. Polymyositisis frequently reported in HCV infection, either without[77, 78] or with CG [79], being associated with interstitiallung disease in some patients. The presence of genomic HCVRNA, but not replicative intermediates in muscle specimens,a picture suggestive of an autoimmune HCV-triggeredprocess, has been detected in patients with polymyositis[80, 81]. Additionally, evidence of complement activationwith membrane attack complex deposition, in addition tocytotoxic T cell activation, has been obtained in a patientwith inflammatory myopathy and muscle HCV RNA [82].

Dermatomyositis has been reported in a few patientswith incidentally discovered HCV infection, as well as insubjects with long-term chronic HCV and hepatocellularcarcinoma. Although the pathogenesis of HCV- or HCV-HCC-related dermatomyositis remains unexplained to date,a role for circulating anti-aminoacyl-tRNA synthetase anti-bodies, including anti-Jo1 and anti-Mi2 autoantibodies, hasbeen suggested [83].

Alternative pathogenic mechanisms, involving HCV-induced oxidative DNA damage, have been advanced inpatients with muscle deposition of HCV-RNA/HCV antigensand inclusion body myopathy, a muscular disorder whoseclassification under inflammatory forms is currently underdebate [84, 85].

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Review Article

HCV and Lymphoproliferation

Anna Linda Zignego,1, 2 Carlo Giannini,1, 2 and Laura Gragnani1, 2

1 Center for Systemic Manifestations of Hepatitis Viruses (MASVE), Department of Internal Medicine, University of Florence,50134 Florence, Italy

2 Istituto Toscano Tumori (ITT), 50139 Firenze, Italy

Correspondence should be addressed to Anna Linda Zignego, [email protected]



Copyright © 2012 Anna Linda Zignego et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Hepatitis C virus (HCV) infection is a serious public health problem because of its worldwide diffusion and sequelae. It is notonly a hepatotropic but also a lymphotropic agent and is responsible not only for liver injury—potentially evolving to cirrhosisand hepatocellular carcinoma—but also for a series of sometimes severely disabling extrahepatic diseases and, in particular, B-celllymphoproliferative disorders. These latter range from benign, but prelymphomatous conditions, like mixed cryoglobulinemia,to frank lymphomas. Analogously with Helicobacter pylori related lymphomagenesis, the study of the effects of viral eradicationconfirmed the etiopathogenetic role of HCV and showed it is an ideal model for better understanding of the molecular mechanismsinvolved. Concerning these latter, several hypotheses have been proposed over the past two decades which are not mutuallyexclusive. These hypotheses have variously emphasized the important role played by sustained stimulation of the immune systemby HCV, infection of the lymphatic cells, viral proteins, chromosomal aberrations, cytokines, or microRNA molecules. In thispaper we describe the main hypotheses that have been proposed with the corresponding principal supporting data.

1. Introduction

Hepatitis C virus (HCV) infection is a major public healthproblem with an estimated 3-4 million people infected eachyear worldwide and about 170–200 million carriers. Theselatter are at risk of developing liver cirrhosis and/or livercancer. More than 350,000 people die from HCV-related liverdiseases each year. Moreover, these estimates do not take intoaccount the extrahepatic aspects of HCV infection.

Early after its discovery, it was shown that HCV is also alymphotropic virus [1]. As a consequence of the lymphaticinfection, several lymphoproliferative disorders (LPDs) havebeen associated with this virus [2], including mixedcryoglobulinemia (MC), B-cell non-Hodgkin’s lymphoma(NHL) [3–10] and monoclonal gammopathies [11–13].

Mixed cryoglobulinemia is the most frequent and wellknown LPD developing during HCV infection. Althoughclinically benign, MC is a prelymphomatous disorder leadingto NHL in about 5–10% of cases. This makes MC a valuablemodel for study of pathogenetic mechanisms of HCV-relatedLPDs [2–14]. MC was previously interpreted as a lymphoma

in situ, being characterized by bone marrow and/or liverinfiltrates closely resembling NHL [15]. Therefore, it washypothesized that HCV may be involved in the pathogenesisof NHL as well [1, 4]. This hypothesis was substantiatedby several observations, including the significantly highprevalence of HCV infection in NHL patients in severalstudies [6, 7, 10, 12, 16–18]. A lot of data are presentlyavailable showing, in most cases, a significant associationwith B-cell NHL, even with a clear south-north gradientand involving different histopathological types of lymphoma,the most strictly associated being the lymphoplasmacytic,marginal zone and diffuse large B-cell lymphoma [19]. Acase-control study has shown that HCV infection increasesthe risk for NHL involving the liver and major salivaryglands by about 50-fold (i.e., a risk higher than that forhepatocellular carcinoma) and the risk for NHLs at othersites by about 4-fold [20].

The observation of the effect of viral eradication usingantiviral agents strongly supports the etiopathogenetic linkbetween HCV infection and lymphomagenesis. Analogouslywith what has been reported for MC, in the case of low


Inhibition ofapoptosis

Sustainedantigenic

stimulationLymphotropism

HCVproteins

Cytokines

HCV-relatedLPDs

Chromosomalaberrations

CD81-E2interaction

Host geneticpredisposition

Figure 1: Pathogenesis of HCV-related lymphoproliferative disorders (LPDs). Main working hypotheses and their principal interconnec-tions.

grade B-cell lymphoma—and especially in cases of spleniclymphoma—clinical remission following effective antiviraltherapy in HCV-associated cases has been observed [21–23].

Interestingly, in a recent Japanese study involving about3,000 HCV-infected patients observed during a long-termfollow-up, it was shown that the annual incidence oflymphoma was 0.23% and the cumulative rate of lymphomadevelopment after 15 years was 2.6% in both the untreatedand non-responder patients with persisting infection versus0% in treated patients achieving viral eradication, stronglysuggesting that antiviral therapy protects against the devel-opment of lymphoma [24].

2. Mechanisms ofHCV-Related Lymphomagenesis

Several hypotheses, frequently interconnected with eachother, have been proposed in regard to the possiblemechanisms of HCV-related lymphomagenesis (Figure 1).These include a key role played by the sustained antigenicstimulation of the B-cell compartment, the role of viral lym-photropism and viral proteins, chromosomal aberrations,cytokines, and microRNAs.

2.1. The Role of Sustained Antigenic Stimulation. SustainedHCV-driven antigenic stimulation has been suggested toplay a key role in inducing B-cell clonal expansion char-acterizing these disorders (Figure 2). The presence in theliver of lymphatic structures resembling lymphatic folliclesis characteristic of HCV infection. It has been suggested thatthey represent an important site of B-cell clonal expansion,especially in patients with MC, where they have been foundin almost all cases [25]. Furthermore, B lymphocytes isolated

from hepatic follicles produced rheumatoid factor (RF)that most frequently display the WA cross-reactive idiotype,considered to be characteristic of MC [25]. In particular,it was observed that intrahepatic B-cell clonalities wereinvariably associated with extrahepatic manifestations ofHCV infection, including high serum levels of RF activity,cryoglobulins, monoclonal gammopathy of undeterminedsignificance (MGUS), and frank B-cell NHL [26]. The keyrole of antigen-driven stimulation in HCV-related lympho-proliferation was also supported by a study investigatingmutations in the V(H) and V(K) genes of the B-cell cloneinducing a frank NHL in an MC patient and producingan IgM homologous to a protein with RF specificity. Theobservation of an IgH ongoing mutation process and theexpression of an Ig antigen receptor significantly homolo-gous to an anti-HCV protein suggested that both MC andNHL were antigen-driven LPDs sustained by HCV [27]. Theanalysis of bone marrow specimens from the same patienttaken at different times during the evolution from MC toNHL showed a marked reduction in intraclonal diversity atthe stage of overt NHL, indicating a minor dependence ofthe cells on the antigen-driven mechanism. Such a progres-sive independence from the initial etiologic agent may bededuced also by the effects of viral eradication in patientswith variable severity of the HCV-related LPD (see alsothe following). Analogously with Helicobacter pylori-relatedlymphomagenesis, it is conceivable that, during the multisteplymphomagenic process, progressive independence from theantigen-driven mechanism will develop, possibly due to theoccurrence of chromosomal translocations or other geneticaberrations [14, 28] (Figure 2).

It has also been suggested that the same HCV antigensmay be involved in the induction of both MC and lymphoma[27, 29]. The viral antigen/s responsible for B-cell clonal


B-cellinfection

HCV-inducedmutagenesisHCV

proteins

Genetic and/orenvironmental factors

Mixed cryoglobulinemia

Dep

ende

nce

on

an

tige

nic

sti

mu

lati

on

Additional genetic aberrations

+ prolonged B-cell survival

SustainedB-cell activation

t(14;18)/others?Bcl-2 overexpression

B-cell apoptosis inhibition

Direct/indirect/others?

Cytokines(BAFF)

Malignant NHL

E2-CD81binding

Figure 2: HCV-related LPD pathogenesis is a multifactorial and multistep process. Current data suggest that the starting points of thisprocess are represented by the cooperation between a sustained and persistent stimulation—by direct or indirect action of viral particlesor proteins—and antiapoptotic mechanisms acting on B-cell compartment. A predisposing genetic background would be responsible forthe final evolution to a particular LPD (namely, MC). The progressive addition of genetic aberrations would lead to a frank neoplastictransformation, gradually making the process less dependent on the etiologic agent.

expansion are not perfectly defined yet. However, De Re andcolleagues showed that, in patients with MC and immuno-cytoma, the B-cell receptor (BCR) of the monoclonal,overexpanded B-cell population, as well as the IgM-RF+component of the cryoprecipitate, showed cross-reactivityagainst HCV NS3 antigen [30].

Other studies have focused on the possible role ofproteins of the HCV envelope and mainly on HCV E2 protein(Figure 2). It has been shown that E2 interacts with thetetraspanin CD81, present also on the B-cell surface. Thisbinding has been suggested to be responsible for sustainedpolyclonal B-cell activation essentially by lowering the B-cellactivation threshold [31, 32].

Data supporting the hypothesis that some HCV-associated lymphomas could originate from B cells thatwere initially activated by the HCV-E2 protein have beenprovided. In fact, Quinn and coworkers showed that theimmunoglobulin from one of two HCV-associated lym-phomas they tested bound the E2 protein in a manner iden-tical to a bona fide human anti-E2 antibody, hypothesizingthat the B-cell activation derived from the dual binding ofE2 to a cognate BCR and to the CD81 molecule, which is acomponent of a signaling complex [33].

Interestingly, the HCV-E2 protein appears to mimichuman Ig. In fact, it was observed that the N-terminal regionof E2 is antigenically and structurally similar to humanIg variable domains and could represent a target for anti-human IgG antibodies [34]. Consistently, the analysis ofthe CDR3 sequences of the IgM-RF+ purified from thecryoprecipitate in MC patients allowed the identification ofHCV-E2 as the antigen driving the production of IgM-RF[35].

2.2. The Role of Viral Lymphotropism and Viral Proteins. Thepotential role of viral lymphotropism in the pathogenesisof HCV-related LPDs has been emphasized since the firstevidence of the presence of viral replication in lymphaticcells [1] (Figure 2). The association between viral infectionof peripheral blood mononuclear cells (PBMCs) and thepresence of LPDs was initially shown in patients with MC,where more evident infection of PBMCs in comparisonwith HCV-positive patients without MC was observed [4].HCV infection was then observed by Galli et al. in bonemarrow cells from all patients with MC versus 43% ofpatients without MC [36]. After these pioneering reports,a large amount of data correlating the presence of HCVin the lymphatic compartment and the development ofautoimmune/lymphoproliferative disorders has been pro-duced. In a study using the model of injection of lymphoidcells from HCV-positive patients into SCID mice, it wasshown that the samples derived from HCV patients withmalignant LPD were characterized by positivity for HCVreplicative intermediates, stronger signals when tested forHCV genomic sequences, and successful serial passage ofinfected cells in different animals [37]. In addition, Sungand coworkers showed the establishment of B-cell lines per-sistently producing infectious virus from an HCV-positivelymphoma [38].

One interesting point about the actual dimension of lym-phocyte infection in HCV+ subjects has been provided by Paland coworkers who showed HCV infection in 85% of lymphnode specimens tested by in situ hybridization and HCVreplication in 50% of cases by detection of HCV replicativeintermediate [39]. Interestingly, quasispecies analysis in onecase indicated that 68% of variants circulating in serum


were also present in lymphoid tissues, and only 40% ofserum variants were identified in liver, documenting a majorcontribution of lymphoid replication to HCV viremia [39].

The existence of lymphotropic viral quasispecies has alsobeen demonstrated by elegant studies evaluating HCV IRESsequence in liver and PBMC and its ability to drive viralgenome translation in different cell types [40, 41].

The M. Lai’s group, using a model of in vitro HCVinfection of B-cells, showed that the viral infection mayinduce an enhanced mutation rate of immunoglobulin genesand some oncogenes, possibly through induction of error-prone DNA polymerase and activation-induced cytidinedeaminase (AID), suggesting that HCV may cause tumorsby a hit-and-run mechanism [42]. More recently, Ito andcoworkers observed a dramatically increased expression ofAID in the B-cells of HCV patients, suggesting that this maybe a key factor in the lymphomagenetic process mediatedby HCV [43]. Furthermore, a significantly higher expressionof several lymphomagenesis-related genes in the CD19+ ofHCV patients than in controls was also shown [43].

In regard to viral proteins, particular attention hasbeen focused on the HCV core protein due to previouslyshown pleiotropic effects on different cell signaling pathwaysmodulating cell viability and proliferation [44]. Focusing onanimal models, core transgenic mice developed lymphomawith a high frequency (80%) at ages over 20 months [45].The core mRNA was shown in the enlarged lymph nodesof the transgenic mice which developed lymphoma. Inanother transgenic model, where the IFN signaling wasdisrupted, the inducible and persistent expression of theHCV core in the context of all structural proteins wasassociated with the development of lymphoid disordersincluding frank lymphoma, suggesting a synergistic actionof the viral proteins with IFN signaling impairment inpromoting lymphomagenesis [46].

More recently, the expression of the whole HCV genome,restricted to the B-cell compartment, resulted in a highprevalence of diffuse large B-cell lymphoma (DLBCL)(up to29%) within 600 days after birth [47]. Interestingly, the HCVcore gene was expressed in all lymphomas. A wide analysisof the cytokine and chemokine pattern showed elevatedlevels of serum IL-2Rα in mice with lymphoma, directlyoriginating from lymphoma tissue [47].

The expression of the HCV core in primary B cells byan adenoviral vector significantly inhibited B-lymphocyteapoptosis and induced a dramatic down regulation of MHCclass II molecules. Moreover, genes associated with leukemiaand B-lymphoma were consistently up regulated by the HCVcore in this model [48].

Finally, in a study performed on both B-cell linesexpressing the HCV core protein and in primary B-cellsfrom patients with LPDs, it was possible to show the alteredexpression of some isoforms of genes of the p53 family, theDNp63 and DNp73, previously shown to be overexpressed inhuman cancers, including lymphoma [49, 50].

Although ectopic protein expression in both in vivo andin vitro models does not perfectly reproduce the actualsituation during chronic viral infection, the consistencyand coherence of the data accumulated until now suggest

a potential lymphomagenic effect of the core protein whenexpressed as a single protein or in the context of other viralproteins.

2.3. Chromosomal Aberrations. Interesting data are availableabout the role played by chromosomal aberrations in HCV-related LPDs. The most investigated genetic aberration wasthe (14;18) translocation—t(14;18)—that was found to besignificantly associated with type II or monoclonal MC.The presence of t(14;18) in MC was correlated with theoverexpression of the antiapoptotic bcl-2 gene in B-cells,resulting in an imbalance of the Bcl-2/Bax ratio and abnor-mal B-cell survival [51, 52] (Figure 2). The regression of theexpanded B-cell clones following effective antiviral treatmentand, in some relapsing patients, a new expansion of thesame clones were also shown [53]. Furthermore, a long-termfollow-up study allowed the identification of occult HCVpersistence limited to the lymphatic compartment in somepatients resulting sustained viral responders after antiviraltherapy [54, 55]. More interestingly, such a persistent occultlymphatic infection was associated with the initial diagnosisof MC, the persistence of some MC symptoms after therapy,and the persistence of expanded t(14;18)+ B-cell clones[54, 55]. The observation of the possibility, even if rare, ofa persisting MC disease in spite of complete viral eradicationsuggested the existence of points of no return in the evolutionof the HCV-related lymphoproliferation.

Recently, Goldberg-Bittman et al. reported an increasedrate of aneuploidy in chronically infected HCV subjectsversus healthy controls, with values similar to an NHLgroup. This observation suggests that HCV patients couldbe more prone to develop a lymphatic malignancy alsobecause of bearing such alterations of the ploidy grade[56]. An important contribution to understanding the set ofchromosome instability associated with HCV infection wasprovided by a study of Machida and coworkers. The authorsobserved a reduced expression of Rb protein—responsiblefor cell cycle arrest in case of DNA abnormalities—in variousconditions including HCV-infected PBMCs isolated frompatients, hepatocyte models infected in vitro with HCV ortransfected with the viral core protein alone, and transgenicmice expressing core protein. In fact, lower levels of Rbcould easily lead to skipping the mitosis checkpoints andcontribute to generation of polyploid cells, a conditionfavoring neoplastic transformation [57].

2.4. Cytokines, Chemokines, and HCV-Related LPDs. Cytok-ines and chemokines are essential mediators of the immuneresponse. A disturbance of the equilibrium between activat-ing and repressing effects of these soluble molecules maybe responsible for several autoimmune/lymphoproliferativedisorders. Numerous reports have suggested that cytokinesand chemokines are key factors in the pathogenesis ofHCV-related LPDs. The MC model, as prelymphomatouscondition, has been widely used to investigate the cytokinepattern characteristic of HCV-related LPDs. The role of Th1cytokine profile (IFNγ and TNFα) and some chemokines(MIP-1α, MIP-1β, CXCL10, and CXCR3) in the pathogenesisof HCV-MC has been suggested by the elevated expression


of these mediators observed in vasculitic lesions of MCpatients [58]. CXCL13 was another chemokine reportedas upregulated in MC patients [59]. This chemokine, alsoknown as BCA-1 (B-cell-attracting chemokine-1) or BLC(B-lymphocyte chemoattractant), is a major regulator of B-cell trafficking, and its expression has been found to besignificantly enhanced in microdissected samples from liverbiopsy of patients with active cutaneous vasculitis. Antonelliand coworkers showed that serum concentrations of differentcytokines and chemokines are significantly modified in MCpatients [60] and have recently investigated the potentialrole of CXCL-10 and CXCL-11 in the pathogenesis of MC[61, 62]. In fact, high serum concentration of these solublefactors has been shown in HCV patients with MC whencompared to patients without MC and healthy controls.

A growing body of evidence has accumulated in recentyears showing the involvement of the B-cell-activating factor(BAFF or BLyS) in the pathogenesis of HCV-related LPDs(Figure 2). This B-cell-specific cytokine, belonging to theTNF-α family, is essential for B-lymphocyte developmentand survival. Several reports have shown a higher BAFFserum concentration in HCV patients than in healthycontrols and, more significantly, in HCV patients with LPDs(for review, see [63]). The mechanisms of the enhancedserum concentration of BAFF in HCV-LPD patients have notbeen elucidated yet. A possible explanation has been recentlysuggested by the analysis of the polymorphic variants of theBAFF gene promoter. A particular allelic variant (−871 T),reported to induce an increased transcriptional activity of theBAFF gene [64], was significantly more frequent in patientswith HCV-related MC than in HCV patients without MC.The genetic data were corroborated by the presence of higherlevels of the cytokine in the serum of MC patients [65, 66].

Concerning HCV-positive subjects with NHL, Libra et al.showed an increase in the circulating levels of IL-1β [67]. Aswell, high serum osteopontin (OPN) levels were associatedwith B-cell NHL and HCV infection. Interestingly, thehighest serum OPN concentrations were found among HCV-infected patients with concomitant type II MC with andwithout B-cell NHL [68].

2.5. MicroRNAs and HCV-Related LPDs. Increasing evidencesupporting the role of microRNA (miRNA) deregulationin the pathogenesis of chronic hepatitis C infection andrelated disorders has been provided in the last years (forreview: [69]). MiRNAs are small RNA molecules (19–22-mer noncoding RNAs) able to induce translational inhibitionof target genes by partial base complementarity to thetarget mRNA 3′UTR. A liver-specific miRNA, miR-122, hasbeen shown to be important for HCV replication [70].It has been shown that interferon treatment can regulatemiR-122 and other miRNA levels and that these lattermediate—at least in part—the antiviral effects of IFN [71].These in vitro data have been partially corroborated bythe observation of modified levels of miR-122 in patientsunresponsive to the IFN therapy [72]. A few data are stillnow available concerning the involvement of miRNAs in thepathogenesis of HCV extrahepatic disorders. An interestingreport recently analyzed the global miRNA expression profile

in HCV-related and unrelated splenic marginal zone lym-phomas (SMZLs). By using large-scale miRNA expressionprofiling analysis, Peveling-Oberhag and coworkers [73]quantitatively evaluated the expression of 381 miRNAs inmicrodissected SMZLs (HCV-positive and -negative) andin normal splenic tissues. The difference in expressionprofiles arose between SMZLs and normal spleens for 12miRNAs, most of which were previously involved in variousmechanisms of tumor formation for both lymphomas andother tumors. Only one miRNA, miR-26b, known to showtumor-suppressive activity, was significantly down regulatedin HCV-positive lymphomas compared to HCV-negativeSMZLs, suggesting a possible specific mechanism by whichthe virus might unfold its oncogenic potential in malignantlymphoma.

3. Conclusions

In conclusion, as summarized in Figure 2, current datasuggest that HCV lymphomagenesis is a complex, multistep,multifactorial process, probably based on sustained B-cell-activation and the inhibition of B-cell apoptosis within abackground of predisposing genetic factors and evolvingthrough the progressive addition of genetic aberrationswhich allow the process to become progressively less depen-dent on the etiologic agent.


The authors declare the absence of any conflict of interests.

Acknowledgments

This work was supported by grants from the “AssociazioneItaliana per la Ricerca sul Cancro” (AIRC) Investigator Grantno. 1461, “Istituto Toscano Tumori” (ITT), “FondazioneIstituto di Ricerche Virologiche Oretta Bartolomei Corsi,”“Ente Cassa di Risparmio di Firenze,” and “Fondazione Cassadi Risparmio di Pistoia e Pescia.” Authors thank Mrs. MaryForrest for the precious help in editing the paper.

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Review Article

Pathogenetic Mechanisms of Hepatitis C Virus-InducedB-Cell Lymphomagenesis

Fabio Forghieri, Mario Luppi, Patrizia Barozzi, Rossana Maffei, Leonardo Potenza,Franco Narni, and Roberto Marasca

Section of Hematology, Department of Oncology, Hematology, and Respiratory Diseases, University of Modena and Reggio Emilia,41100 Modena, Italy

Correspondence should be addressed to Roberto Marasca, [email protected]



Copyright © 2012 Fabio Forghieri et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Hepatitis C virus (HCV) infection is probably the most common chronic viral infection and affects an estimated 180 million peopleworldwide, accounting for 3% of the global population. Although the liver is considered to be the primary target, extrahepaticmanifestations are well recognized among patients with chronic HCV infection. Epidemiological studies have clearly demonstrateda correlation between chronic HCV infection and occurrence of B-cell non-Hodgkin’s lymphomas (B-NHL). The clinical evidencethat antiviral therapy has a significant role in the treatment at least of some HCV-associated lymphoproliferative disorders,especially indolent B-NHL, further supports the existence of an etiopathogenetic link. However, the mechanisms exploited by HCVto induce B-cell lymphoproliferation have so far not completely clarified. It is conceivable that different biological mechanisms,namely, chronic antigen stimulation, high-affinity interaction between HCV-E2 protein and its cellular receptors, direct HCVinfection of B-cells, and “hit and run” transforming events, may be combined themselves and cooperate in a multifactorial modelof HCV-associated lymphomagenesis.

1. Introduction

Hepatitis C virus (HCV) is an enveloped positive, single-stranded RNA virus, belonging to the Flaviviridae fam-ily [1]. During its replicative cycle it goes through anegative-stranded RNA, but not DNA, intermediate, sothat integration of HCV nucleic acid sequences into thehost genome seems unlikely. The HCV genome encodes asingle polyprotein precursor of approximately 3000 aminoacids, which is proteolytically processed by viral and cellularproteases to produce structural (nucleocapsid, E1, and E2)and nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B,NS5A, and NS5B). The HCV envelope proteins consist oftwo heavily glycosylated proteins, E1 and E2, which act asthe ligands for cellular receptors [1, 2].

Human CD81 is the first identified necessary receptorfor HCV cell entry, which can directly bind with HCV E2protein [3, 4]. CD81 is a widely distributed cell-surface

tetraspanin that participates in different molecular com-plexes on various cell types, including hepatocytes, B-lymp-hocytes, T-lymphocytes, and natural killer cells. It has beenproposed that HCV exploits CD81 not only to invadehepatocytes but also to modulate the host immune responses[5].

Infection with HCV affects an estimated 180 millionpeople, accounting for 3% of the global population [6,7]. HCV is a well-recognized etiologic agent of chronichepatitis. Although the natural history of HCV infectionis highly variable, an estimated 15% to 30% of patientsin whom chronic infection develops have progression tocirrhosis over the ensuing three decades, and these latterpatients warrant surveillance for complications, includinghepatocellular carcinoma (HCC), which develops in 1%–3%of such patients per year [6, 7]. Indeed, the risk of HCC inthe HCV-infected population is 23–35 times higher than innoninfected healthy individuals [8, 9].


Although the liver is considered to be the primarytarget of HCV infection, extrahepatic manifestations, such asmixed cryoglobulinemia (MC), which is a systemic immunecomplex-mediated disorder characterized by B-cell prolifer-ation that may evolve into overt B-cell non-Hodgkin’s lym-phoma (B-NHL) in about 10%–20% of patients several yearsafter diagnosis, are often recognized among patients withchronic HCV infection [10–12]. Moreover, epidemiologicalevidences strongly suggest a close link between chronic HCVinfection and de novo B-NHL, not complicating the courseof MC [13–16]. The possible pathogenetic mechanisms ofHCV-induced B-cell lymphomagenesis are reviewed.

2. Epidemiologic Association ofHCV and B-NHL

Evans and Mueller proposed that either epidemiologic orvirologic guidelines need to be fulfilled to support anetiologic role for a virus in a given human cancer [17].Suggested epidemiologic guidelines included the following:(a) the geographic distribution of viral infection shouldcoincide with that of the tumor; (b) the presence of viralmarkers should be higher in case subjects than in matchedcontrol subjects; (c) viral markers should precede the tumor,with a higher incidence of tumors in persons with the markerthan in those without; (d) prevention of viral infectionshould decrease tumor incidence [17]. Suggested virologicguidelines included the following: (a) the virus should beable to transform human cells in vitro; (b) the viral genomeshould be demonstrated in tumor cells and not in normalcells; (c) the virus should be able to induce the tumor in anexperimental animal [17].

As far as the association between HCV infection andoccurrence of B-NHL is concerned, most of the epidemi-ologic guidelines for causality from Evans and Mueller aremet. HCV is associated with certain B-NHL types, especiallyin geographic areas with HCV endemicity, like Italy, Japan,and Egypt, where prevalence rates range from 20% to 40%[14, 15, 18–21], whereas in nonendemic areas, as NorthernEurope, North America and United Kingdom, the prevalenceof HCV infection in B-NHL is far less than 5% [22–24].The possibility is raised that in these latter geographic areaswhere HCV prevalence among subjects not affected with B-NHL is low, the spread of the virus may be recent, thus notallowing the full consequences on B-NHL development tobe observed. Moreover, studies from areas with low HCVprevalence may not have included sufficient numbers ofpatients to detect a significant association between HCVand B-NHL [16]. Taken together, the epidemiologic analysesdemonstrated that the prevalence of HCV infection inpatients with B-NHL is approximately 15% [25]. The preva-lence of anti-HCV antibodies and/or HCV RNA sequences issignificantly higher in patients with B-NHL than in patientswith other lymphoid malignancies or in age matched healthysubjects. Furthermore, HCV infection often precedes byyears the occurrence of lymphomas [26]. In a recent meta-analysis focusing on 15 studies, the pooled relative risk (RR)of all B-NHL among HCV-positive persons was found to be

2.5 (95% confidence interval (CI), 2.1–3.1) in case-controlstudies and 2.0 (95% CI, 1.8–2.2) in cohort studies [27].Another meta-analysis reviewed data from 23 studies (4,049NHL patients and 1,813,480 controls) and found a strongerassociation (odds ratio 5.70) [28]. It should be noted that RR,although moderate (2-3 on average) in comparison to HCVand HCC association, were similarly increased for all majorB-NHL subtypes and primary sites of presentation [16, 29].Only slightly higher RR for extranodal compared with nodalB-NHL were reported for HCV-positive patients, but thisdifference was largely due to the early studies. Moreover,extensive studies did not demonstrate clear differences onthe association between HCV and major histologic B-NHL subtypes, either indolent, namely, follicular, marginalzone (MZL), lymphoplasmacytic, and chronic lymphocyticleukemia/small lymphocytic lymphoma, or aggressive diffuselarge B-cell (DLBCL) and Burkitt lymphoma [16, 29, 30].In fact, earliest studies suggesting a stronger associationof HCV with certain subtypes, such as lymphoplasma-cytic/Waldenstrom lymphomas, were performed mainly inHCV-infected subjects with MC, a subset of patients inwhich these lymphoma subtypes have been reported to behighly prevalent [16, 29]. Conversely, one of the largest case-control studies to date found a higher OR (3.5 versus 2.3)for aggressive versus indolent lymphomas, respectively, andsuggested that previous data may have also been influencedby the relatively poorer prognosis associated with aggressivelymphomas [14]. Patients with HCV-related DLBCL mayhave more aggressive clinical features at presentation incomparison to HCV-negative patients [31, 32].

The possible association between specific viral geno-types and malignant lymphoproliferative disorders remainsa controversial issue. There are at least six major HCVgenotypes whose prevalence varies geographically. Genotype1 accounts for the majority of infections in North America,South America, and Europe [7]. Various clinical studiesfailed to demonstrate a link between specific viral genotypesand B-NHL, but it should also be noted that this issuewas not specifically addressed in several other series. Luppiet al. documented an unexpectedly lower prevalence ofHCV genotype 1b/II in patients with B-NHL. Conversely,the prevalence of genotypes 2a/III and 2b/IV was higherin patients with B-NHL than in either hemodialysis orchronic liver disease patients, thus suggesting that differentHCV variants may show greater lymphotropism [33]. Recentepidemiologic evidence from a multicenter retrospectivestudy also suggested that genotype 2 may be more prevalentand carcinogenic in lymphoma patients [34]. In details,HCV-positive patients were classified as cancer patients (129patients, including 53 hematologic malignancies and 76solid tumors), immunocompetent (333 subjects) and HCV-HIV coinfected (102 patients). Genotype 1 predominated(84%) in immunocompetent as compared to patients withHCC (74%, P = .08) or lymphoma (59%, P = .001).By contrast, genotype 2 was more prevalent in patientswith lymphoma (24%), compared to immunocompetent(8%, P = .003), yielding a 3-fold increase in cancerrisk among HCV-infected patients than other genotypes[34]. Interestingly, Pellicelli et al. [19] observed that DLBCL


patients had a higher prevalence of genotype 1 and a shorterduration of HCV infection, as compared to patients withindolent, low-grade B-NHL, who showed a higher prevalenceof genotype 2 and longer duration of HCV infection. BecauseHCV genotype 2 is associated with a longer duration ofviral infection, it has been speculated that over time itmay induce a persistent chronic immunostimulation of B-cells. On the contrary, direct lymphocyte transformationcould be hypothesized for HCV genotype 1 in aggressivelymphomas, on the basis of the shorter duration of viralexposure [19]. Future perspective studies enrolling a largenumber of patients are warranted to further investigate thedifferent distribution and carcinogenic potential of differentHCV genotypes.

The regression of HCV-related B-NHL following antivi-ral therapy probably represents the strongest argument infavor of an etiologic link between HCV infection and certainhuman lymphomas [16, 26]. Several clinical trials showedthat antiviral therapy, mostly based on peg-interferon andribavirin, resulted in either complete or partial remissionsof lymphoma in HCV-positive but not HCV-negative B-NHL patients [29, 35–37]. A systematic review has shownthat complete responses were achieved in 75% of theHCV-positive cases [38]. Lymphoma regression was usu-ally positively correlated with viral load reduction [29].These trials have been conducted in asymptomatic indolentlymphomas during a phase in which no other therapeuticintervention was administered. For aggressive lymphoma orsymptomatic indolent lymphoma, HCV eradication aloneis not an option. These patients require systemic therapywith rituximab and chemotherapy-based regimens as firsttreatment. Nevertheless, antiviral therapy to eradicate HCVmay be an option after successful lymphoma therapy.Whether HCV eradication after-chemoimmunotherapy mayimpact future survival outcome remains uncertain [29].Regarding this topic, La Mura et al. retrospectively analyzed343 patients affected with NHL [39]. Twenty-five of the 69HCV-positive subjects received antiviral therapy (interferonand ribavirin) following antineoplastic treatment, in orderto eradicate HCV infection. Overall survival (OS) wasslightly better in HCV-infected NHL patients treated withantiviral therapy compared with untreated, even if withoutstatistically significance. Conversely, disease-free survival(DFS) was significantly improved in treated versus untreatedpatients. A sustained virologic response was obtained in8/25 (32%) HCV-positive NHL patients who underwentantiviral treatment. None of the patients who eradicatedHCV infection had a lymphoma relapse at followup, whereas5/17 of those who did not respond to antiviral therapy expe-rienced relapses. At multivariate analysis, the independentfactors related to a better DFS in this series were antiviraltherapy and indolent histology at the onset of lymphoma[39]. Antiviral treatment may be a strategy to reinforce theresults of successful chemoimmunotherapy regimens, butfuture prospective studies are needed to further investigatethis clinical issue. Of interest, a recent study has shown thatHCV-infected patients who had received interferon therapyand had experienced a sustained virologic response had ahazard ratio of lymphomagenesis that was significantly lower

than patients who had not received antiviral treatment [40].These data suggest that antiviral treatment may also beefficacious in preventing lymphomagenesis in HCV-infectedpatients. Moreover, it should be of interest to investigatethe impact of newer directly acting antiviral agents, suchas protease inhibitors telaprevir and boceprevir [11, 41–43],on the future prevalence and clinical outcome of B-NHLin patients with chronic HCV infection. While reactivationrisk of hepatitis B virus (HBV) after chemoimmunotherapyis well recognized and prophylactic antiviral therapy tosuppress HBV-DNA is widely recommended, the issue ofHCV reactivation in lymphoma patients undergoing anti-neoplastic treatments is lesser understood [29, 44]. However,a significant proportion of patients with HCV-positive NHL,when treated with conventional chemoimmunotherapy, maydevelop liver toxicity due to either direct cytotoxicity orincreased drug toxicity from suboptimal drug metabolism[29, 45]. The addition of rituximab to chemotherapy doesnot seem to impact significantly on liver toxicity [45]. HCV-RNA levels appear to increase during chemoimmunotherapyas a result of viral reactivation, but HCV-RNA levelssubsequently decrease at 6 months posttreatment, oftenwithout major clinical consequences to most patients [44].Nevertheless, it should also be noted that massive livernecrosis may occur in HCV-positive lymphoma patients onwithdrawal of chemotherapy or reduction of corticosteroids,suggesting an immune-mediated mechanism of hepaticdamage [44, 46]. Without initial liver dysfunction, HCV-positive patients with NHL could experience a similaroutcome compared with their HCV-negative counterparts,when treated with conventional chemoimmunotherapy [44,47]. A protective role of antiviral prophylaxis to suppressHCV replication during antineoplastic treatments has notyet been defined [29, 44]. Prospective studies and longerfollowups are necessary to ascertain whether HCV-positiveB-NHL patients have inferior outcome or whether therewould be long term consequences of chemoimmunotherapyon the progression of liver disease [47]. Patients with HCVinfection and lymphoma are recommended to be carefullymonitored for hepatotoxicity and HCV-RNA levels. Further-more, hematologists and hepatologists should work closelytogether in order to optimize the management of HCVinfection throughout lymphoma treatment and improveclinical outcome [29].

3. Mechanisms of HCV-InducedLymphoproliferation

The biological rational for investigating a causal associationbetween HCV infection and the occurrence of B-NHL isbased on epidemiological and clinical observations. Never-theless, limited information are so far available about the bio-logical mechanisms of HCV-induced lymphoproliferation.Evidences from experimental studies suggest that severaldifferent mechanisms may be involved in HCV-mediated B-cell transformation [16, 29, 48].

Similarly to the association of Helicobacter pylori infec-tion and gastric MALT lymphoma, the concept of chronic


Chronic antigen stimulation

HCV antigen

A

B

C

D

Ig surface B-cell

B-cell

B-cell

B-cell

High-affinity binding

between HCV-E2 and CD81

“Hit and run”Transforming events

HCV-E2

CD81Mutations in protooncogenesand tumor suppressor genes - T-cell activation

- Overexpression ofantiapoptotic Bcl-2 proteins

- Activation and proliferationof B cells

AID

Double strand breaks

Mutator phenotype

HCV-associatedlymphomagenesis

Direct infection of B cells by HCV

HCV

Figure 1: (A–D) The different oncogenetic mechanisms are not mutually exclusive, but they may be integrated and cooperate in amultifactorial pathogenetic model of HCV-associated B-cell lymphoproliferation.

antigen stimulation leading to a monoclonal malignantproliferation may also be applied to HCV (Figure 1(A))[49, 50]. Interestingly, HCV-associated B-NHL generallyoriginate from germinal center (GC) or post-GC B-cells,suggesting that lymphomagenesis occurs when B-cells expe-rience somatic hypermutation and proliferate in responseto an antigen [51, 52]. Further evidence comes from theantibody response and immunoglobulin variable (Ig VH)gene usage in patients with chronic HCV infection andHCV-associated B-NHL. In three out of five HCV-positivenodal MZLs, Marasca et al. revealed the usage of the VH1-69 gene with similar CDR3, indicating a highly biased andnonrandom use of the VH segments in this subtype oftumors [53]. These data indicated the role of a commonantigenic epitope involved in the selection and in theexpansion of the B-cell clone at the origin of neoplasticcells. The VH1-69 immunoglobulin segment is expressed inthe restricted repertoire of fetal liver B lymphocytes and isthought to be involved in natural immunity. A productiveVH1-69 rearrangement is present in 1.6% of normal Blymphocytes in adults. VH1-69 is rearranged in 10% to20% of B-cell chronic lymphocytic leukemia and a VH1-69monoclonal rearrangement is also present in the majorityof patients with type II MC, a typical HCV-related disorder[53]. Further experimental sequencing of clonal Ig variableregions from both MC and HCV-associated B-NHL patientsdocumented restricted IgV gene repertoire, with expressionof VH and VL genes (VH1-69 and Vκ3-A27), suggestingexposure and response to a common antigen [54–56]. Ofnote, HCV-E2 protein is the primary target of antibodyresponses against HCV [57]. Quinn et al. obtained thecloning of the B-cell receptor from one HCV-positive DLBCL

and its expression as a soluble immunoglobulin [58]. Theimmunoglobulin rescued was shown to bind the HCV-E2glycoprotein in a manner identical to a bona fide humananti-E2 antibody, suggesting that some HCV-associated B-NHL may originate from B-cells that were initially activatedby HCV-E2 protein [58]. Similarly, in a reported case ofan HCV-associated plasma cell leukemia, immunoblottingshowed that the monoclonal IgG-kappa detected in theserum was directed against a viral protein, namely, theHCV core protein [59]. These and other studies suggest anindirect, antigen-driven lymphomagenetic role of HCV, withHCV-E2 protein recognized as one of the most importantantigens involved in chronic B-lymphocyte stimulation [16,26, 29].

A second mechanism, potentially involved in HCV-associated lymphomagenesis, derives from the high-affinitybinding between HCV-E2 and one of its receptors, thetetraspanin CD81, expressed on B-cells (Figure 1(B)) [16].CD81 is known to form B-cell costimulatory complex withCD19, CD21, and interferon-inducible Leu-13 (CD225)proteins. This complex reduces the threshold for B-cellactivation via the B-cell receptor by bridging antigen specificrecognition and CD21-mediated complement recognition[60, 61]. It was reported that engagement of CD81 on humanB-cells by a combination of HCV E2 protein and anti-CD81mAb leads to the proliferation of naive B-cells, and E2-CD81 interaction induces protein tyrosine phosphorylationand hypermutation of the immunoglobulin genes in B-cell lines [62]. In addition to direct effects on B-cells,engagement of CD81 on T-cells lowered the threshold forinterleukin-2 production, resulting in strongly increased T-cell proliferation. This could lead to T-cell activation in


response to suboptimal stimuli and bystander activationof B-cells [63]. Taken together, these results suggest thatCD81 engagement on B- and T-cells may lead to director indirect activation [16]. Chronic B-cell proliferation, inresponse to antigenic stimulation or polyclonal activation,may predispose to genetic lesions such as translocationand/or overexpression of the antiapoptotic protein Bcl-2[64]. In a recent study, human Burkitt’s lymphoma cell line(Raji cells) and primary human B lymphocytes (PHB) weretreated with HCV-E2 protein and HCV particles producedby cell culture (HCVcc) [65]. The results showed that bothE2 and HCVcc triggered phosphorylation of IkBα, withsubsequent increased expression of NF-kB and NF-kB targetgenes, such as antiapoptotic Bcl-2 family proteins (Bcl-2and Bcl-xL). Either Raji cells or PHB cells were protectedfrom FAS-mediated death. In addition, both E2 protein andHCVcc increased the expression of costimulatory moleculesCD80, CD86, and CD81 itself, and decreased the expressionof complement receptor CD21. The effects were dependenton E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments.Moreover, an E2 mutant that loses the CD81 binding activitycould not trigger the responses of both Raji cells and PHBcells. Of note, the effects were not associated with HCVreplication in cells [65]. Hence, E2-CD81 engagement plays arole in activating B-cells, protecting B-cells from activation-induced cell death, and regulating immunological function.These latter mechanisms may contribute to the pathogenesisof HCV-associated B-cell lymphoproliferative disorders [65].Moreover, the interaction between HCV-E2 and CD81 on B-cells has been shown to stimulate the enhanced expression ofactivation-induced cytidine deaminase (AID) and to inducedouble-strand DNA breaks in the IgVH gene locus, therebycontributing to a mutator phenotype that increases the riskof B-cell malignant transformation [66].

Another oncogenetic mechanism that has been proposedis the direct infection of B-cells by HCV (Figure 1(C))[16, 29, 67]. In the early 1990s, the presence of HCV-RNA was demonstrated by PCR not only in serum/plasmaand liver tissues but also in peripheral blood mononuclearcells (PBMCs), especially in B-cells, of patients infectedwith HCV [68–71]. Nevertheless, although HCV has beendetected in lymphocytes from HCV infected patients andpatients with MC, only in a minority of cases RNA-negativestrands, the HCV replicative intermediates suggestive ofviral replication, were also detected in the cells [72–74].Detection of negative-strands HCV-RNA in PBMCs bypolymerase chain reaction, may also be due to eithercontamination or passive absorption of circulating HCV,thus potentially leading to false positive results [75, 76].Marukian et al. showed that culture-grown HCV replicatedin hepatoma cells, but no HCV replication was detectedin B- or T-cells, monocytes, macrophages, or dendriticcells from healthy donors [77]. Furthermore, Stamataki etal. have provided experimental evidence that HCV mightinfect B-cells, but B-cells were not able to support activeviral replication [78]. Overall, these results should indicatethat PBMC may not be permissive to HCV replication[16].

However, it has been reported that HCV may infectand replicate only in a relatively rare subset of B-cells, suchas CD5+ B-cells. These cells have been shown to expresshigh levels of CD81 and to expand in HCV-infected liver[79]. Alternatively, B-cells may need another event, suchas coinfection with another virus, namely, Epstein-Barrvirus (EBV), to become permissive for HCV infection andreplication [16, 80]. Neither HCV-RNA nor viral proteinshave generally been detected in lymphomatous cells in vivo,with a few exceptions, for example a primary DLBCL ofthe liver, found to harbor viral nucleic acids by in situhybridization and a mantle cell lymphoma case, from whicha lymphoma cell line could be established in vitro [26, 81,82]. Moreover, Sung et al. established a B-cell line (SB)from an HCV-infected B-NHL, whose virions could infectprimary human hepatocytes, PBMCs, and an established B-cell line (Raji cells) in vitro [83]. Further studies providedevidence that HCV replicates in various hematopoietic celltypes, including peripheral dendritic cells, monocytes, andmacrophages [84–86]. Overall, despite the evidence thatHCV can infect B-cells, the results about its capacity toreplicate in B-cells and other blood mononuclear cells andto induce direct malignant lymphoproliferation still appearhighly conflicting [16, 29].

A Japanese group recently established HCV trans-genic mice that expressed the full HCV genome in B-cells (RzCD19Cre mice) [87]. Interestingly, RzCD19Cremice with substantially elevated serum-soluble interleukin-2 receptor α-subunit (sIL-2Rα) levels developed B-NHL.Another mouse model of lymphoproliferative disorder wasestablished by persistent expression of HCV structuralproteins through disruption of interferon regulatory factor-1(irf-1 −/−/CN2 mice). Irf-1 −/−/CN2 mice showed extremelyhigh incidences of lymphomas and lymphoproliferativedisorders. Moreover, these mice showed increased levelsof interleukin (IL)-2 and IL-10, as well as increased Bcl-2 expression, which promoted oncogenic transformationof lymphocytes. In this mouse model, the overexpres-sion of apoptosis-related proteins and/or aberrant cytokineproduction were the primary events involved in inducinglymphoproliferation [87].

A recent study found that peripheral blood B-cellsfrom patients with chronic HCV infection were infectedand also had enhanced gene expression associated with B-NHL development when compared to healthy controls [88].Furthermore, HCV has been found to induce high mutationfrequency of cellular genes (immunoglobulin heavy chain,Bcl-6, p53 and beta-catenin genes), in B-cell lines andPBMCs in vitro, by inducing double strand breaks andby activating error-prone-polymerases and AID [89]. Thesemutations of cellular genes are amplified in HCV-associatedB-NHL in vivo, suggesting that HCV-induced mutationsin proto-oncogenes and tumor suppressor genes may leadto oncogenetic transformation of the infected B-cells. Theso-called mutator phenotype induced by HCV acute andchronic infection in B-cells may be considered a “hit andrun” mechanism of cell transformation (Figure 1(D)) [89].

It has been proposed that HCV uses B-cells as reservoirsfor persistent infection, which could result in the enhanced


expression of lymphomagenesis-related genes, particularlyAID, which is thought to be crucial for the initiation andprogression of B-NHL [67]. Other studies suggested thatthe evolution from lymphoproliferation to malignancy mayrequire a second transforming event such as the antiapop-totic Bcl-2 rearrangement. The t(14;18) translocation isindeed significantly associated with chronic HCV infectionand particularly with MC [90, 91]. Although the role of viruspenetration and replication in B-cells has still to be fullyclarified, several evidences suggest that the presence of HCVvirus or HCV proteins in these cells represents an oncogenicstimulus [16, 29].

4. Conclusion

Epidemiological studies have clearly demonstrated a cor-relation between chronic HCV infection and occurrenceof B-NHL. The clinical evidence that antiviral therapyhas a significant role in the treatment and prevention ofsome HCV-associated lymphoproliferative disorders, espe-cially indolent B-NHL, further supports the existence of anetiopathogenetic link. The mechanisms exploited by HCVto induce B-cell lymphoproliferation differ from the clas-sical mechanisms of herpesviral-induced lymphomagenesis,which require the maintenance of either EBV or humanherpesvirus-8 genomes in the transformed B-cells as clonalepisomes, together with the expression of an array of latentand, to a lesser extent, of lytic proteins [92]. It is conceivablethat the different mechanisms proposed, namely, chronicantigen stimulation, high-affinity interaction between HCV-E2 protein, and its cellular receptors, direct HCV infectionof B-cells and “hit and run” transforming events, are notmutually exclusive, but they may be combined themselves ina multifactorial model of HCV-associated lymphomagenesis(Figure 1(A–D)) [16, 26, 29, 67].


The authors indicated that there are no potential conflicts ofinterests.

Acknowledgments

This work was supported by Associazione Italiana perla Ricerca sul Cancro, Milan, Italy GRANT No. (AIRCIG10621-2010).

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Review Article

Immunological HCV-Associated Thrombocytopenia:Short Review

Dimitrios Dimitroulis,1 Serena Valsami,2 Paraskevas Stamopoulos,1 and Gregory Kouraklis1

1 Second Department of Propaedeutic Surgery, Laiko Hospital, Athens University Medical School, Sakellariou 4 Street,Alimos, 17455 Athens, Greece

2 Blood Transfusion Department, Areteion Hospital, Athens University Medical School, 11528 Athens, Greece

Correspondence should be addressed to Dimitrios Dimitroulis, [email protected]



Copyright © 2012 Dimitrios Dimitroulis et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Infection with Hepatitis C virus (HCV) is affecting about 3% of the world’s population, leading to liver damage, end-stage liverdisease, and development of hepatocellular carcinoma, being thus the first indication for liver transplantation in the USA. Apartfrom the cirrhotic-liver-derived clinical signs and symptoms several conditions with immunological origin can also arise, suchas, glomerulonephritis, pulmonary fibrosis, and thrombocytopenia. HCV-related autoimmune thrombocytopenia shows specificpathogenetic characteristics as well as symptoms and signs that differ in severity and frequency from symptoms in patients thatare not HCV infected. Aim of this short paper is to estimate the epidemiological characteristics of the disease, to investigate thepathogenesis and clinical manifestation, and to propose treatment strategies according to the pertinent literature.

1. Introduction

Hepatitis C virus (HCV) infection is an infectious diseaseaffecting more than 170 million individuals worldwide, rep-resenting approximately 3% of the world’s population. Inthe United States HCV is the most common chronic blood-transmitted infection with about 1.6% of the American pop-ulation being infected [1, 2]. After acute infection about 80%of these individuals develop chronic hepatitis, with manyand life-threatening both hepatic and extrahepatic compli-cations. From the hepatic complication development of livercirrhosis, portal hypertension, end stage liver disease, andhepatocellular carcinoma are combined with high mortalityand morbidity and are definitely treated with liver trans-plantation. The incidence of HCV-related liver dysfunctionis increasing with time and HCV infection is now the leadingcause of liver transplantation in the United States [3–5].

Apart from these very serious complications HCVinfection is also associated with extrahepatic disorders aswell as with several immune-related conditions, such as,glomerulonephritis, Sjogren syndrome, vitiligo, pulmonaryfibrosis, thyroid disease, and cutaneous vasculitis [6–9].Hematological disorders are also common in HCV-infected

individuals ranging from lymphoproliferative diseases toautoimmune thrombocytopenic purpura (AITP) and auto-immune hemolytic anemia (AIHA), with the last two havingprobably an autoimmune underlying mechanism [6, 9, 10].AITP is characterized by accelerated platelet destruction andvariably impaired platelet production resulting in thrombo-cytopenia while in AIHA the red blood cells are affected.AITP is a classical paradigm of immune thrombocytopeniain infected individuals as it is associated also with HIV orHelicobacter pylori infections [11–15].

Aim of this paper is to evaluate the actual prevalenceof AITP in the HCV-infected population and to distinguishprobable subpopulations with different characteristics (e.g.,differences between treated and untreated HCV carriers).Beside that we tried to evaluate proposed underlying mech-anisms and treatment strategies as these parameters aredescribed in the pertinent literature.

2. Epidemiology

The attempt to identify the exact prevalence and other epi-demiological characteristics of AITP among HCV-infectedindividuals and especially to distinguish these characteristics


between subgroups has objective difficulties, as the publishedstudies in the pertinent literature are mainly retrospectiveand have several limitations. This question is also approachedfrom two different directions. Six cross-sectional studiesapproached the problem by identifying serological evidenceof HCV infection in patients with clinical diagnosis of AITP[16–21]. In a total of 799 patients with AITP 159 were HCV-infected (20%) with ranges from 10 to 36%. AITP patientswith concomitant HCV infection were significantly older incomparison with not infected (54.9± 8 years versus 40.3± 8years, P < 0.001), while there was not any distributionbetween sexes.

On the other hand some investigators approached theproblem trying to identify AITP cases among HCV-infectedindividuals. Pockros et al. identified 7 AITP cases among3440 new HCV patients over a 56-month period. The esti-mated prevalence of this hematologic disorder among thesepatients was much greater than would be expected by chance(P < 0.00001) [22]. Similar results provided also the studyby Dufour et al., where 7 out of 4345 HCV patients fulfilledthe criteria for AITP diagnosis, thus calculating the annualincidence of HCV-infection-associated AITP as being 53cases/100000/year [23]. Nagamine et al. showed also a higherincidence of low platelet count among HCV-infected patientscompared to patients infected with hepatitis B virus [10].

A very interesting and large study is that by Chiao et al.,who determined the incidence of AITP and AIHA among120691 HCV-infected and 454905 matched HCV-uninfectedUSA veterans who received diagnosis during the period 1997to 2004 [24]. The results of the study showed that theoverall incidence rate of AITP was higher among the HCV-infected compared with the HCV-uninfected individuals(30.2 per 100000 person years versus 18.5 per 100000 personyears resp.). The one year cumulative incidence for AITP ascalculated in the study among the HCV-infected populationand HCV-uninfected population was 27.4 and 12.5 per100000, respectively. In the above mentioned study there wasalso an attempt of differentiation in subgroups, showing thatthe incidence rate of AITP was slightly higher among HCV-infected individuals who received treatment in contrast toHCV-infected and untreated but not in a statistically sig-nificant manner. Interferon-α, a treatment regimen againstHCV infection, has several serious side effects, such as,thrombocytopenia that can even lead to quit from treatment[25]. These treatment adverse effects could explain theseresults.

3. Pathogenesis Symptomatology

Several pathogenic mechanisms have been proposed forAITP related to chronic HCV infection. One potent mech-anism is the specific bind between HCV and human CD81receptor on the platelet membrane, thus causing auto-antibody production against this complex leading to phago-cytosis of platelets [26]. Beside that HCV infection causesa dysregulation of the host immune system stimulatingnonspecific autoimmunity or triggering the production ofspecific autoantibodies against platelet membrane glycopro-teins, there has been a controversy in the pertinent literature

regarding the published data with respect to the presenceof these specific antibodies in patients with HCV-relatedAITP [10, 22, 27]. Though, in one report 66% of patientsinfected with HCV had detectable antiplatelet glycoproteinantibodies. However, the presence of these antibodies did nothave any clinical significance and did not affect the plateletcount [28].

There a further mechanism has also been proposed bywhich HCV can directly affect megacaryocytes and thereforecauses their depletion. The fact that both AIHA and AITPare autoimmune hematological disorders but HCV infectionmainly causes AITP to support the hypothesis that beside ageneralized autoimmune reaction there must be a specificantiplatelet autoimmune reaction. This hypothesis has ledChiao et al. to the proposal that AITP in the setting of HCVinfection should be renamed as “HCV-related thrombocy-topenia” in accordance to “HIV-related thrombocytopenia”[24]. At this point it should be mentioned that HCV-infectedpatients have further reasons to develop thrombocytopenia,such as, liver damage, impaired thrombopoietin production,portal hypertension, splenomegaly, or treatment adverseeffects.

Signs and symptoms of AITP in the setting of HCV infec-tion are more or less similar in comparison with noninfectedindividuals, but they are presented with differentiations inseverity and frequency. So Rajan et al. published a studywhere symptoms and signs of thrombocytopenia were lessfrequent in HCV-positive patients with AITP than in HCVnot infected but major bleeding was more frequent ininfected individuals (25 versus 10%, P = 0.0059) [29]. Thisfinding was also verified in the study of Chiao et al.,where 60% of the HCV-infected patients had at least oneepisode of bleeding, epistaxis, or purpura [24]. On theother hand Pawlotsky et al. could not demonstrate anydifferences between these two groups. This study, however,had limitations, such as, evaluating patients with very lowplatelets count [20].

4. Treatment

Before choosing the optimal treatment modality for HCV-related AITP one should take into consideration two majorparameters, raising thus a difficult therapeutic challenge.First these patients have a double disorder, and the attemptto cure one of them could enhance progress of the other.In addition timing to start treatment should include thefact that these patients have less frequent symptoms, butthey bleed more severe. Beside these two parameters HCV-associated thrombocytopenia might be related to immuneand/or toxic mechanisms as well as impaired platelet pro-duction as described in the section of pathogenesis. So itis often difficult to estimate the correct diagnosis for thelow platelet count in order to provide optimal treatmentstrategies. Although there are no specific guidelines regard-ing treatment of AITP in HCV-infected patients intravenousimmunoglobulin (IVIg) is first-line treatment regimen,similar to noninfected patients [30]. Here we should pointout that unlike in primary ITP the use of steroid therapyin HCV-related thrombocytopenia was never popular due


to worsening of liver function tests, rising viral load, andin some cases even causing an elevation in serum bilirubin[31]. Dufour et al. demonstrated in their study that responserate to steroids was significantly lower in HCV-positivethan HCV-negative patients (P = 0.02). Moreover, it isrecommended in the same study that steroid treatmentshould be avoided as much as possible as this can increaseviral load and develop liver damage [23]. This later studyshows similar results with previous analyses. Furthermore,in one Japanese study none of the 10 HCV-positive patientstreated with prednisone achieved any response [32]. Rajanet al. found that treatment with IVIg was proven effective inincreasing platelet counts in 90% of cases of HCV-infectedpatients but could not achieve long-term response for thesepatients [29]. Furthermore, patients who are Rh positive,who do not present with severe anemia and still preservetheir spleens are also candidates for anti-RhD treatment. Theduration of response to anti-RhD therapy is usually longer incomparison to IVIg treatment but can occasionally result inmild hemolytic anemia [33, 34].

After failure or long-time dependence to steroids or theabove-mentioned classical forms of conservative treatmentsplenectomy is proposed as second-line therapy in severalstudies. Response to splenectomy is similar for both theHCV-infected and for the HCV seronegative patients [16,32]. At this point it should be mentioned that, before propos-ing splenectomy as second-line therapy, we should verycarefully examine the status of the patient’s liver function,the stage of probable liver cirrhosis and the presence of portalhypertension or portal vein thrombosis.

Apart from classical forms of first- and second-line ther-apeutic proposals for HCV-related AITP further regimenshave been used for selected cases. Pockros et al. reportedremarkable responses under cyclophosphamide in two severeAITP cases. Due to its toxicities, though, cyclophosphamideshould be limited to severe and refractory cases [22]. Dufouret al reported in their study that two out of four patientswith relapsing AITP despite steroids and IVIg experienceda sustained response after rituximab treatment [23]. Rit-uximab is a monoclonal antibody reacting with the CD20antigen which is present on B cells. Efficacy of this antibodyhas been reported in several autoimmune conditions, suchas refractory severe AITP associated with HCV infectionor HCV-related-mixed cryoglobulinemia [35–37]. However,due to its immunosuppressive properties rituximab shouldbe proposed only for refractory or associated with severebleeding cases. Further immunosuppressive agents thathave also been used are azathioprine, cyclosporine A, andmycophenolate mofetil [38].

Antiviral therapy was also proposed in several studiesfor nonresponders to classical regimens. Approximatelyhalf of adult patients with HCV-related AITP treatedwith interferon-α responded with a rise in platelet count.Furthermore, responders to IFN-α could be distinguishedfrom nonresponders by a decrease of the HCV RNAload and hepatic enzymes. Iga et al. reported significantimprovement of platelets counts in twelve HCV-infectedpatients who were complete responders to IFN-α treatment,but no raise in the platelet count was seen in patients

where IFN-α therapy failed as estimated by the viral load[27, 39].

New prospective in the treatment of HCV-related AITPis given with the use of thrombopoiesis-stimulating agents.Eltrombopag and romiplostim are small molecule throm-bopoietin (TPO) receptor agonists proven to be effective inraising platelet count in patients with relapsing or refractoryITP [40, 41]. The response rate of the patients treated withhigh dose of eltrombopag was 70–80% compared with 11%response rate that was seen in patients who received placebo.Furthermore, McHutchison et al. described a rise in thenumber of platelets in HCV-infected patients with AITPto that point that these patients were eligible to continueantiviral chemotherapy without any dose reduction orinterruption [42]. A third confirmatory study was performedby Bussel et al. comparing for a period of six weeks 50 mgof eltrombopag against placebo. Beside the encouragingresponse rates of this study some patients developed signif-icant liver enzyme elevations requiring cessation of medica-tion. Hepatotoxicity has been reported in patients receivingeltrombopag. In these cases discontinuation of the drug isadvised. It is worth mentioning that to date hepatotoxicityhas not been observed in patients treated with romiplostim[34]. A further concern regarding adverse effects of thesenew agents is the development of venous thromboembolicevents (VTEs). Nevertheless no significant increase in inci-dence of VTEs has been reported in patients who receivedeltrombopag or romiplostim compared with the incidenceof this phenomenon in ITP patients. These events alsowere not related to the number of platelets. However, theseagents should be used with caution in patients with pre-disposing factors for arterial or venous thromboembolism[43]. Another worrisome safety issue of TPO mimetics isthe development of bone marrow fibrosis with increasedreticulin deposition. Reticulin fibrosis appears to be a rare,dose depended, and reversible side effect of TPO mimeticsthat resolves after discontinuation [34].

As a conclusion one could say that the multifactorialpathogenesis of HCV-related autoimmune thrombocytope-nia is much more reflected on the option for the best treat-ment modality. A clear diagnosis is sometimes difficult tobe made and in these cases the treatment regimens arecontroversial. Further studies are needed as new pharmaco-logical agents in combination with former conservative andinterventional therapeutic strategies should fulfill a doublepurpose: raise platelet count and allow continuation ofantiviral treatment.

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Review Article

Hepatitis C Virus Infection and Mixed Cryoglobulinemia

Gianfranco Lauletta,1 Sabino Russi,1 Vincenza Conteduca,1 and Loredana Sansonno2

1 Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, Liver Unit,University of Bari Medical School 70124, Bari, Italy

2 Department of Biomedical Sciences, University of Foggia, 71122 Foggia, Italy

Correspondence should be addressed to Gianfranco Lauletta, [email protected]

Received 11 May 2012; Accepted 11 June 2012


Copyright © 2012 Gianfranco Lauletta et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Hepatitis C virus (HCV) chronic infection is recognized as the major cause of mixed cryoglobulinemia (MC). Its persistencerepresents a continuous stimulus for host immune system with production of circulating immune complexes (ICs), one-third ofthem with cryoprecipitate property. Several factors contribute to the biological activities of ICs, many of which are not completelyknown. Among them, complement factors play a crucial role in the cold-insoluble ICs-mediated vasculitis, involving primarilysmall blood vessels in different tissues including skin, kidney, peripheral, and central nervous system. Liver represents the majortarget of HCV infection with inflammatory infiltrates, resembling secondary lymphoid follicles. Cytokine like CXCL13 contributeto B-cell homing in intraportal lymphoid aggregates, in which B-cell clonal selection may arise. B-cell clonal expansion startsas an antigen-driven event and expands towards indolent and malignant B-cell proliferation. Occurrence of intrahepatic B-cell clonalities correlates with extrahepatic clinical manifestations of HCV infection. In this context, cryoglobulinemic patientsshould be considered a peculiar HCV-infected population that needs a clinical multidisciplinary approach and more articulatedtherapeutic measures.

1. Introduction

Hepatitis C virus (HCV) is a Flaviviridae family mem-ber, genus Hepacivirus, infecting about 200 million peopleworldwide [1]. About 80% of HCV-infected patients developchronic hepatitis. Among them, 10–20% evolve into cir-rhosis, while 1–5% of cirrhotic patients display an hepa-tocarcinoma [2]. Although HCV is primarily hepatopathic,its clinical feature is characterized by the emergence ofseveral extrahepatic manifestations. Mixed cryoglobulinemia(MC), recognized as the most common HCV-induced extra-hepatic disease, is an immune-complex-mediated vasculitisinvolving small vessels characterized by an underlying B cellproliferation [3]. Since B-cell clonal expansion is hallmark ofMC, B-cell malignant evolution may reflect the occurrenceof additional genetic accidents [4].

Here, we will discuss the currently accepted pathogeneticmechanisms that characterize cryoglobulinemic vasculitiswith its peculiar clinical manifestations, the molecular events

proposed to explain the potentially malignant evolution, andthe current therapeutic approaches.

2. The Virus

HCV genome is about 9,600 kb length and encodes for asingle protein from an open reading frame of over 9024nucleotides. This single polyprotein is subsequently cleavedinto several structural and nonstructural proteins. Thestructural proteins are represented by core and two envelopeproteins (E1 and E2), starting from the 5′ end [1]. Theion channel protein p7 derives from E2 cleavage [5] andis followed by the six nonstructural proteins, namely, NS2,NS3, NS4A, NS4B, NS5A, and NS5B. In addition, anotherprotein called F or ARFP can be produced from a frame-shift of the core protein [6]. At the 5′ and 3′ ends ofHCV genome there are two untranslated regions (UTR);the 5′UTR is a highly conserved region constituted by 341nucleotides that contains an internal ribosome entry site


(IRES) for translation. The 3′UTR is constituted by 200 to235 nucleotides and contains a variable region, a poly U/UCstretch and a highly conserved 98 nucleotide sequence [7].

During the replicative stage, HCV genomic RNA is tran-scribed into a complementary RNA strand. This “negative”strand constitutes a template for a new genomic synthesisand its identification represents a convincing evidence ofactive replication [8]. Viral proteins are the result of a co- andpost-translational cleavage of a single polyprotein, while hostpeptidases catalyze the cleavage of structural proteins. HCVparticles form a membrane-associated replication complex;after genome amplification and protein expression, progenyvirions are assembled and released [9, 10] (Figure 1).

3. The Cryoglobulins

Cryoglobulins are immunoglobulins (Igs) characterized byinsolubility at low temperature (below 37◦C) and redissolv-ing after warming. The first observation of a cryoprecipi-tation was registered in the serum of a patient affected bymultiple myeloma in 1933 [11], even if the term “cryoglob-ulin” was introduced by Lerner and Watson in 1947 [12].Meltzer and Franklin first described the cryoglobulinemicsyndrome in 29 patients associating cryoglobulin productionto a symptomatologic clinical triad characterized by purpura,arthralgias, and weakness [13], with increased serum levels ofrheumatoid factor (RF) and/or organ dysfunction.

On the basis of their immunochemical composition,cryoglobulins are classified as single (type I) or mixed (typeII and III) [14]. Type I cryoglobulinemia consists of amonoclonal Ig, more frequently of IgM or IgG isotype.IgM cryoglobulins occurs in almost 6% of malignant IgMparaproteinemias, whereas IgG cryoglobulins characterizealmost 2% of all myelomas. Type I IgA cryoglobulins are rare[15]. Type II MC accounts for 50–60% of all cryoglobulins.It comprises an IgM monoclonal component, frequentlymounting light k chains, and polyclonal IgG. IgM moleculesdisplay a rheumatoid factor activity capable of reacting withintact IgG and/or its F(ab)2′ fragment [16]. No monoclonalcomponent is contained in type III MC that accounts for 30–40% of cryoglobulins. Some authors have noted that type IIIMC may represent a transition form evolving into type II MC[17].

Mixed cryoglobulins are potentially present in the courseof connective tissue and autoimmune diseases, and chronicinfections [18, 19]. The term “essential” defines cryo-globulinemic syndromes without an underlying identifiabledisease. It is now accepted that the majority of them occursin HCV chronically infected patients [20] as the result ofspecific interactions between the virus and the host immunesystem [21]. The clinical picture is characterized by thecutaneous manifestations ranging from palpable purpura oflower limbs to chronic torpid cutaneous ulcers more frequentin the supramalleolar regions. Skin reactions include Ray-naud’s phenomenon, livedo reticularis, urticarial, and edema(Figure 2). Arthralgias more frequently involve the handsand knees symmetrically. Weakness is nearly always present.Kidney, liver, and nervous system are frequently involved.

Renal injury may complicate MC in almost 30% of casesand in 20% of whom nephropathy is present at the diagnosis[22–24]. Clinical features like hypertension, proteinuria,microhematuria, red blood cell casts, and renal failure havean indolent course in about 50% of cases. Less commonare nephritic (14%) or nephrotic (21%) syndromes [25].A defined picture of cryoglobulinemic glomerulonephritisevolve into chronic renal failure in 14% of cases after a meanfollowup of 6 years [26].

Although kidney involvement is a common feature ofsystemic vasculitis, cryoglobulinemic nephropathy is consid-ered as a distinct clinical and pathological entity and theetiological role of HCV has been extensively investigated[27]. Type I membranoproliferative glomerulonephritis ispredominantly associated with HCV infection [28, 29]. Themechanism of HCV-induced renal damage is unclear. HCVcore protein resulted homogeneously distributed along theglomerular capillary wall and tubulo-interstitial blood ves-sels [30] in association with an anticore activity, suggestinga major role of these immune complexes in the pathogenesisof renal damage [31].

The involvement of the nervous system in the course ofHCV-related MC ranges from 17% to 60% [32]. Sometimes,peripheral neuropathy can represent the first clinical sign ofcryoglobulinemia [33]. Peripheral nervous system involve-ment presenting with sensory-motor neuropathy especiallyof the lower limbs, is often characterized by paresthesias withloss of strength, pain, and burning sensations [34]. Less fre-quent is central nervous system involvement, characterizedby transient dysarthria, hemiplegia, and confusional state[35].

Liver is involved in almost 70% of cases, often with ahistopathologic picture of chronic active hepatitis with orwithout cirrhosis [36, 37].

Less common clinical pictures of cryoglobulinemicvasculitis are represented by gastrointestinal (2–6%) andpulmonary (5%) involvement. Intestinal ischaemia may arisewith acute abdominal pain; intestinal perforation is alsodescribed as well as symptoms that mimic cholecystitisand/or pancreatitis [38]. Interstitial pneumopathy may char-acterize patients displaying dyspnea and dry cough, whereasan acute alveolar haemorrhage with haemoptysis, respiratoryfailure, and a radiologic demonstration of multiple infiltratesis rare [39, 40].

4. HCV Chronic Infection and MC

After the identification of HCV as the etiologic agent of non-A, non-B chronic hepatitis and the availability of a serologictest for the demonstration of IgG anti-HCV in the early1990s, several authors described an intriguing associationbetween HCV infection and “essential” MC, apart from somegeographical differences [36, 41, 42]. These association wassubsequently confirmed by detection of viral genome in seraof cryoglobulinemic patients with a selective concentrationin cryoprecipitates [21, 43]. Incidence of HCV infection inMC ranges from 40 to 90% [22]. Otherwise, HCV-negativeMC accounts for about 5–10% [44].


C NS5a NS5b

E2

Endocytosis

Uncoating

RNA replicative strands

Translation

ER lumen

Cytoplasm

Golgi

Virion assemblyMaturation

Exocytosis

Negative Positive

E1

p7

NS2NS3

Figure 1: HCV life cycle in host cell. During the replicative stage, after endocytosis, HCV genomic RNA is transcribed into a complementary(negative) RNA strand. After genome amplification and structural and nonstructural viral protein expression, progeny virions are assembledand released.

Cryoglobulins classification

Type I: Monoclonal cryoglobulins (IgM or IgG, rarely IgA)

Type II: Mixed cryoglobulins with a monoclonal

Type III: Policlonal mixed cryoglobulins (IgG + IgM)

component (IgMk + IgG)

Type II/III: Policlonal IgG + oligoclonal IgM

(a) (b)

(c) (d)

Figure 2: Clinical aspects of cryoglobulinemia. (a) Cryoglobulins classification; (b) lower limbs purpuric manifestations; (c) cutaneousulcers; (d) livedo reticularis.


Core protein

IgG

IgM

C1q

gC1qR

Endothelial cell

Figure 3: Pathogenetic model of cryoglobulinemic tissue damage.HCV core protein, which has been detected in cryoprecipitateimmune complexes, interacts with C1q protein and the receptorfor the globular domain of C1q protein (gC1q-R) on the surfaceof both circulating blood and endothelial cells. Cryoprecipitatingimmune complexes, including gC1qR complexed to HCV core andC1q proteins, bind in turn IgM molecules with rheumatoid factoractivity linked to anti-HCV IgG.

The intrinsic mechanism by which HCV promotes cryo-globulin production is unclear. Virus persistence, therefore,may represent a continuous stimulus for host immunesystem unable to produce neutralizing antibodies [45, 46].In this context, cryoglobulins may represent the product ofvirus-host interactions in HCV-infected patients, whereasthe production of IgM molecules with RF activity is a crucialevent in the cryoprecipitating process [22]. The majority ofthese IgM molecules are almost always associated with lightchain cross-idiotype 17.109 and heavy chain cross idiotypeG6 [47]. These cross-idiotypes are considered as the productof a restricted expression of germline genes [19].

It has been hypothesized that the composition of ICsin the course of chronic HCV infection includes IgM-17.109 RF molecules which bind anti-HCV IgG [48]. Amongviral antigens, the core protein plays a crucial role incryoglobulins constitution being the relevant ligand forIgG [31]. Interaction between HCV and lymphocytes iscapable of modulating cell functions; in particular, an invivo activation and expansion of CD5-positive B cells hasbeen considered the major source of IgM RF moleculesin type III MC [49, 50]. Therefore, it has been postulatedthat an initial activation of these cells may be followedby the emergence of a dominant clone that synthesize amonoclonal RF supporting the development of type II MCafter a transition phase in which an IgM clonal heterogeneitymay define a type II-type III variant [17]. In a subsetof HCV-positive patients with MC, a clonal expansion ofIgM+CD27+ B cells expressing hyper-mutated RF-like Ighas been demonstrated in peripheral blood in associationto VH1–69/JH4 and VH3–20 gene segment restriction [51].These findings have been interpreted as a B-cell proliferationinduced by specific antigen stimulation, thus sustaining thenotion that persistent B-cell stimulation may represent a firststep to malignant evolution.

A crucial role in the composition of cryoprecipitatingICs is played by complement system. Generally, complementbinding to setting up ICs decreases the size maintainingthem in solution [52]. Mean levels of C3 and C4 fractionsin the soluble phase of MC patients’ sera correlate to verylow amounts in cryoprecipitates thus suggesting the existenceof two different compartments characterized by a distinctcomplement activation [22]. On the contrary, C1q proteinand C1q binding activity result significantly enriched in thecryoprecipitates [31]. These data support the hypothesis thatan efficient engagement of C1q protein by cryoglobulins mayrepresent a crucial factor in the pathogenetic pathway of MC.

HCV-encoded core protein interacts directly with thereceptor for the globular domain of C1q protein (gC1q-R)representing an efficient way to affect the host T- and B-cell immunity. This interaction has been considered capableof modulate T-cell immune response and, on the otherhand, circulating HCV core protein engagement with gC1q-R expressed on the surface of B-lymphocytes may represent adirect way by which the virus can affect host immunity [53–55]. The wide expression of gC1q-R on the surface of bothcirculating blood immunocytes and endothelial cells maydetermine a specific binding to HCV core protein-containingICs.

Recently, it has been demonstrated that MC patientsdisplay higher levels of soluble gC1q-R that reflects a higherspecific mRNA expression in blood mononuclear cells [56].It was also demonstrated that soluble gC1q-R circulates as acomplexed form containing both C1q and HCV core proteinin two different binding sites of the molecule (Figure 3).

C4d, a low-molecular-weight fragment derived fromthe cleavage of C4 complement fraction following classiccomplement pathway activation, results are lower in MCpatients’ sera than in chronic HCV carriers or in healthysubjects [56]. Otherwise, C4d fragment deposits characterizealmost all skin biopsy samples of cryoglobulinemic vasculitis.These data lead to hypothesize that low circulating C4d levelsare the result of sequestered fragments in the vascular bed.In vitro experiments showed a peculiar property of MCpatients in that, in step with HCV core inhibition of theperipheral blood lymphocytes (PBL) proliferative response,large amounts of soluble gC1q-R were found in culturesupernatants. It can be inferred that gC1q-R synthesis andits release from PBL are HCV core mediated and negativelyregulated by cell proliferation [56].

In conclusion, in the presence of high levels of circulatinggC1q-R, HCV core protein can exacerbate the inflamma-tory condition by activation of complement cascade thusdetermining endothelial cell activation starting an in situinflammatory response. From a biological point of view,clinical response to antiviral therapy is characterized bya significant reduction of soluble gC1q-R associated toincreased levels of C4d and lower viral load [56].

5. HCV Infection and Lymphoid Cells

HCV is capable of directly modulate B- and T-cells functions[57]. The monoclonal IgM RF production can be consideredas the expression of a single dominant clone following the


DC

Antigenic stimulus

Macrophages

T cell

B cellStromal

cell

Parenchymalcell

CXCR5

Inflamed blood vessel

CXCL13

Initiation of inflammationProgression to

chronic inflammationOrganization of secondary

lymphoid follicles

FDC

Secondary B-cellfollicle with germinal

centre

CXCL13LTα1β2

LTβR

CXCL13: CXC motif chemokine ligand 13CXCR5: chemokine receptor 5

LTβR: lymphotoxin-β receptor

LTα1β2: lymphotoxin-α1β2DC: dendritic cellFDC: follicular dendritic cell

cc

Figure 4: Schematic representation of chronic inflammation and organization of secondary lymphoid follicles in HCV chronic infection.The ability of HCV to chronically persist in the host may represent a continuous stimulus for the immune system resulting in B-celloligo/monoclonal expansions with selective advantage to clones depending on antigen stimulation. Some chemokines may play a crucialrole in the establishment of an adequate microenvironment for activation and expansion of B-lymphocytes in response to signals providedby antigen-presenting cells. Among them, CXC motif chemokine ligand 13 (CXCL13) and its chemokine receptor 5 (CXCR5) are importantfor secondary lymphoid tissue development and distribution of lymphocytes within microenvironments. CXCL13 is released by endothelialand stromal cells mediated by lymphotoxin-β receptor (LTβR) signaling and contributes to lymphoid homing in the liver by the creation ofa favourable microenvironment sustaining focal B-cell aggregation similar to lymphoid follicles.

initial stimulation, thus supporting type II MC development[17, 50]. The ability of HCV to chronically persist in the hostmay represent a continuous stimulus for the immune systemresulting in B-cell oligo/monoclonal expansions (Figure 4)[4].

HCV recognizes different binding molecules on cellssurface that are not completely identified. Among them, themost known are CD81 [58], scavenger receptor class B type I[59], and low-density lipoprotein receptor [60]. MC patientsare distinctly characterized by higher levels of cell-associatedviral load, because a significant enrichment of HCV RNA inPBL has been demonstrated [61]. This peculiar feature maybe considered as the result of a higher density [62] and/orpolymorphism of receptor genes [63, 64], whereas directinfection and replication of HCV in B cells may promotelymphocyte proliferation [65].

The presence of HCV minus-strand RNA is the key factorto demonstrate an active viral replication in cells, whereasthe presence of plus-strand RNA may indicate a possiblecontamination by circulating virions. By means of a highlyspecific and sensitive method for the detection of HCV RNAminus strand an active viral replication in lymphoid cellsfrom MC patients has been demonstrated [66]. These resultssuggest that there is a direct correlation between HCV activeinfection of lymphoid cells and MC. In a cohort of MCpatients, PBL may be considered another HCV productive

infection compartment, in addition to the liver, representinga circulating reservoir of HCV infection [67].

Although no specific viral protein has been indicated asBCR ligand [68], analysis of Ig variable gene (IgV) sustainan antigen-driven B-cells expansion. IgV heavy and lightchain genes are always mutated as occurs in germinal orpost-germinal center origin of B cells [69, 70]. The presenceof hypermutated IgV genes capable of recognizing a singleepitope suggests that they arise randomly from the B cellpool [70] selected for non-self-antigens. Otherwise, most B-cell expansions show a CDR3 with significant homology toRF-CDR3 [68, 70], suggesting a distinct pathogenesis sincethese B-cell clonalities derive from precursors with auto-IgG specificity [71]. It has been demonstrated that in someHCV-positive MC patients, BCR recognize IgG-Fc and HCV-NS3 domains, suggesting that its specificity derives from across-reactivity between a virus-associated epitope and IgGautoantigen [72]. This mechanism may also contribute to thevirus enrichment on the lymphoid cells in MC patients, thusconditioning RF B cells to undergo cell cycle and secrete RFmolecules [73].

6. HCV and Lymphoproliferation

In the course of B-cell proliferation, several mutants mayderive from IgV genes somatic mutations. By means of


polymerase chain reaction technique (PCR) directed againstthe variable-determining-joining region (VDJ), it is possibleto identify the combination of N regions along with differentDH and JH regions. This unique combination represents aclonal marker of cell progeny. The application of this methodleads to the demonstration that B-cell clonal expansions arepresent in the liver tissue of almost 90% of HCV-positiveMC patients if compared with blood and bone-marrowcompartments [73].

HCV chronic infection is characterized by the devel-opment of inflammatory infiltrates involving the portaltracts. These infiltrates often appear as follicle-like structuresresembling a germinal center functionally active [74, 75].VDJ pattern obtained from these patients resulted in oligo-clonality to monoclonality, suggesting that intrahepatic B-cell expansions raise from very few or single cells. In addition,each focus may derive from different B cell of the polyclonalrepertoire, resulting in the development of unrelated clones.

The occurrence of intrahepatic B-cell clonal expansionsprofoundly influenced the clinical spectrum of HCV infec-tion, since it was associated invariably with extrahepaticmanifestations including cryoglobulinemia, high serum lev-els of RF activity, monoclonal gammopathy of undeterminedsignificance, and also B-cell non Hodgkin lymphoma. Clonalexpansions display a restricted V gene usage, thus confirm-ing a direct relation with clinical manifestations [76]. Inaddition, sequence analyses of IgH CDR3 gene segmentsof intraportal B-cell clonalities revealed a wide range ofvariations, suggesting that they are the result of an antigen-driven response [77]. These findings lead to hypothesize thatB-cell clones start expanding in the liver as a consequenceof an upregulated IgH-VDJ mutational activity and thenmigrates in the circle and also bone marrow [76].

However, the relationship between emergence and per-sistence of intrahepatic or circulating B-cell clones andHCV infection remains unclear. Several pieces of evidenceindicate that some chemokines can play a crucial role inthe establishment of an adequate microenvironment foractivation and expansion of B-lymphocytes in response tosignals provided by antigen-presenting cells [78]. Amongthem, CXC ligand 13 (CXCL13), also known as B-cellattracting chemokine 1 or B-lymphocyte chemoattractant,is important for secondary lymphoid tissue developmentand distribution of lymphocytes within microenvironments[79]. High serum levels of CXCL13 protein in MC patientsparalleled those of specific mRNA expression in liver andskin tissues, suggesting that this chemokine may representa key factor in the pathogenesis of cryoglobulin-induceddamage [80]. CXCL13 contributes therefore to lymphoidhoming in the liver by creating a local microenvironmentsustaining focal B-cell aggregation similar to lymphoidfollicles (Figure 3).

In addition, B-cell enrichment may be the result ofthe presence of some signals enhancing cell survival [81].B-lymphocyte activating factor (BAFF), also known as B-lymphocyte stimulator (BLyS), is expressed and secretedby activated monocytes, macrophages, and dendritic cells.Serum BAFF levels results increased in patients with chronicHCV infection, as well as in other autoimmune diseases

like systemic lupus erythematosus and rheumatoid arthritis,and this was correlated to autoimmune and vasculitic man-ifestations. The increased levels of BAFF may modulate thesensitivity of B cells to apoptosis prolonging their survival,thus representing another possible factor in the clonal B-cellexpansion [82].

7. Management of MC

The main goals of the therapy of MC are represented by: (a)eradication of HCV infection; (b) deletion of the underlyingB-cell clonal expansions; (c) depletion of cryoproteins.

Conventionally, in the pre-HCV era, management ofMC was based on the use of corticosteroids and immuno-suppressive drugs. Following the empirical observation in1987 of the effectiveness of recombinant IFN-α in 7 patientswith “essential” MC [83], and the subsequent demonstrationof the pathogenetic role of HCV [21], IFN-α became arational therapeutic strategy. The introduction of pegylatedIFN-α changed the therapeutic scenario of chronic hepatitisC increasing virological responses [84, 85] as well as theintroduction of ribavirin (RBV), a nucleoside antimetaboliteagent [86]. This combination, now considered the standardof care (SoC) for HCV management [87], has been shown tobe effective in a remarkable proportion of HCV-related MCpatients, resulting in a complete clinical response and sus-tained virological response (SVR) in 78% of the patients [88].In addition, serum levels of C3 and C4 complement fractionsnormalized in 80% and cryoglobulins disappeared in 56%of the patients. Even when the antiviral treatment results inresolution of vasculitis, no or only partial improvement inneuropathy and glomerulonephritis is observed, suggestingthat the clinical outcome may be conditioned by factors otherthan the virus [22].

It should also be emphasized that the occurrence ofB-cell clonal expansions is able to influence the clinicalexpression of HCV infection, in that it is consistentlyassociated with extrahepatic manifestations, like MC [76,89, 90]. Enrichment of B-cell clones in at least threedifferent compartments, namely, liver, bone marrow, and thecirculation, and expansion of RF-synthesizing B cells are thebiological hallmark of MC [22]. Consequently, deletion ofB-cell clonalities may provide a rational way to treat MC. Itis well known that CD20 antigen, a transmembrane protein,is selectively expressed on pre-B and mature lymphocytes,and that CD20-positive cells are remarkably expanded andactivated in patients with MC [61, 91].

Since rituximab (RTX), a chimeric moAb specificallydirected to CD20 antigen, has been shown to be thera-peutically effective in autoimmune and lymphoproliferativedisorders [92–94], it seemed logical to propose its use inHCV-related MC patients refractory to, or relapsing after,conventional antiviral therapy. The first papers about the useof RTX in HCV-related MC [95, 96] showed that it is aneffective, safe, and well-tolerated treatment for type II MCpatients, including those resistant to, or frequently recurringafter, previous treatments. However, a not negligible draw-back is the frequently increased viremia in the responders.


On these bases, several subsequent papers have addressed theissue of the use of RTX, alone or in combination with steroids[97, 98]

In our own study [99], a triple therapeutic combination(pIFN-α plus RBV plus RTX), designated with the acronymPIRR, was administered to 22 HCV-positive MC patients,whereas 15 additional patients with the same pathologyreceived, by comparison, pIFN-α plus RBV with the exclu-sion of RTX. Followup was protracted for 36 months fromthe end of treatment. Results showed a complete responsein 54.5% of patients treated with PIRR, and only in 33.3%of those who were given pIFN-α plus RBV without RTX(P < 0.05). Even more interesting were the observationsthat: (a) in the large majority (83.3%) of the respondersbelonging to the PIRR-treated group, a conversion of B-cellpopulations from oligoclonal to polyclonal was recorded inthe liver, bone marrow, and peripheral blood compartments;(b) compared with 40% of the control group, in all patientsof the PIRR group the CR was maintained throughout thefollow-up period. Whether RTX should be administered topatients with cryoglobulinemic vasculitis as first- or second-line therapy remains to be established [100].

Of particular interest is the question about MC patientsthat do not obtain an SVR or those patients showing a con-tinuous cryoglobulin production despite virus eradication.In the first case the use of the new direct-acting antivirals(DAAs) like Telaprevir or Boceprevir (recently approved bythe FDA for the treatment of HCV genotype 1 chronicinfection) may represent a further therapeutic option [101].Persistence of MC vasculitis in patients achieving a SVRrepresents an emerging picture following antiviral and B-celldepletive combined therapies [102, 103]. In these patients adifferent immunochemical structure of circulating immune-complexes may be postulated; the use of corticosteroids,cyclophosphamide, RTX, or ofatumumab (an IgG1k fullyhumanized CD20 MoAb) may be considered [104].

Therapeutic apheresis is a palliative procedure that can beextremely useful for the treatment of severe, life-threateningvasculitis [100] as well as for the treatment of chronic legulcers in patients resistant to other therapies [105].

Others additional therapeutic approaches for MC havebeen proposed, like tyrosine kinase inhibitor imatinib,antiangiogenic drugs like thalidomide, bortezomib (a protea-some inhibitor), and IL-2, but future controlled studies arerequired to establish if these agents will improve MC therapy[106, 107].

8. Conclusions

Although the major role of HCV in the production of cryo-globulins and systemic vasculitis has been clearly established,there are several aspects in the pathogenesis of MC that stillrequire further investigations. Particularly interesting is theB-cell expansion process that starts as a consequence of viralpersistence, with preferential involvement of RF-producing Bcells. This process seems to occur in a microenvironment likeintraportal lymphoid follicles as a result of a distinct selectionprocess probably supported by cytokine signaling sustainingB-cell activation and proliferation.

In this context, some viral proteins like core protein, maydirectly modulate the mechanism underlying ICs depositionin the vascular bed leading to cryoglobulinemic vasculitisand promote proliferation signals of B cells supporting anactive viral replication. In addition, host’s genetic factorsmay represent a crucial factor for the clinical outcome ofHCV chronic infection. These complex relations representthe biological basis for a more appropriate treatment ofthe cryoglobulinemic vasculitis that include antiviral therapyand B-cell depletion even if further studies are necessaryfor the relapsed-refractory cases in which other pathogeneticmechanisms, often antigen-independent, are involved.

Acknowledgments

This study was supported in part by a grant from theItalian Medicines Agency (AIFA), funds for independentstudies, 2007, Contract no. FARM7SJX and by a grant fromUniversity of Bari, Italy.

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Review Article

HCV Proteins and Immunoglobulin Variable Gene (IgV)Subfamilies in HCV-Induced Type II Mixed Cryoglobulinemia:A Concurrent Pathogenetic Role

Giuseppe Sautto, Nicasio Mancini, Laura Solforosi, Roberta A. Diotti,Massimo Clementi, and Roberto Burioni

Laboratorio di Microbiologia e Virologia, Universita Vita-Salute San Raffaele, Via Olgettina, 58, 20132 Milano, Italy

Correspondence should be addressed to Nicasio Mancini, [email protected]

Received 13 February 2012; Accepted 2 April 2012


Copyright © 2012 Giuseppe Sautto et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

The association between hepatitis C virus (HCV) infection and type II mixed cryoglobulinemia (MCII) is well established, butthe role played by distinct HCV proteins and by specific components of the anti-HCV humoral immune response remains to beclearly defined. It is widely accepted that HCV drives the expansion of few B-cell clones expressing a restricted pool of selectedimmunoglobulin variable (IgV) gene subfamilies frequently endowed with rheumatoid factor (RF) activity. Moreover, the sameIgV subfamilies are frequently observed in HCV-transformed malignant B-cell clones occasionally complicating MCII. In thispaper, we analyze both the humoral and viral counterparts at the basis of cryoglobulins production in HCV-induced MCII, withparticular attention reserved to the single IgV subfamilies most frequently involved.

1. Introduction

Mixed cryoglobulinemia (MC), an immune complex (IC)-mediated systemic vasculitis mainly involving the smallblood vessels, has been observed in a wide variety ofdiseases, including malignancies, chronic infections, andsystemic autoimmune disorders [1, 2]. In symptomatic MC,the presence of cold-precipitable immunoglobulins (cryo-globulins) is frequently associated with the developmentof vascular, renal, and neurological lesions [3–5]. The vastmajority (50–90%) of patients with symptomatic type IImixed cryoglobulinemia (MCII), characterized by lympho-proliferation and by the deposition of mono/oligoclonalIgM antibodies (Abs) with rheumatoid factor (RF) activitybound to oligo/polyclonal IgG, are infected with hepatitis Cvirus (HCV) [6]. Consistently, more than 40% of chronicallyHCV-infected patients present MCII, that in a relevantnumber of patients (10–60%) will eventually develop insymptomatic cryoglobulinemia [7, 8].

It has been demonstrated that antiviral treatment sig-nificantly induces remission in HCV-associated MCII and

that this effect is highly correlated with effective suppressionof viral replication, supporting a direct role of HCV inthe pathogenesis of this lymphoproliferative disorder [9].Furthermore, MC should not be considered an in situ oroccult B-cell lymphoma, as evidences indicate that its B-cellclonal expansion does not still display the molecular featuresof a true neoplastic process [10]. As a matter of fact, inmore than 50% of symptomatic patients the clinical course isrelatively benign, but 5–10% of patients with cryoglobuline-mic vasculitis develop B-cell malignancies, in particular B-cell non-Hodgkin lymphomas (B-NHL), as compared with0.2–2.6% of the overall HCV-infected population [11–15].A possible role of chronic immune stimulation associatedwith persistent infection in the pathogenesis of these malig-nancies has been hypothesized and further confirmed bythe sequence analysis of tumor-related immunoglobulin (Ig)gene rearrangements, evidencing a preferential use of thesame Ig heavy and light chain variable regions (VH and VL)genes associated with anti-HCV response and with MCII[16–18].


In this paper, after reviewing the main viral featuresassociated with MCII, we will overview the main IgV genesubfamilies described in patients with HCV-related MCIIand will evidence their correlation with the anti-HCVhumoral response and with the MCII-related neoplasticcomplications.

2. The Liver as a Lymphoid Organ

It is well known that the liver is the main target organ ofHCV infection. Within the inflamed liver, particularly in theearliest stages of the disease, there is an accumulation ofmyeloid and lymphoid cells, including follicular dendriticcells, T and B lymphocytes [19]. Local activation of thesecells is thought to play an essential role in perpetuating thechronic inflammatory process and enhancing liver damage[20]. Moreover, intrahepatic B-cell proliferation is oftenassociated with extrahepatic manifestations of HCV infec-tion, including high serum levels of RF activity, cryoglobu-lins, monoclonal gammopathy of undetermined significance(MGUS), and frank B-NHL, indicating that it has a directrole in HCV-related systemic complications (Figure 1(a))[21].

Immunohistochemical studies showed that T and B cellsfrequently accumulate in the portal tracts and organizefollicle-like structures (foci), mainly consisting in B cells sur-rounded by T-cell zones at the periphery. Within these focithere are obvious germinal centres (GCs) where activation,proliferation, differentiation, and maturation of B cells andAb production may occur similarly to lymphoid organs [22].This process is rarely observed in patients with other typesof hepatitis and seems to contribute to the pathogenesis ofHCV immune-related disorders, as suggested by functionaland molecular analyses showing that these structures arecharacterized by B-cell oligo/monoclonal expansion [23].Indeed, these clonal expansions have been observed inthe liver of almost 50% of HCV-infected patients and,less frequently, in their blood and bone marrow [21]. Inparticular, Sansonno et al. observed a monoclonal patternonly in HCV-infected patients with MC, while Magaliniet al. observed it both in MC and non-MC HCV-infectedpatients, supporting the view that HCV per se is able toderange the functions of the immune system [24, 25]. Fur-thermore, isolated intrahepatic B lymphocytes were shownto produce IgM molecules with RF activity, supporting theintrahepatic origin of HCV-related autoimmune processes[24, 26]. Interestingly, these intrahepatic focus-forming Bcells frequently express a restricted repertoire of VH andVL subfamily genes, as discussed more in detail below.Each single focus may derive from a single B cell, with theresult that distinct foci contain unrelated B-cell clones withsingle-antigen (Ag) specificity (Figure 1(a)) [27]. Althoughcontinuous viral antigenic stimulation is probably the mainfactor determining the formation of intrahepatic GCs inHCV-infected patients, the reason why, differently fromother hepatotropic viruses, HCV preferentially induces theirformation in the liver is currently uncertain. It mightbe related to the unique virological properties of HCV,

including preferential induction of an autoreactive humoralimmune response.

Taken together, these observations suggest that, duringHCV infection, the liver acts as an important secondarylymphoid organ where autoimmune processes may originate(Table 1). The main viral factors and the specific componentsof the anti-HCV humoral response involved in this processwill be reviewed in the following paragraphs.

3. HCV Proteins and B Cells

HCV is an enveloped, positive-stranded RNA virus, charac-terized by an extreme variability, even within a single host.On the basis of some conserved regions it can be dividedin seven genotypes and numerous subtypes [28, 29]. AllHCV genotypes have been associated to MCII, althoughseveral reports describe its higher prevalence among patientsinfected with genotypes 1 and 2a [19, 24, 30–34]. However,the differences in the geographical distribution of HCVgenotypes may bias this apparent correlation.

HCV genome is approximately 9600 nucleotides longand encodes a polyprotein precursor of about 3000aminoacids. It is cleaved by viral and host proteases, resultingin a series of structural (core, E1 and E2) and nonstructuralproteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B)[35]. Some HCV proteins have been demonstrated to directlyactivate important proinflammatory cascades in monocyteand T cells. This activation may serve to lower activationthreshold thus enhancing cellular response to Ags, includingauto-Ags [36]. This may provide B cells with a pro-inflammatory environment and a myriad of costimulatorysignals promoting clonal expansion. Moreover, sequencedata on BCR, the presence of HCV Ags in the cryoprecipi-tates, and a reported correlation between HCV viral load andclinical manifestations of cryoglobulinemia in some patientssupport the model of an Ag-driven origin for HCV-relatedlymphoproliferative disorders [30]. In this paragraph weinvestigate this last point with particular attention to thedifferent HCV proteins possibly involved.

3.1. Core Protein. Several reports describe the presence ofHCV RNA and HCV proteins in the cryoprecipitate ofpatients with HCV-related MCII. In particular, the coreis supposed to be the most involved viral protein incryocrit formation, as demonstrated in the skin and renaltissues of HCV-infected patients with MCII-associated activevasculitis and nephropathy, respectively [37]. Nonenvelopedcore protein is overproduced during virogenesis, and inMCII patients its plasmatic levels have been correlated tocryoglobulinemia-associated symptoms [37, 38]. Indeed, ithas been demonstrated that HCV core protein participates inthe formation of immune complexes (ICs) and suppresses T-cell response by interacting with the globular domain of C1qcomplement receptor (gC1qR) (Figure 1(b)) [39, 40]. Thisinteraction may play a key role in determining complementactivation, a crucial interdependent regulator of the size andsolubility of immune aggregates [41].

Sansonno et al. demonstrated the presence of high levelsof HCV core protein in the cryoprecipitates of patients with


HCV

HCV-driven stimulation and expansion of a restricted B-cellrepertoire

B BB

BB

BB

BCR

RF activity

Extrahepatic manifestations:↑serum RF activity, cryoglobulins,MGUS, B-NHL (Ag-independent)

Oligo/monoclonal expansion:-Liver-Bone marrow-Blood

Ag-dependentstimulation

E2 and NS3 molecular mimicry of Ig domains

RF producing B-celltargeting shared structures:-E2/IgV- NS3/Fc

HCV/E2

HCV/NS3

B

Ag-drivenstimulation

Core, anticore IgG and RF IgM deposition pathways

HCV/core

Anticore IgG

RF IgM

Anticore IgG

HCV/core

RF IgM

C1q

gC1qRendothelium,

neutrophilgranulocyte

Proliferation and prooncogenic signals

CD81

B

B BB

BB

B

HCV/E2

HCV/NS3

HCV/core

Oligo/monoclonal expansion,

lymphoma

BCR

Immortalizationantiapoptotic and

proliferation signals

(a)

(b)

(c)

(d)

Super-Ag?

↑CD5 + and cryoglobulins?,

HCV entry andreplication?

natural Ab WA-Id

Figure 1: Proposed etiopathogenetic mechanisms involved in the origin of HCV-induced MCII. (a) Direct involvement of HCV infectionand of specific HCV Ags in the emergence and maintenance of B-cell expansions, more frequently occurring in the liver and mostlyinvolving RF-producing B cells. This B-cell repertoire is therefore limited and likely coded by few germline genes. These clonal expansions areinvariably associated with extrahepatic manifestations, including high serum levels of polyclonal rheumatoid factor activity, cryoglobulins,monoclonal gammopathy of undetermined significance (MGUS), and eventually frank B-cell non-Hodgkin lymphoma (B-NHL). (b) Thewide expression of gC1qR on the surface of blood cells, like neutrophil granulocytes, as well as of endothelial cells favors their specificbinding to immune complexes containing HCV core protein and may determine their cold precipitation. Alternatively, IgM moleculesare good acceptors of C1q, whose binding site is on their Fc portion and, if endowed with RF activity, may precipitate in presence ofIgG molecules with specific anticore activity. (c) HCV/E2- and HCV/NS3-induced proliferation and expansion of B-cell clones producingcross-reactive Ig recognizing structures shared between these Ags and discrete Ig regions (i.e., Fc or IgV domains). (d) HCV might initiate amultistage process of lymphomagenesis by replicating in lymphoid cells and expressing proteins that associate with host cell-encoded tumor-suppressing proteins, thereby abrogating their cell-cycle checkpoint functions and predisposing the cell to genetic instability. Alternatively,HCV/E2 binding to CD81, as part of the CD19/CD21/CD81, might provide a strong proliferation signal. Moreover, HCV/E2 could behaveas a B-cell super-Ag and directly stimulate proliferation and oligo/monoclonal expansion through its direct binding to BCRs encoded byspecific IgV subfamilies.

MCII [42]. These cold-insoluble precipitates included poly-clonal IgG and monoclonal IgM molecules with RF activity.In particular, the polyclonal IgG component showed specificreactivity against HCV core protein and, in turn, was linkedthrough its Fc portion to IgM with RF activity. Importantly,cryoprecipitation was directly correlated with anticore IgGconcentration in the cryoprecipitate, thus inferring that itsproduction is dependent on their selective binding to the Agin the presence of IgM molecules with RF activity. Thus, IgMRF acts as an incomplete cryoglobulin, precipitating at lowtemperature, probably following a conformational changeinduced by their binding to IgG with anti-core reactivity.

In the same report, the above-described HCV coreprotein interaction with the gC1qR was proposed as anotherpathogenetic mechanism, making T cells unable to suppressRF producing B-cell clones generated by chronic antigenicchallenge. The presence of gC1qR on the surface of bothcirculating blood cells and endothelial cells may favor theirspecific binding to HCV core protein-containing ICs [5, 43].Moreover, IgM molecules are good acceptors of C1q andindeed can favour indirect binding of HCV core protein toendothelial cell surface (Figure 1(b)) [44].

Finally, HCV core protein has also been shown topromote immortalization in different cell lines, as well as


Table 1: Schematic representation of viral and humoral factors implicated in the instauration of HCV-related MCII and B-NHL.

Implicated factors Associated mechanisms References

HCV proteins

Core(i) Complement activation(ii) Cryoprecipitation(iii) ↓ T cell response

[37, 39, 41–43, 45]

E2

(i) Molecular mimicry of IgV domain(ii) Induction of RF(iii) Proliferation and transformation signals

following interaction with CD81/BCR(iv) Super-Ag?

[46, 47, 52–59]

NS3(i) Molecular mimicry of IgG-Fc domain(ii) Oncogenesis

[69, 71]

IgV subfamilies

VH1-69

(i) Expansion of CD5+ B cells?(ii) WA with RF activity(iii) Expansion of natural IgM

expressing B cells

[52, 55, 76, 78, 79,97–99]

Heavy chains VH4-34 Naturally autoreactive [79]

VH4-59, VH3-7,VH3-21, VH3-23,

VH3-48 and VH3-30Mono/oligoclonal expansion following HCVinfection

[75, 77, 100–102]

Light chainsVκ3-15 [80]

Vκ3-20 WA with RF activity[77, 80, 97, 103,104]

being capable of blocking c-myc induced apoptosis andindeed could have a direct role in the pathogenesis of HCV-related lymphomas [45].

3.2. E2 Envelope Glycoprotein. HCV/E2 envelope glycopro-tein is another viral protein possibly involved in the devel-opment of HCV-related lymphoproliferative disorders [46].A hint suggesting HCV/E2 role comes from the reportedmolecular mimicry of its N-terminal hypervariable region(HVR1) with some conserved motifs in selected human Igvariable domains, as well as with the T-cell receptor (TCR)alpha and beta chains (Figure 1(c)) [47]. This molecularmimicry would make anti-HVR1 Abs potentially capableof cross-reacting with other Abs or with TCR. Indeed, theexpression of proteins structurally similar to host defenseproteins and immunomodulators is an important immune-evasion strategy leading to persistence, already described forother viruses [48–51]. Confirming this, Ferri et al. demon-strated a low pattern of HVR1 mutations in HCV-positivepatients with MCII and presenting a monoclonal IgMexpansion with RF activity [52]. This low variability in themain target of anti-HCV humoral response was interpretedas a clear sign of impaired response, as already observedin agammaglobulinemia or in otherwise immunosuppressedpatients [53, 54]. Interestingly, a direct correlation has beenobserved between the degree of similarity of HCV/E2 HVR1to Ig and TCR molecules and the degree of immune escapeand persistence in humans and experimentally infectedchimpanzees [47]. This indicates that variation in HVR1sequence is not only correlated with escape from neutralizing

Abs but also to an improvement in Ig similarity consistentwith a model of immune evasion through mimicry. Overall,this mimicry could determine a chronic stimulation inducedalso by self-Ags, thus leading to the HCV-related lymphopro-liferative disorders.

As a matter of fact, in HCV-associated B-NHL, malignantmonoclonal B cells often secrete IgM endowed with RFactivity which feature high sequence homology with anti-HCV/E2 Abs [55]. Thus, it has been suggested that theseforms of B-NHL may originate from monoclonal prolifer-ation of RF-secreting B cells stimulated as reported above(Figure 1(c)) [52]. Consistently with this observation, Igscloned from a patient with an HCV-associated B-NHL wereshown to bind to HCV/E2 [56].

Finally, it has been postulated that HCV may also exert adirect stimulating activity on B cells following HCV/E2 inter-action with the cellular receptor CD81 during the viral par-ticle internalization [57, 58]. In fact, the CD81 tetraspaninon B-cell surface may provide a strong stimulatory signal ifactivated as part of a complex (CD19/CD21/CD81 complex),together with the activation of BCRs belonging to restrictedVH subfamilies and recognizing HCV/E2. Indeed, the abilityof E2 to directly engage CD81 in addition to the bindingof specific anti-HCV/E2 BCRs may create a powerful stim-ulatory signal, promoting proliferation (Figure 1(d)). As apossible consequence of this mechanism, CD81 is upregu-lated in HCV-infected patients, with a further upregulationin patients with MCII. However, conflicting results have beenpublished showing CD81 downregulation, whereas CD19receptor was upregulated on peripheral B lymphocytes in


HCV-infected patients with MCII or B-NHL. In addition,HCV/E2 binding to CD81 induces double-strand DNAbreaks and hypermutation, specifically in the VH gene ofB cells. This process was demonstrated to be dependenton activation-induced cytidine deaminase and related to anincrease in the production of TNF-alpha [59, 60].

3.3. NS3 Protein. It is known that NS3 can induce animportant humoral and cellular immune response [61–68]. A possible role of NS3 in inducing autoreactive Absthrough molecular mimicry has been hypothesized [69]. Inparticular, IgM reacting against the NS3 helicase domain ispresent in the cryoprecipitate of all chronically HCV-infectedpatients with a bone marrow monoclonal B-cell patternand of a consistent portion (36%) of HCV-related MCIIpatients [69]. These IgM Abs recognize epitopes locatedwithin a region (1238–1279), previously indicated as one ofthe two immunodominant regions for B cells on NS3 (1250–1334 and 1359–1449). Importantly, these IgM Abs show alsoreactivity against human IgG and in particular against aunique peptide (Fc345–355) corresponding to the IgG CH3domain (Figure 1(c)).

On the other hand, the region of NS3 encompassingresidues 1406–1415 has been demonstrated to be also aCD8+ T cell epitope [70].

The possible role of HCV/NS3 protein in the patho-genesis of HCV-associated MC is also suggested by anotherstudy evidencing NS3 deposits in the kidney of viremic HCV-positive patients with membranoproliferative glomeru-lonephritis associated with cryoglobulinemia and presentinga mild polyclonal B lymphocytosis [71].

In addition, NS3 has been demonstrated to promoteoncogenic transformation and to interact with p53 andinterfere with apoptosis, thus contributing singularly orsynergistically with the HCV/E2 and core proteins to thedevelopment of HCV-related lymphomas [11, 72–74].

4. Biased IgV Subfamily Use andLymphoproliferation

As evidenced above, understanding the nature of the Bcell-stimulating Ag resulted to be extremely difficult. Onereason relates to the difficulty in isolating expanded cellsthat are mostly confined to restricted areas of the liver or ofthe bone marrow. Another reason relates to the complexityof analyzing the BCR specificity of oligoclonal expandedcells, which are present at low concentrations among tissueresident, nonspecific, B-cell populations. A further reasonis the difficulty in obtaining reliable information fromcomparing the sequences of autoreactive BCRs or Abs withthose of BCRs or Abs of known specificity.

Some data could be obtained from the cloning of the VHand VL genes into an expression vector and from the testingof their biological activity. As an example, in several studiesthe use of phage display technology allowed the selection ofdistinct autoreactive Abs which were further characterizedin terms of Ag specificity and biological activity [75]. Thesestudies demonstrated that the VH and VL sequences ofthe cryoprecipitable monoclonal IgM match those of the

IgV genes of monoclonal B cells isolated from patientswith MCII [55, 76]. This confirms that in patients with anestablished monoclonal pattern, the IgM component of ICsrepresents the circulating counterpart of the BCR expressedon the surface of expanded B cells and, therefore, can beexploited to identify the putative Ag involved in inducing andmaintaining B-cell activation.

Interestingly, several other studies demonstrated therecruitment of selected IgV gene subfamilies, the presenceof IgV genes mutations compatible with a GC or post-GCderivation, a replacement/silent mutation ratio consistentwith the maintenance of a functional structure of the BCR,and the presence of intraclonal heterogeneity evidencingan ongoing Ag-induced maturation [21, 24, 55, 76–80].Conceivably, the similarity in the structure of the variableBCR region and the restricted recruitment of certain IgVsubfamilies, both for heavy and light chains, may accountfor selection of B cells expressing specific and commonreactivity.

In this paragraph we will review the V gene subfamiliesmost frequently observed in HCV-related lymphoprolifera-tive disorders.

4.1. VH1-69

4.1.1. Molecular Characteristics of VH1-69 Subfamily-DerivedAbs. Approximately 1.7% of peripheral blood B cells ofhealthy individuals express the distal VH1-69 gene, asexpected for a random use of the total repertoire offunctional VH gene regions [81]. Furthermore, this genesegment is expressed in the restricted repertoire of fetalliver B lymphocytes and is thought to be involved innatural immunity [82, 83]. Although minimally used in adultlife, this VH subfamily is highly represented in anti-HCVhumoral immune response, particularly that directed againstthe HCV/E2 glycoprotein [84–88].

Interestingly, this subfamily is highly represented alsoin broadly neutralizing humoral responses directed againstother enveloped viruses, such as influenza viruses andHIV [89–92]. In particular, several groups have describedthe heterosubtypic activity of VH1-69-derived monoclonalantibodies (mAbs) directed against the hemagglutinin (HA)stem region of influenza A viruses [89, 90, 93, 94]. Thissuggests the presence of a conserved motif in this Ab sub-family determining the observed peculiar features. Indeed,crystallization studies demonstrated that these mAbs interactwith the Ag only through the VH1-69-derived heavy chainCDR1 and CDR2 regions, but not with the CDR3 thatusually confers Ag specificity to an Ab [95]. In particular,the distinct hydrophobic CDR2 loop encoded by VH1-69may confer antiviral activity by binding to hydrophobic viraltargets [90]. Another peculiar feature of the anti-HA VH1-69-derived mAbs was their extremely long CDR3 region[90]. Interestingly, a very long CDR3 and high levels ofsomatic mutations were also observed in VH1-69-derivedmAbs directed against other viruses, such as HIV [96].Moreover, similar characteristics were observed also for anti-protein Abs produced in the context of chronic systemicautoimmune diseases [96]. Conversely, for HCV infection,


a recent paper reported a shorter CDR3 length of the Abrepertoire in people who spontaneously resolved an acuteHCV infection compared to healthy individuals and thosewith chronically evolving HCV infection, and the authorsexplain this difference suggesting a mobilization of the Abrepertoire due to clonal selection [76].

4.1.2. VH1-69 and HCV-Related Cryoglobulinemia and Lym-phomas. The VH1-69 germline gene is commonly reportedin monoclonal IgM observed in HCV-related lymphopro-liferative disorders as well as in normal B cells respondingto the HCV/E2 viral antigen [52]. Furthermore, analysis ofthis V region sequence in HCV-infected cryoglobulinemicpatients revealed that it undergoes somatic mutation, pre-sumably during affinity maturation. This observation hascorroborated the hypothesis that HCV-associated MCII andlymphomas may originate in B cells responding to HCV/E2glycoprotein, the most involved in stimulation of VH1-69 expressing B cells [79]. Studying patients with HCV-associated type II MC, Carbonari et al. reported that upto 98% of their circulating B cells expressed the VH1-69gene and that it was frequently associated with the Vκ3-20light chain gene [78]. This pairing was also frequently foundamong B-cell chronic lymphocytic leukemia (B-CLL) clones[105]. Indeed, it has been described that most commonlythe association of these two subfamilies forms the WA cross-reactive idiotype (Id) endowed with RF activity [52, 97].Moreover, it has been observed that the VH1-69 expression isfrequently associated with DH3-22 and JH4 rearrangements,especially in patients with HCV-associated MCII [77, 98].

Preferential use of this gene has also been seen in 10–20% of patients with CD5+ B-CLL, and polyclonal activationand expansion of CD5+ B cells occur during interactionbetween HCV and lymphocytes and are associated with HCVinfection and HCV-related MCII [19]. The circulating innateCD5+ cells are in fact believed to be equivalent to murineB-1 cells, which have restricted receptor gene segment usageand are primary source of auto-Abs (IgM). But, in thisregard, there are conflicting data, as other groups reportedno correlation between the increase of CD5+ B cells andthe presence of cryoprecipitate or RF in patients with HCV-related lymphoproliferative disease [77, 80, 99, 100].

Furthermore, it has been observed that the majority ofVH1-69-expressing B cells in HCV positive patients hada memory phenotype and express modestly somaticallymutated IgM, indicating that a clonal population of memoryVH1-69 expressing B cells progressively invades the circu-lating B-cell compartment of patients with HCV-associatedMCII [99]. It has also been reported that the peripheral B-cell repertoire of HCV patients may be represented almostcompletely by VH1-69 monoclonal B cells [78, 106]. More-over, some of these clones have CDR3 sequences identical toRF IgMs isolated from patients with MALT neoplasms, withMCII-associated splenic lymphoma, and with leukaemia-like B-cell monoclonal expansion [78, 106]. Thus, originallynonneoplastic VH1-69 B cells responding to Ag stimulationcould evade the homeostatic mechanisms that regulate theAg-driven clonal expansion, and subsequent genetic events

may cause further escape from control and lead to absolutelymphocytosis.

Finally, recent evidences suggest that somatic hypermu-tation, as well as class switching, may significantly alterthe germline-determined original BCR reactivity [107, 108].This may explain why VH1-69-derived IgG Abs are oftenendowed with a broadly neutralizing anti-HCV activity,whilst their IgM counterpart may feature autoreactive activ-ity. In this regard, Racanelli et al. identified the pauciclonalityof the peripheral memory B-cell population as a distin-guishing feature of patients who spontaneously resolved anacute HCV infection compared to those chronically evolvingHCV infection. This finding, also observed in patientswith preneoplastic HCV-associated lymphoproliferative dis-orders, suggests that the B-cell clones potentially involved inclearance of the virus may also be originally more prone tofeature autoimmune characteristics and more susceptible toundergo abnormal expansion [76].

4.2. Other VH Subfamilies. B cell mono/oligoclonal expan-sion of clones expressing VH subfamily gene segments otherthan VH1-69, such as VH3-7, VH3-21, VH3-23, VH3-30,VH4-34, VH3-48, and VH4-59, seems to be implicated inHCV-related lymphoproliferative disorders [75, 77, 100].Moreover, as in the case of VH1-69, monoclonal expansionof B-cell clones expressing most of these VH subfamilies is acommon feature of a wide variety of autoimmune disordersand lymphomas [75, 100, 101, 108].

4.2.1. VH3-21 and VH3-23. Our group previously reportedthat the biased VH1-69 gene use in anti-HCV/E2 responsemay selectively expand B-cell clones reacting against thisspecific VH subfamily. In particular, it has been observedthat the immune repertoire of a patient with HCV-associatedMCII contains IgM clones able to react specifically againstanti-HCV/E2 Abs belonging to VH1-69 subfamily derivedfrom the same patient. Indeed, we found that 61% of IgMsreactive to anti-HCV/E2 VH1-69-Fab fragments belongedonly to two VH subfamilies, VH3-23 (39%) and VH3-21 (22%), that are frequently described in autoimmunedisorders [75, 101].

Furthermore, the mutational pattern of selected anti-HCV/E2 IgMs showed that almost all clones featured ahigh homology to the germline. More in details, differentlyfrom VH3-21 subfamily, VH3-23 clones did not featurepolyreactivity but showed a binding bias toward the VH1-69-derived IgG1 Fabs. These data suggest that VH3-23 IgM maybe naturally prone to recognize specific VH regions withinthe VH1-69 subfamilies [75, 102].

Overall, the HCV/E2-driven stimulation of the immunesystem may cause the expansion of specific B cells expressingVH1-69-derived Abs recognized by some natural IgM Absubfamilies. This could lead to the formation of circulatingICs, and the cross-linking of BCR by auto-Abs may allow achronic activation and a clonal expansion of anti-HCV/E2 Bcells.

4.2.2. VH4-34. The VH4-34 gene is used in about 5% ofhealthy adult B lymphocytes and is frequently found in


diffuse large-cell lymphoma, primary central nervous systemlymphoma, B-CLL, and several autoimmune disorders [79].

Furthermore, it is well known that, independently fromthe associated DH and JH gene segments, as well as from thesubfamily and isotype of the paired light chain, the VH4-34 isa naturally autoreactive subfamily. Interestingly, the VH4-34gene is found in virtually all cases of cold agglutinin disease,where the red blood cell I/I Ags bind to the FR1 domainof selected Ig subfamilies, including VH4-34, with a minorinvolvement of the CDR3 region [109]. Indeed, the restrictedusage of VH genes and the binding outside the CDRsare characteristics of B-cell super-Ags that are supposed todirectly activate B cells [110]. As previously mentioned, thiscould be the case of the HCV/E2 glycoprotein, due to itsability to stimulate the expansion of a restricted set of VHand VL subfamily expressing B cells. Moreover, as previouslydescribed, it directly provides a strong proliferation signalto B cells through its interaction with CD81 as part of theBCR complex. A similar behavior has also been describedfor staphylococcal enterotoxins A and D, that function ashuman B super-Ags rescuing B cell-expressing VH3 andVH4 (including VH4-34) genes inducing cell survival in invitro experiments, and has been suggested also for gp120 ofHIV [96, 111]. Moreover, certain portions of the FRs seemto be important for super-Ag binding, and these would bepreserved in a super-Ag selective pressure [109].

Therefore, the high frequency of the VH4-34 gene usageand the intrinsic molecular features of its FR and CDRdomains suggest a possible role of yet unknown B-cell super-Ag in driving HCV-related lymphoproliferative disorders[79].

4.3. VL Subfamilies. In the majority of clonal B-cell expan-sions following HCV infection there is a major involvementof Vκ-expressing B cells, as demonstrated by the highlyskewed κ/λ ratio and as corroborated by the usage of theVκ genes belonging to restricted subfamilies, as Vκ3-15and Vκ3-20 [80]. As previously mentioned, the Vκ3-20germline gene expression during HCV humoral immuneresponse is frequently associated with the VH1-69 expressionin the context of HCV-associated MCII and lymphomas[77, 80, 103].

Giving this restricted Vκ usage in HCV-positive subjectswith a related MCII as well as in those evolving in a B-NHL,some reports suggest an immune attack targeted on idiotypicdeterminants, as a possible passive or active immunotherapyfor HCV-related autoimmune diseases and B-cell lym-phomas [104]. In particular, De Re et al. after immunizationof an experimental model with a VH4-59/Vκ3-20 scFv mAb,cloned from a patient with a HCV-related MCII and B-NHL, demonstrated the possible induction of anti-Id Absdirected against conserved Vκ epitopes. This finding opensto possibilities of a potential therapeutic application of Absreactive with shared Id for patients with HCV-associatedB-cell lymphoproliferative diseases, obviating the need toproduce an anti-Id Ab or to use a different Id vaccine for eachpatient [103]. Consistently, this approach has been applied insome types of B-cell tumors and some autoimmune diseases[112].

5. Conclusions

Several viruses are involved in the development of systemicautoimmune-related damage. Over the last few years severalstudies have firmly demonstrated the close interplay betweena primarily hepatotropic virus, such as HCV, and B cells andthe role of this interaction in the occurrence of HCV-relatedautoimmune preneoplastic lymphoproliferative disorders, asMCII. The HCV-induced stimulation of distinct specific B-cell clones expressing specific BCRs, derived from a limitednumbers of V gene subfamilies, clearly underlines the roleof specific HCV Ags in the initiation of this pathogeneticmechanism. However, there are still many dark areas left,such as the molecular factors determining the breaking of“self tolerance” and those leading from the clonal expansionof a limited number of HCV-specific clones to the neoplasticimmortalization of some of them.

Overall, these mechanisms may be seen as escape strate-gies put forth by HCV to evade the immune response inthe course of a persistent infection [47, 113]. This point ofview, as well as the approaches followed in the study of HCV-related autoimmune disorders, may be important also in theevaluation of other autoimmune diseases not yet related withan infectious etiology or associated to a given pathogen. Thestudy of the systemic and local B-cell repertoire in the courseof diseases, as systemic sclerosis, multiple sclerosis, or specificclinical forms of atherosclerosis, and its comparison withthe specific V gene repertoire induced by a given pathogenmay be a new way for investigating their real causes openingnew doors on the comprehension of their pathogenesis[114–119].

Authors’ Contribution

G. Sautto and N. Mancini contributed equally to the paper.

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Review Article

Autoimmunity and Extrahepatic Manifestations inTreatment-Naıve Children with Chronic HepatitisC Virus Infection

Giuseppe Indolfi,1 Elisa Bartolini,1 Biagio Olivito,2

Chiara Azzari,2 and Massimo Resti1

1 Paediatric and Liver Unit, Meyer Children University Hospital of Florence, Italy2 Immunology Unit and Immunology Laboratory, Meyer Children University Hospital of Florence,Department of Sciences for Woman and Child’s Health, University of Florence, Florence, Italy

Correspondence should be addressed to Giuseppe Indolfi, [email protected]

Received 4 January 2012; Accepted 21 February 2012


Copyright © 2012 Giuseppe Indolfi et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Hepatitis C virus (HCV) infection has been associated with autoimmunity and extrahepatic manifestations in adults. Few dataare available on these topics in children. Nonorgan specific auto-antibodies development is part of the natural course of chronichepatitis C in children. Smooth muscle autoantibody is the most common autoantibody found, while liver-kidney microsomaltype-1 antibody positivity is the most peculiar autoimmune feature of children with HCV infection. The clinical significanceof non-organ specific autoantibodies in the course of paediatric chronic hepatitis C is still debated. Autoantibody positivitycan be considered neutral for most patients, while it can be associated with negative connotations for others, especially thosepositive for liver-kidney microsomal type-1 autoantibody. Subclinical hypothyroidism but not autoimmune thyroiditis has beendemonstrated in HCV infection in children, while only few cases of HCV-associated membranoproliferative glomerulonephritishave been described. Single reports are available in the literature reporting the anecdotal association between chronic hepatitisC and other extrahepatic manifestations such as myopathy and opsoclonus-myoclonus syndrome. Despite the low incidence ofextrahepatic manifestations of chronic hepatitis C in children, overall, available data suggest a careful monitoring.

1. Introduction

Since its discovery in 1989 [1], hepatitis C virus (HCV)has been associated with autoimmunity and extrahepaticmanifestations [2]. Data on these topics in children arescarce, but the incidence of extrahepatic manifestations isoverall lower in children with chronic hepatitis C whencompared to adults [2–6]. The purpose of the present articleis to summarize the current knowledge on autoimmunityand extrahepatic manifestations in treatment-naıve childrenwith chronic HCV infection.

2. Nonorgan Specific Autoantibodies

Nonorgan specific autoantibodies (NOSAs) developmentis considered part of the natural course of chronic HCV

infection in children [3–6]. Different mechanisms havebeen implicated in the development of NOSAs duringchronic hepatitis C [7]. The high prevalence of NOSAsin adults is considered the clear evidence of the alteredimmune system homeostasis in chronically infected patients.The characteristic lymphotropism of HCV could be oneof the bases of the increased production of autoantibod-ies. It has been hypothesized that HCV interacting withB lymphocytes can lower the B-cell activation thresholdfavouring autoantibodies production, and that HCV, suchas other hepatitis viruses, triggers autoimmune responsevia a molecular mimicry mechanism. Molecular mimicryoriginates when the target of the immune response to amicroorganism shares similarities with a “self” antigen,and the original antimicrobial immune response becomescross-reactive with the “self,” that is, autoimmune. By


Table 1: Studies investigating the prevalence of nonorgan specific auto-antibodies in children with chronic hepatitis C.

n NOSAs SMA ANA LKM-1 Dilution threshold of positivity Sample/s used

Bortolotti et al. 1996 [3] 40 32.5% 17.5% 7.5% 10% 1 : 20 Single point determination

Gregorio et al. 1998 [4] 51 65% 51% 10% 8% 1 : 10 Sequential serum samples

Muratori et al. 2003 [5] 47 34% 17% 9% 15% 1 : 10 Single point determination

Gehring et al. 2006 [6] 39 8% 5% — 2% 1 : 40 Two serum samples

Note: NOSAs, nonorgan specific autoantibodies; SMA, smooth muscle autoantibody; ANA, antinuclear antibody; LKM-1, liver kidney microsomal type-1auto-antibody.

a complementary mechanism, HCV can induce cellularinjury determining the release of “self” antigens that arenormally protected from the immune system but whenreleased are able to elicit an autoimmune response [8, 9].

The prevalence of NOSAs in children with chronic HCVinfection has been investigated in few studies, and wideranges of positive results have been found (Table 1) [3–6]. The heterogeneity of the prevalence estimations amongdifferent studies is due probably to technical differencesin the laboratory methods used and to the fluctuatingbehaviour of autoantibodies. For these reasons studies basedon determinations of NOSAs on serial samples [4], usinglower dilution thresholds of positivity [4, 5] and more sen-sitive laboratory methods [4], had results higher than thosebased on single point determinations [3, 5], using higherthresholds of positivity [3, 6] and less sensitive methods.

Firstly, in 1996 Bortolotti et al. [3] analyzed the preva-lence of NOSAs in forty Italian children with chronicHCV infection. About one third of the children studiedhad circulating NOSAs smooth muscle autoantibody (SMA)being the most common autoantibody found. Interestingly,in this cohort, patients with liver-kidney microsomal type-1 (LKM-1) autoantibody were more often infected by HCVgenotypes 1 and 2, while no difference was found betweenautoantibodies positive and negative cases with respect toclinical features, γ-globulins levels, and liver histology. Fewyears later, in 1998 Gregorio et al. [4] in a cohort of fifty-one children with chronic infection using a lower dilutionthreshold of positivity (1 : 10) and a homemade substrate forindirect immunofluorescence found an higher prevalence ofautoantibodies positivity (65%). SMA was again the mostcommon autoantibody detected being present in 51% of thepatients. Twenty-nine percent of the patients enrolled werepositive for antibodies to gastric parietal cells [4]. Muratori etal. in 2003 [5] found NOSAs in 34% of forty-seven children,and they did not find any difference in clinical, virological,and biochemical parameters between NOSAs-positive and -negative children. Confirming previous findings in adults,NOSAs negative children in this cohort had significantlyhigher HCV viral load than those with NOSAs. As apossible explanation the authors speculated that the unbal-anced immune reaction determining autoimmunity createsa cytokine environment which hampers viral replication [5].In all the studies examined NOSAs titres were usually lower,and the antigenic specificity by indirect immunofluorescencewas different than that usually found in children withautoimmune hepatitis (AIH) [3–5]. Antinuclear antibodies

(ANA) presented with a speckled rather than homogeneouspattern and SMA reacted only with vessels, sparing glomeru-lar and tubular structures which are additional targets inAIH type 1. Not all the children studied presented LKM-1positivity reacting with the cytochrome P45IID6 (CYP2D6)that is the common target of LKM-1 autoantibody in type 2AIH.

In the studies by Gregorio and Muratori [4, 5] childrenwith chronic hepatitis C were enrolled together with childrenwith chronic hepatitis B and with other chronic liver diseasesas controls. Overall, NOSAs were more common in childrenwith chronic hepatitis C than in children with other liverdisorders of similar severity [4, 5], suggesting that thepresence of autoantibodies is not just a consequence ofthe chronic liver damage. It is noteworthy that LKM-1reactivity was not found in any of the controls suggestingthat LKM-1 positivity, even if not the most common, wasthe most peculiar autoimmune feature of children withchronic hepatitis C. Furthermore, LKM-1 prevalence inHCV-infected children was consistently higher than thatfound in studies involving HCV-infected adults [2].

2.1. Clinical Significance of NOSAs. The clinical significanceof NOSAs in the course of chronic hepatitis C is stilldebated [7]. The real challenge for clinicians and scientistsis to understand whether and to what extent HCV-inducedautoimmunity contributes to liver damage distinguishingtwo theoretically possible populations of children: (1) chil-dren with HCV infection and histologically silent HCV-induced autoimmunity and (2) children with HCV infectionand HCV-driven autoimmune liver damage.

As first hypothesis, NOSAs reactivity in children withhepatitis C could be considered a simple consequence ofhepatocellular damage without pathogenic significance. Thishypothesis is supported by the observation that differentchildren despite NOSAs presence and persistence havenormal serum aminotransferase level, will not develop otherfeatures of autoimmunity such as increased γ-globulinslevels, and usually have autoantibodies titres lower than thatobserved in AIH and with a different pattern as detectedby indirect immunofluorescence [3–5]. On the other handautoimmunity could have pathogenetic implications in liverdamage of chronically infected children. Some data sup-porting this hypothesis are available. In adults some studiesshowed that the presence of autoantibodies was associatedwith elevated levels of γ-globulins, immunoglobulins, alka-line phosphatase, and γ-glutamil transpeptidase [10, 11].


Furthermore, LKM-1 positive HCV-infected children havebeen demonstrated to have a more advanced liver diseasewhen compared with LKM-1 negative peers [12].

Positivity for LKM-1 autoantibody in chronically infect-ed children appears to have important clinical implicationin view of the significant biochemical deterioration observedin LKM-1 positive patients who developed marked increasesin aminotransferase activity when treated with interferon-α[3–5, 12]. This is a hot topic given the recent approval ofthe combined treatment with pegylated interferon-α and rib-avirin by FDA and EMA for children older than three years.It has been hypothesized that in LKM-1 positive childreninterferon-α may amplify the autoimmune response target-ing CYP2D6 and thereby trigger acute LKM-1 mediatedliver damage. It is important that treatment of LKM-1/HCVpositive patients is decided after thorough investigations toexclude AIH. The issue of immunosuppressive therapy inthese children is debated as it can improve clinical andbiochemical parameters in selected patients, but it favourspersistent HCV replication.

3. Thyroid

Few data are available regarding natural history of thyroiddysfunction and thyroid autoimmune disease in childrenwith chronic HCV infection [4, 6, 13]. Gregorio et al. testedthe presence of antithyroglobulin and antithyroperoxidase(TPOA) in chronic HCV positive children before and aftertreatment with interferon-α with no positive result [4].Ghering et al. investigating thyroid function and prevalenceof autoimmune phenomena in chronic HCV-infected chil-dren treated with interferon-α, found a strong correlationbetween treatment and emergence of thyroid antibodies [6].Before treatment all children had normal thyroid function,one child had an isolate TSH elevation and a further onehad borderline TPOA levels [6]. Indolfi et al. in a case-control study enrolling a cohort of untreated children withvertically acquired chronic HCV infection (n = 36), showeda high prevalence of subclinical hypothyroidism (11%)and of autoimmune thyroiditis (5.6%) [13]. Subclinicalhypothyroidism was not related to length of infection, or todifferent HCV genotypes, but it was related to the presenceof active liver disease. Subclinical hypothyroidism was notfound in children with apparent virus clearance but only inthose with chronic infection and persistent viraemia eventhough no correlation was found between development ofsubclinical hypothyroidism and levels of viraemia. No childwith subclinical hypothyroidism in this study presentedantithyroid autoantibodies suggesting that the mechanismof subclinical hypothyroidism in HCV-infected children isnot antibody mediated [13], although autoimmunity cannotbe excluded absolutely as in adults autoimmune thyroiditiswithout detectable circulating anti-thyroid antibody titreshas been demonstrated and autoimmunity can be executedby autoreactive T cells also in the absence of detectableautoantibodies [14]. The possible role in the genesis ofsubclinical hypothyroidism during chronic HCV infectionof the impaired hepatic metabolism of thyroid hormones

[12] and of the thyroid infection by HCV [15] remainsspeculative.

4. Kidney

Membranoproliferative glomerulonephritis is the most com-mon renal disease associated with HCV infection in adults[2]. To the best of our knowledge only three cases ofHCV-associated membranoproliferative glomerulonephritisin children have been reported [16–18]. The specific patho-genesis of the glomerular injury caused by HCV infection isnot known, but it is thought to result from the depositionof circulating immune complexes of HCV and anti-HCV onthe glomerular capillary in the mesangium [2]. In one of thethree children described, successful antiviral monotherapywith pegylated interferon-α resulted in HCV RNA clearanceand disappearance of proteinuria [16].

5. Anecdotal Observations

Single reports are available in the literature reporting theanecdotal association between chronic hepatitis C and extra-hepatic manifestations. Mohan et al. described a 15-year-oldboy with diabetes, chronic HCV infection, and an inflamma-tory myopathy presumed to be associated with HCV infec-tion [19]. The potential association between inflammatorymyopathy and HCV infection has been infrequently reportedin adult patients [20], and an autoimmune mechanism hasbeen hypothesized. Ertekin and Tan reported a 9-year-oldboy with progressive cerebellar ataxia, action myoclonus,palpebral flutter, vomiting, headache, and opsoclonus, diag-nosed with opsoclonus-myoclonus syndrome [21]. Thisrare neurologic disorder characterized by multidirectionalchaotic eye movements, myoclonus in the limbs, and ataxiamay be associated with viral infections and for the first timewith hepatitis C [21]. The exact immunopathogenesis ofthe disease is undefined [22], but as for other extrahepaticmanifestations of HCV infection, the underlying mechanismwas postulated to be immune system dysregulation.

6. Conclusions

The wide range of extrahepatic manifestations of HCVin adults suggests that HCV chronic infection should beconsidered a systemic disease. An important role in the devel-opment of extrahepatic manifestations of HCV is thought tobe played by geographic, genetic, or environmental cofactorsas well as by the disease activity [2]. Chronic hepatitis Cis much different in children when compared to adultswith regard to disease activity, response to treatment, andextrahepatic manifestations. Mixed cryoglobulinaemia, forexample, is the most documented extrahepatic manifestationof HCV infection in adults and has never been described inchildren. Although discussed, the association between mixedcryoglobulinaemia and severe liver damage has been shownby different epidemiological studies [2, 23]. The naturalhistory of chronic hepatitis in children is usually of a milddisease [24], and the low incidence of severe liver damage


during childhood could be partly responsible for the absenceof mixed cryoglobulinaemia in children. Despite the lowincidence of extrahepatic manifestations in children, overall,available data suggest a careful monitoring.

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Review Article

Cytokines and HCV-Related Disorders

Poupak Fallahi,1 Clodoveo Ferri,2 Silvia Martina Ferrari,1 Alda Corrado,1

Domenico Sansonno,3 and Alessandro Antonelli1

1 Department of Internal Medicine, School of Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy2 Department of Internal Medicine, Rheumatology Unit, School of Medicine, University of Modena and Reggio Emilia,Via del Pozzo, 71, 41100 Modena, Italy

3 Department of Internal Medicine and Clinical Oncology, University of Bari, Piazza Giulio Cesare, 11, 70124 Bari, Italy

Correspondence should be addressed to Alessandro Antonelli, [email protected]

Received 5 January 2012; Accepted 17 February 2012


Copyright © 2012 Poupak Fallahi et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cytokines are intercellular mediators involved in viral control and liver damage being induced by infection with hepatitis Cvirus (HCV). The complex cytokine network operating during initial infection allows a coordinated, effective development ofboth innate and adaptive immune responses. However, HCV interferes with cytokines at various levels and escapes immuneresponse by inducing a T-helper (Th)2/T cytotoxic 2 cytokine profile. Inability to control infection leads to the recruitment ofinflammatory infiltrates into the liver parenchyma by interferon (IFN)-gamma-inducible CXC chemokine ligand (CXCL)-9, -10,and -11 chemokines, which results in sustained liver damage and eventually in liver cirrhosis. The most important systemic HCV-related extrahepatic diseases—mixed cryoglobulinemia, lymphoproliferative disorders, thyroid autoimmune disorders, and type2 diabetes—are associated with a complex dysregulation of the cytokine/chemokine network, involving proinflammatory andTh1 chemokines. The therapeutical administration of cytokines such as IFN-alpha may result in viral clearance during persistentinfection and reverts this process.

1. Introduction

Cytokines are small soluble proteins secreted by immune sys-tem cells and other cells and are part of an intercellular com-munication system responsible for immune response [1].These proteins play their role by binding specific cell recep-tors that either induce or inhibit cytokine-regulated genes.During viral infection, various cytokines play a role both inviral clearance and tissue damage [1].

2. Cytokines

Over 100 different cytokines have been reported, which areclassified according to their primary role. In relation to theirfunctions, cytokines can be classified in subgroups: (a) pro-inflammatory cytokines (interleukin (IL)-1, IL-6, tumor ne-crosis factor (TNF)-alpha); (b) T-helper (Th)1 cytokines,which are produced by Th1-activated lymphocytes (inter-feron (IFN)-gamma, IL-12, IL-18); (c) Th2-type cytokinewhich plays a role in the inhibition of cytokines derived from

Th1 cell which turns out to downregulate the function of Th1immune responses, inhibiting antigen-presenting capacity ofmacrophage and promoting B-cell proliferation and there-fore antibody production (IL-10, IL-4, IL-5, IL-13); (d) Th17cytokines which are important for the differentiation of Th17lymphocytes. IL-23, together with IL-6 and transforminggrowth factor (TGF)-beta, leads to the differentiation of Th0to Th17 cells which carry out the function of secreting IL-17A, IL-17F, TNF-alpha, and IL-1 thus leading to proinflam-matory reaction [1].

3. Chemokines

Chemokines are a large multifunctional family of cytokines(chemotactic cytokines) that induce the migration of cellsto sites of infection or injury. Functionally chemokines fallinto two main categories: homeostatic or proinflammatory.Homeostatic chemokines are produced constitutively; theseare generally involved in lymphocyte trafficking, immunesurveillance, and localization of lymphocytes with antigen


in the lymphatic system [2]. Other chemokines are onlyproduced by cells during infection or following a proinflam-matory stimulus and prompt the migration of leukocytes toan injured or infected site. Such inflammatory chemokinescan also activate cells to raise an immune response.

Chemokines are structurally related, because most ofthem contain four invariant cysteine residues. Depending onthe arrangement of the first two of these cysteines, chemok-ines are divided into four subfamilies: CXC (alpha), CC(beta), C (gamma), and CX3C (delta) [3]. Chemokines areproduced as propeptides and are cleaved during secretion toproduce an active mature protein [4] that functions by acti-vating G-protein-coupled receptors. The receptors for thesechemokines have been termed accordingly as CXCR, CCR,CR, and CX3CR [5].

4. Hepatitis C Virus (HCV) andImmune Response

HCV is a hepatotropic, noncytopathic virus of the familyFlaviviridae, which induces both acute and chronic necro-inflammatory liver disease [6, 7]. HCV escapes immune con-trol in 60–85% of cases. When infecting the liver parenchy-ma, HCV continuously releases viral particles into the bloodstream. The first line of defense that HCV will encounterincludes natural killer (NK) cells and natural killer T (NKT)cells [8]. These cells are activated by type 1 IFN (alpha andbeta) released by infected liver cells. NK and NKT cellsconstitute a relevant source of IFN-gamma and TNF-alpha[9]. These cytokines inhibit viral replication without de-stroying liver cells. NK cells are activated by IL-12 releasedfrom dendritic cells (DCs) and thus become empowered toeliminate infected cells [10]. NK cells may also induce partialor total DCs maturation [11].

DCs can process viral antigens and present them to spe-cific immune system cells via class I and class II major histo-compatibility complex (MHC) molecules. DCs capture viralparticles through Toll-like receptors (TLRs). Upon activa-tion, DCs secrete several types of cytokines (IL-12, TNF-alpha, IFN-alpha, IL-10) that will regulate and polarize theresponse of adjacent cells [12]. Mature DCs leave the liverafter viral epitope collection and head for lymph nodes,where they will activate T cells in the specific immune system[13].

Cytokines released in the liver parenchyma induce che-mokine release by liver cells, including IFN-gamma-induci-ble protein (IP-10/CXCL10), IFN-gamma-induced monoki-ne (MIG/CXCL9), IFN-inducible T-cell alpha chemoattrac-tant (I-TAC/CXCL11), macrophage inflammatory protein(MIP)-1alpha (MIP/CCL3), and MIP-1beta/CCL4, which re-cruit [14] specific cells capable of infection control.

Mature DCs and immature T cells, both of which expresschemokine receptor CCR7, are recruited towards lymphnodes by secondary lymphoid-tissue cytokine (SLC/CCL21)[13]. In the lymph node, T cells expressing T-cell receptors(TCRs) appropriate for the recognition of epitopes presentedby DCs in their MHC molecules are activated. The inter-action between the TCR and MHC-viral epitope complexresults in specific T-cell activation. Certain specific CD8

T cells, cytotoxic T lymphocytes (CTLs), become cytolytic,secrete type 1 cytokines, and travel to the infected liver [15–17]. Specific CD4+ T cells will regulate the adaptive responseby secreting Th1 cytokines (IL-2, IFN-gamma, TNF-alpha)to facilitate a cell-mediated immune response and Th2 cytok-ines (IL-4, IL-10, IL-13) to regulate the humoral immunity[18]. It is widely accepted that adaptive immune responseplays a key role in the control of HCV infection.

5. Cytokines and HCV Chronic Infection

HCV manages to escape immune response. To this end theyinterfere with various immune mechanisms including cytok-ine activity modulation.

5.1. Innate Immunity. A primary cell defense mechanismduring initial infection is the synthesis of antiviral type 1IFN-alpha/beta [19]. On binding its receptor, IFN-alpha/beta activates a number of intracellular mechanisms that canprevent viral replication and spread to other liver cells. HCVis a good inducer of IFN-alpha/beta expression [20]. Howev-er, HCV seems to be, at least in part, unresponsive to IFN-alpha/beta effects and may effectively replicate in the liverdespite such gene induction. HCV can block type 1 IFN in-duction; this possibly results from the fact that nonstructuralproteins (NS 3 and NS5A), and structural protein E2 mayboth potentially block the expression and transcription ofIFN-alpha/beta-induced genes. HCV NS5A protein inducesIL-8 expression, which is associated with IFN-alpha inhibi-tion [21].

The outcome of a viral infection depends on the interplaybetween the host capacity to trigger potent antiviral respons-es and viral mechanisms that counteract them. AlthoughToll-like receptor (TLR)-3, which recognizes virally deriveddouble-stranded (ds) RNA, transmits downstream antiviralsignaling through the TIR adaptor Trif (TICAM-1), viralRNA-sensing RIG-like helicases (RLHs) use the mitochon-drial-bound CARD protein Cardif (IPS-1/MAVS/ISA). Theimportance of these two antiviral signaling pathways is re-flected by the fact that both adaptors are inhibited throughspecific cleavage triggered by the HCV serine protease NS3-4A [22, 23].

NK cells and NKT cells exert their antiviral actionthrough direct, non-MHC-restricted cytotoxic mechanismsand IFN-gamma production [24]. In addition, they allowmaturation for DCs favoring the development of Th1/Tcy-totoxic (Tc)1 responses [10]. However, they do not seem toplay a significant role in acute HCV infection [25]. It has beensuggested that HCV can block NK cells and NKT cells func-tions thus preventing antiviral cytokines such as IFN-gammafrom being produced, via an interaction between HCV E2protein and NK-cell CD81 molecule [26].

During chronic infection with HCV, a decrease in IFN-alpha production by plasmacytoid DCs has been reported[27], such as a decrease in IL-12 production by myeloid DCs[28]. In fact, HCV structural proteins can interact with TLR-2 in monocytes and induce IL-10 production, which inhibitsIL-12 and IFN-alpha production in DCs [29]. However,other studies reported an increased IFN-alpha production,


especially in patients who fail to respond to exogenous IFN-alpha, in whom IFN-stimulated genes (ISGs) are highly acti-vated [30].

It has been also suggested that DCs cytokine profilecannot polarize T-cell responses towards a Th1/Tc1 response[31] and contributes to inadequate NK cells and NKT cellsactivation. However, other studies have shown that a pro-gressive liver injury in chronic hepatitis C infection correlateswith increased intrahepatic expression of Th1-associated cy-tokines [32, 33].

5.2. Adaptive Response. HCV CD4+-T cells play a key rolein adaptive response in that they provide help in activatingcytotoxic and humoral responses. They can secrete Th1-cytokines including IFN-gamma, which favors neutrophiland macrophage recruitment and leads to inflammatory re-sponse. They also may release Th2 cytokines such as IL-4and IL-10, which limit Th1 cytokine-mediated response andfavor the development of humoral response [34]. A multis-pecific, strong, sustained, CD4+-T-cell-specific Th1 responsemay be seen in infections with HCV infection evolving toresolution [35]. However, when infection becomes chronic,a weak CD4-T-specific response with few specificities andscarce type 1 cytokine production is observed [36].

CD8+ CTLs can clear viruses using apoptosis-related cy-tolytic mechanisms and mechanisms mediated by type 1 cy-tokines (IFN-gamma, TNF-alpha). In chronic infection withhepatitis B virus or HCV, specific CTLs are few and engagefew specific targets; they also display anergic characteristicswith reduced type 1 cytokine secretion [37]. Another poten-tial mechanism of blocked type 1 cytokine production resultsfrom regulatory T-cell activity. These cells can release IL-10and TGF-beta and inhibit proliferation and cytokine synthe-sis in T cells, either directly or through other cytokines, inhepatitis C [38].

Cytokines produced by T cells play a role in the regulationof humoral responses; nevertheless, these responses cannotcontrol chronic viral hepatitis, even though they play a rolein the pathogenesis of extrahepatic manifestations [18].

6. Cytokines and Liver Damage

When specific immune response fails to control viral rep-lication, the infected liver cells secrete IFN-gamma-in-duced chemokines such as CXC chemokine ligand CXCL9,CXCL10, and CXCL11, which result in the migration ofnonspecific mononuclear cells into the liver [39], which areunable to control infection but result in sustained liver dam-age [40]. Inhibition of these chemokines limits nonspecificcell migration and hence reduces the inflammation [41].The recruitment of persistent mononuclear infiltrates leadsto the development of chronic inflammation, which resultsin sustained liver damage. Finally, chronic inflammation in-duces regenerating mechanisms in the liver parenchyma.Several factors influence this process, including cytokinessuch as IL-6, TNF-alpha, TGF-beta, hepatocyte growth fac-tor, and epidermal growth factor. These and other factorsactivate transcription factors such as nuclear factor-κB, signaltransducer, and activator of transcription 3 which initiate the

gene expression cascade leading to hepatocyte proliferation[42].

Persistent inflammation also activates hepatic stellatecells, myofibroblasts, and fibroblasts, which favors the devel-opment of liver fibrosis. The activation of these cells is regu-lated by pro-inflammatory cytokines such as TGF-beta, IL-6, TNF-alpha, CCL21, and platelet-derived growth factor,among other stimuli [43].

7. HCV-Related ExtrahepaticDiseases (HCV-EHDs)

HCV is known to be responsible for both hepatic and HCV-EHDs. The most important systemic HCV-EHDs are HCV-related mixed cryoglobulinemia (MC) (MC+HCV) and lym-phoproliferative disorders, while the most frequent and clin-ically important endocrine HCV-EHDs are autoimmunethyroid disorders (AITDs).

8. Cytokines, Cryoglobulinemia, andLymphoproliferation

MC is a distinct syndrome clinically characterized by pur-pura, weakness, arthralgia, and involvement of one or moreorgan systems, including membranoproliferative glomeru-lonephritis, peripheral neuropathy, skin ulcers, liver damage,and diffuse vasculitis. Cryoprecipitable immunocomplexes,namelymixed (IgG-IgM) cryoglobulins, are the serologicalhallmark of the disease: IgG is the autoantigen and IgM,with rheumatoid factor (RF) activity, the autoantibody. MCis classified in type 2 and type 3 according to the presence ofpolyclonal or oligo-monoclonal IgMs. Because expansion ofRF-producing B cells is the underlying disorder of MC, thiscondition is considered a “benign” B-cell lymphoprolifera-tive disease [44, 45].

The mechanism(s) responsible for the lymphoprolifera-tion surrounding MC remain unknown. Due to geographicalheterogeneity in prevalence of MC+HCV, it is conceivablethat unknown genetic and/or environmental factors mayinfluence the development of this syndrome [46]. Severaldata are consistent with the possibility that chronic stimula-tion of B cells by viral epitopes could play an important role[47–49].

A wide body of evidence, in addition, strongly suggeststhat a key factor in the pathogenesis of MC+HCV is repre-sented by the inhibition of the apoptosis of B cells, leadingto their progressive accumulation. First, this is suggestedby the histopathological characteristics of liver and/or bonemarrow lymphocyte infiltrates in MC patients [50], as wellas by the high prevalence of bcl-2 rearrangement (t(14;18) translocation) in patients with MC, with regression oftranslocated B-cell clones after successful antiviral therapy[45, 51, 52].

Furthermore, B-lymphocyte stimulator (BLyS) serumlevels are significantly correlated with B-cell proliferationduring chronic HCV infection. These results strongly suggesta role for BLyS in the induction and expression of HCV- B-cell proliferation [53–55]. Chemokine CXCL13, also known


as BCA-1 (B cell-attracting chemokine-1) or BLC (B-lym-phocyte chemoattractant), is a major regulator of B-celltrafficking. HCV infection may be associated with B-celldysfunction and lymphoproliferative disorders, includingMC+HCV. The results by Sansonno et al. [56] indicate thatupregulation of CXCL13 gene expression is a distinctivefeature of HCV-infected patients. Higher levels of this che-mokine in the liver as well as in the skin of patients withactive MC+HCV vasculitis suggest a possible interrelationbetween these biologic compartments.

Recently, Saadoun et al. [57] studied the local immuneresponse in the liver, which is considered the principal sitefor immune reactions involved in MC pathogenesis. In thatstudy, the cytokine profile of liver-infiltrating T lymphocytesfrom MC+HCV patients and without MC (of type 2) werecompared. They showed that, although no differences werefound in the proportion of CD4+, CD8+ liver T cells, theability of freshly isolated liver T cells to produce type 1 cy-tokines in response to stimulation with phorbol myristateacetate and ionomycin for 6 hours was significantly higher inMC+HCV patients than in HCV-infected controls withoutMC, whereas production of type 2 cytokines by these cellswas similar (IL-4) or reduced (IL-10).

This agrees with previous data obtained in peripheralblood mononuclear cells [58], ruling out the possibility ofa discrepancy between the response of peripheral and liverT cells. Interestingly, in both studies by Saadoun et al. andLoffreda et al. [57, 58], a reduced expression of IL-10 (astrong inhibitor of IFN-gamma production) is demonstratedregardless of the different sources. These observations sug-gest that the evolution of HCV infection toward MC is char-acterized by a strong Th1 response.

Several studies have shown an increased expression ofIFN-gamma [59] and IFN-gamma-inducible chemokines[60], in particular CXCL10, in hepatocytes and in lympho-cytes of HCV-infected patients [61, 62], directly related withthe degree of inflammation and with an increase of circulat-ing levels of IFN-gamma and CXCL10 [14, 63–66].

Furthermore, it has been shown that NS5A and coreproteins, alone or by the synergistic effect of cytokines, suchas IFN-gamma and TNF-alpha, are capable of upregulatingCXCL10 and CXCL9 gene expression and secretion in cul-tured human hepatocyte-derived cells [67], suggesting thatCXCL10 produced by HCV-infected hepatocytes could playa key role regulating T-cell trafficking into a Th1-type inflam-matory site as the liver tissue during chronic HCV infection,by recruiting Th1 lymphocytes that secrete IFN-gamma andTNF-alpha, which induce CXCL10 secretion by hepatocytes,thus perpetuating the immune cascade [68].

Furthermore, we have recently shown that circulatingCXCL10, CXCL11, IFN-gamma-inducible (Th1) chemoki-nes are higher in patients with MC+HCV than in chronichepatitis C (CHC) patients. Moreover, our studies demon-strate markedly high serum levels of CXCL10 and CXCL11in patients with MC+HCV compared to healthy controlsin particular in the presence of active vasculitis. A strongrelationship between circulating IFN-gamma and CXCL11was shown, strongly supporting the role of a Th1 immuneresponse in the pathogenesis of MC+HCV patients [69–74].

For comparison the prototype Th2 chemokine (C-Cmotif) ligand 2 (CCL2) was not significantly different inpatients with MC+HCV and active vasculitis than in MCpatients, and it suggests that the Th1 CXCL10 chemokine isspecifically involved in the appearance of vasculitis in thesepatients [74].

The pro-inflammatory cytokines IL-1beta, IL-6, andTNF-alpha have also been evaluated in MC+HCV patients.In fact, MC+HCV patients show significantly higher meanIL-1beta, IL-6, and TNF-alpha levels than the controls or theHCV patients. If the importance of IL-1beta and IL-6 in thepathogenesis of MC is confirmed, these results will open theway for the evaluation of new therapies for MC [75].

On the whole the above-mentioned data underline theimportance of the activation of the Th1 immunity in theimmunopathogenesis of MC+HCV, but suggest a complexdysfunction of the cytokine/chemokine network in these pa-tients, involving also pro-inflammatory cytokines.

9. Cytokines and AITDs Associated withHCV and MC

The pattern of thyroid disorders observed in HCV infectionis characterized by the presence of increased circulating levelsof antithyroid peroxidase antibody (AbTPO) and increasedrisk of hypothyroidism in AbTPO positive subjects [76–80].

This pattern is similar to that observed in IFN-alpha-treated patients, too [81].

Differences in geographical distribution [82], geneticvariability in the populations studied [83], and environmen-tal cofactors, such as iodine intake or other infectious agents[84, 85], could play an important role in the development ofAITD.

Recently it has been shown that high levels of CXCL10are present in patients with autoimmune thyroiditis (AT),in particular in the presence of hypothyroidism [68], andan involvement of Th1 immune response in the inductionof AT [86], Graves’ disease, and Graves’ ophthalmopathy[87] has been shown. Furthermore, the presence of HCV inthe thyroid of chronically infected patients has been recentlydemonstrated [88, 89]; however, other studies are needed tofurtherly confirm this point.

On the above-mentioned bases, it has been speculatedthat HCV thyroid infection may act by upregulating CXCL10gene expression and secretion in thyrocytes (as previouslyshown in human hepatocytes [67]) recruiting Th1 lympho-cytes that secrete IFN-gamma and TNF-alpha, which induceCXCL10 secretion by thyrocytes, thus perpetuating the im-mune cascade, that may lead to the appearance of AITDs ingenetically predisposed subjects.

This hypothesis has been recently confirmed by two stud-ies that demonstrated high serum levels of CXCL10 inMC+HCV patients and showed that CXCL10 is significantlyhigher in the presence of AT compared to MC+HCV patientswithout thyroiditis [90, 91]. For comparison the prototypeTh2 chemokine CCL2 was not significantly different inpatients with MC+HCV in the presence of AT than inMC+HCV patients, and it suggests that the Th1 CXCL10


chemokine is specifically involved in the appearance of ATin these patients [91].

Among the pro-inflammatory cytokines, IL-1beta andTNF-alpha were not associated with the presence of AT inMC+HCV patients, while IL-6 was modestly but significantlyincreased in patients with AT [71, 92].

On the whole the above-mentioned data underline theimportance of the activation of the Th1 immunity in theimmunopathogenesis of AT in patients with MC+HCV.

10. Cytokines and Type 2 Diabetes Mellitus(T2DM) Associated with HCV and MC

Several clinical epidemiological studies since 1994 have re-ported that HCV infection is linked to diabetes [93]. The as-sociation between HCV infection, in patients without cirrho-sis (a well-known risk factor for T2DM), and T2DM has beenfirst studied in two of our studies, in patients with chronicHCV infection (HCV+) associated with MC (MC+HCV)[94] and in patients with HCV-related chronic liver disease[95].

There is one population study (National Health and Nu-trition Examination Survey-NHANES III 1988–1994) thatshowed an adjusted odds ratio of 3.8 for T2DM for those whowere aged >40 years and HCV+ [96] and increased incidenceof T2DM [97].

There have been a few reports, too, that IFN treatmentof HCV infection improves glucose tolerance [94, 98] whenHCV infection is eradicated; however, another study did notconfirm these results [99].

Altogether the above-mentioned data indicate that HCVchronic infection is a risk factor for developing T2DM.

10.1. Mechanism

10.1.1. Insulin Resistance and Steatosis. It is speculated thatinsulin resistance (as a consequence of hepatic steatosis (i.e.,present in about 50% of the subjects with HCV infection)[93] and/or elevated expression of TNF-alpha (strongly cor-related with the degree of liver diseases and the level of insu-lin resistance) [89]) may lead to the development of T2DM[93].

10.1.2. Direct Islet Cell Destruction by HCV. Masini et al.[100] recently demonstrated a direct cytopathic effect ofHCV at the islet cell level.

10.1.3. Possible Autoimmune Induction. The type of diabetesmanifested by patients with HCV chronic infection is not theclassical T2DM. The labelling of HCV+ patients as T2DMis purely conventional and possibly inaccurate: the linesseparating type 1 diabetes from latent autoimmune diabetesin adults (LADA) and from T2DM are fading away as newpathogenetic information is obtained [101].

Three studies have previously reported [94, 95, 102] thatHCV+ patients T2DM were leaner than T2DM controls andshowed significantly lower LDL-cholesterol and systolic anddiastolic blood pressure. Furthermore, MC-HCV+ patients

with T2DM had non-organ-specific autoantibodies morefrequently (34% versus 18%) than nondiabetic MC-HCV+patients [94].

An immune-mediated mechanism for MC-HCV+ asso-ciated diabetes has been postulated [94], and a similar path-ogenesis might be involved in the diabetes of HCV+ patients.This hypothesis is strengthened by the finding that autoim-mune phenomena in T2DM patients are more commonthan previously thought [103]. Since the prevalence of classicbeta-cell autoimmune markers in HCV+ patients has notbeen found to be increased [89], other immune phenomenamight be involved [104].

On the above-mentioned bases, it could be interestingto speculate that HCV infection of beta cells [100] mayact by upregulating CXCL10 gene expression and secretion(as previously shown in human hepatocytes) recruiting Th1lymphocytes that secrete IFN-gamma and TNF-alpha, whichinduce CXCL10 secretion by beta cells, thus perpetuating theimmune cascade that may lead to the appearance of beta cellsdysfunction in genetically predisposed subjects.

This hypothesis has recently been confirmed by a studythat demonstrates higher serum levels of CXCL10 in HCV+patients with T2DM with respect to those without [64, 105].

11. Therapeutic Role of Cytokines inChronic Viral Hepatitis

IFN-alpha is the only cytokine currently used in the treat-ment of chronic viral hepatitis. In CHC, pegylated IFN-alphacombined with ribavirin leads to sustained viral clearance in50% of patients [106]. The most important effect of IFN-alpha is directly antiviral; however, it has also immunomodu-lating actions that favor Th1/Tc1 response restoration [107–109]. On the other hand, ribavirin, a wide-spectrum antivi-ral agent used in combination therapy for hepatitis C, hasimmunomodulating effects that induce type 1 cytokine pro-duction [110]. Sustained viral load reduction with antiviralagents has also been seen to facilitate specific T response re-covery with type 1 cytokine production in hepatitis C [111].

An exogenous administration of Th1-inducing cytokinessuch as IL-12 [112] or anti-inflammatory cytokines such asIL-10 has also been attempted to reduce intrahepatic inflam-mation severity [113]. However, such therapies remain ex-perimental, and their effectiveness is unclear.

From a theoretical standpoint Tc1-associated chemokinereceptors may represent an interesting therapeutic target inthe development of drugs for patients with chronic hepatitisunresponsive to antiviral agents, their aim being a reductionof liver inflammation and progression to fibrosis by blockinginflammatory cell migration into the liver [39, 41].

Treatment-induced and spontaneous clearance of HCVinfection are affected by various host factors. Polymorphismsin the region of the gene IL-28B are associated with HCVclearance, implicating the gene product, IFN-lambda3, inthe immune response to HCV. Although it is not clear howthe IL-28B haplotype affects HCV clearance, IFN-lambda3upregulates IFN-stimulated genes, similar to IFN-alpha and-beta, but via a different receptor. There is also evidence thatIFN-lambda3 affects the adaptive immune response.


It is known that IL-28B may establish a robust T-cell adaptive immune response [114, 115]. This effect mayexplain the relationship between single-nucleotide polymor-phism (SNPs) near IL-28B, adaptive response, and viralclearance [116].

The IL-28B genotype can be considered, along withother factors, in predicting patient responses to therapy withpegylated IFN-alpha and ribavirin [117, 118].

Clinical studies assessing safety and efficacy in the treat-ment of HCV with exogenous IFN-lambda3 are currentlyunderway. Early results suggest that IFN-lambda3 treatmentinhibits HCV replication and is associated with a limitedside effect profile. However, hepatotoxicity in both healthyvolunteers and HCV-infected patients has been described[119].

12. Conclusion

Cytokines are intercellular mediators involved in viral con-trol and liver damage as induced by infection with HCV. Thecomplex cytokine network operating during initial infectionallows a coordinated, effective development of both innateand adaptive immune responses. However, HCV interfereswith cytokines at various levels and escape immune responseby inducing a Th2/Tc2 cytokine profile. Inability to controlinfection leads to the recruitment of inflammatory infiltratesinto the liver parenchyma by IFN-gamma-inducible CXCL9,-10 and -11 chemokines, which results in sustained liverdamage and eventually in liver cirrhosis; however, fibrogen-esis may also follow distinct paths. The most important sys-temic HCV-EHDs—MC, lymphoproliferative disorders, andAITDs—are associated with a complex dysregulation of thecytokine/chemokine network, involving pro-inflammatoryand Th1 chemokines. The therapeutical administration ofcytokines such as IFN-alpha may result in viral clearanceduring persistent infection and reverts this process.


The authors have no conflict of interests to declare.

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