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Background/objectives: To describe frequency and type of ocular manifestations in patients with cryoglobulinemic vasculitis (CV), as well as management approaches and outcomes., Subjects/methods: This was a retrospective, observational, cohort study of patients who were diagnosed with CV at a single center and regularly underwent a comprehensive ocular assessment., Results: Ophthalmologic manifestations were recorded in 16 patients (28%). The diagnoses included dry eye disease and primary Sjögren syndrome in 5 and 2 patients, respectively; peripheral ulcerative keratitis and anterior scleritis in 1 patient each; hyperviscosity syndrome and hypertensive retinopathy in 2 patients each; and Purtscher- like retinopathy in 3 patients. Twelve patients (75%) were anti-HCV/HCV RNA-positive, 11 of whom achieved a sustained virologic response (SVR) following treatment with interferon-α2b plus ribavirin or direct-acting antivirals. All patients were treated with ocular lubricants. Systemic therapeutic measures, including glucocorticoids, immunosuppressive and biologic agents, induced the disappearance or ≥50% reduction of cryoglobulins and major signs of vasculitis in 11 patients (68.7%). In the remaining 5 patients (31.3%), cryoglobulins and CV manifestations remained unchanged or decreased by <50%. The corresponding ophthalmologic assessment showed a variable degree of improvement in the ocular symptoms in all but 2 patients (87.5%). The best corrected visual acuity following treatment improved in 26 eyes, was unchanged in 3 eyes, and worsened in 3 eyes., Conclusions: Eye involvement is not a rare event in CV patients. A timely diagnosis and the correct employment of the available therapeutic measures may result in a favorable outcome of the ocular and extra-ocular manifestations., (© 2023. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Immunoglobulins that reversibly precipitate at temperatures below 37 °C are called cryoglobulins (CGs). Cryoglobulinemia often manifests as cryoglobulinemic vasculitis (CV), whose symptoms range in severity from purpuric eruptions to life-threatening features. The majority of CV patients are infected with hepatitis C virus (HCV), whereas lymphoproliferative disorders or connective tissue diseases (CTD) are commonly diagnosed among patients with CV of non-infectious origin. In the absence of detectable associated disease, cryoglobulinemia is classified as "essential" (EMC). All HCV-positive CV patients should be given direct-acting antiviral agents (DAAs) that are consistently able to induce a sustained virologic response (SVR). Glucocorticoids (GCs) can mitigate CV-associated vasculitis, but they have no role as maintenance therapy. Cyclophosphamide restrains the hyperactive phase(s) of the disease and the post-apheresis rebound of newly synthesized CGs. Its use has been largely replaced by rituximab (RTX) in patients unresponsive to DAAs, patients progressing to B-cell non-Hodgkin lymphoma (B-NHL) and patients in whom CV persists or reappears after clearance of HCV. Therapeutic apheresis is an emergency treatment for CV patients with hyperviscosity syndrome. HCV-positive CV patients are at an increased risk of developing NHL, but the achievement of SVR can effectively prevent HCV-related NHL or induce the remission of an already established lymphoma, even without chemotherapy. The treatment of patients with IgM or IgG monoclonal cryoglobulins and an underlying immunoproliferative disorder is based on the regimens adopted for patients with the same B-cell malignancies but without circulating CGs. For patients with CTD, GCs plus alkylating agents or RTX are similarly effective as first-line therapy and in the relapse/refractory setting. In patients with EMC, treatment should consist of GCs plus RTX, with the dose of GCs tapered as soon as possible to reduce the risk of infectious complications., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The role of CD5 + B cells in patients with HCV infection and HCV-related disorders, including mixed cryoglobulinemia (MC), has been addressed in previous reports with conflicting results. We established a correlation between CD5/CD20 expression on circulating B lymphocytes, characterizing monoclonal B cell lymphocytosis (MBL), and clinical features in a cohort of 45 patients with chronic HCV hepatitis [without MC: 23 patients (MC- group); with MC: 22 patients (MC+ group)], and 45 HCV-negative healthy subjects as controls. By flow cytometry analysis, three B cells phenotypes were singled out: 1) CD5 + CD20 dim (CLL-like phenotype); 2) CD5 + CD20 bright (atypical phenotype); and 3) CD5 - CD20 + phenotype. CD5 + CD20 bright cells were reduced in MC- patients (p=0.049). CD5 + CD20 dim B cells were significantly higher in group B than in the control group (p=0.003). ROC curve analysis in MC+ patients showed the highest positive likelihood ratio at ≥7.35% (p=0.008) for CLL-like phenotype and at ≤63.6% (p=0.03) for the CD5 - CD20 + B cell phenotype. HCV infection was associated with a higher frequency of CLL-like (odds ratio=16, p=0.002) and a lower frequency of atypical (odds ratio: 3.1, p=0.02) and CD5 - CD20 + (odds ratio: 11, p=0.01) phenotypes. The association with higher levels of CLL-like phenotype progressively increased from group of MC- patients (odds ratio: 9.3, p=0.04) to the group of MC+ patients (odds ratio: 25.1, p=0.0003). CONCLUSIONS: The occurrence of a CLL-like pattern may allow to identify HCV-infected patients at risk of developing MC and eventually non-Hodgkin lymphoma, who should require a closer surveillance and a longer follow-up., (Copyright © 2019. Published by Elsevier B.V.)

Cryoglobulins are circulating immunoglobulins that reversibly precipitate at temperatures below 37 °C. Type-II cryoglobulins consist of monoclonal IgM/polyclonal IgG immune complexes (ICs), whereas in type-III cryoglobulins both IgM and IgG are polyclonal. The clinical condition resulting from the presence of cryoglobulins in the blood is called mixed cryoglobulinemia (MC), which can be asymptomatic or manifest as cryoglobulinemic vasculitis (CV). Type-I cryoglobulins, consisting of a single monoclonal isotype, are detected in patients with lymphoproliferative disorders. It is now established that > 90% of MCs are associated with HCV infection. Clinically, the spectrum of symptoms may range in severity from occasional purpuric eruptions to life-threatening features. In addition to the development of liver cirrhosis and hepatocellular carcinoma, the possible progression of HCV-positive CV patients to B-cell non-Hodgkin lymphoma (B-NHL) has been reported. The pathogenetic role played by HCV infection in the onset of B-NHL is suggested by regression of the latter following the achievement of a sustained virologic response (SVR). For several years, interferon-α alone or combined with ribavirin has been the standard of care. However, the rates of clinical, biochemical, and virologic responses have been low, and the occurrence of relapse frequent. The addition of rituximab has resulted in a higher rate of responses. With the advent of direct-acting antiviral agents, SVR has been achieved in ~ 95% of CV patients. However, in a minority of patients, despite SVR, CV may persist or reappear over variable lengths of time from the completion of therapy. The eventual appearance of B-NHL is also possible.

Rationale: Type I cryoglobulinemia is a rare and life-threatening condition. It occurs mainly in B-cell lymphoproliferative disorder. In almost half of the patients, type I cryoglobulinemia is characterized by severe cutaneous involvement., Patient Concerns: A 71-year-old man comes to our observation because of the onset of persistence of paresthesia and painful acrocyanosis in the fingers and toes. IgG-k multiple myeloma treated with chemotherapy and radiation therapy on the left iliac wing characterized his clinical history. At the evaluation of response after the first-line therapy, the patient achieved a very good partial response. At the time of progression, the same myelomatous disease has taken a typical behavior of cryoglobulinemia., Diagnoses: Type I multiple myeloma-associated cryoglobulinemia was diagnosed., Interventions: The patient underwent to an immediate composite therapeutic approach based on prostanoid infusion, plasmapheresis along with second-line chemotherapy., Outcomes: Despite the rapid biochemical response, the ischemia of the feet worsened. Moreover, a bacterial infection overlapped. The surgical amputation of both feet was necessary. Allowing MM cytoreduction continuation the patient's clinical condition became stabilized., Lessons: Though rare, type I cryoglobulinemia can be associated with plasma cell dyscrasias. Any delay in diagnosis and the start of therapy can cause worsening of organ damage and endanger the patient's life. Therapeutic strategies in these cases should be directed to the underlying diseases.

Background: The efficacy and safety of direct-acting antiviral agents (DAAs) were evaluated in a cohort of prospectively enrolled patients with hepatitis C virus (HCV)-related mixed cryoglobulinaemia (MC), an immune complex-mediated vasculitis of small and medium vessels in which the pathogenetic role of HCV has been clearly established., Methods: Twenty-two patients received DAAs. Clinical and laboratory features were recorded at baseline, every 4 weeks until the end of treatment (EoT), and 12 weeks afterwards. Primary efficacy endpoints were (a) sustained virological response 12 weeks after therapy completion (SVR12), (b) regression of symptomatology (clinical response) and (c) cryoglobulin disappearance or cryocrit reduction ≥50% (immunological response). Complete response (CR) was defined as the occurrence of all three primary endpoints; partial response (PR) was defined as the occurrence of SVR12, with or without either immunological or clinical response; and no response was defined as missing the achievement of all three endpoints., Results: All patients reached SVR12. Compared with basal values, mean cryocrit values were significantly decreased at EoT and SVR12. A significant reduction of alanine transaminase and a parallel increase of complement component C4 levels were also detected. Rheumatoid factor activity was significantly reduced at EoT but not at SVR12. At SVR12, a CR was established in 14 patients (63.7%) and a PR in 8 patients (36.3%). In one patient with small lymphocytic lymphoma, the tumour progressed despite viral clearance. Mild adverse events were recorded in nine patients (40.9%)., Conclusions: The response rates induced by the use of DAAs in patients with MC were remarkably higher than those previously achieved with pegylated interferon-α/ribavirin, with or without rituximab. A much longer follow-up is desirable to achieve useful information in terms of persistent viral clearance and clinical response.

Cryoglobulinemic vasculitis (CV) is a small-to-medium-vessel vasculitis that appears in 10-15 % of patients chronically infected with hepatitis C virus (HCV). The classic symptom triad of CV, purpura/asthenia/arthralgia, is accompanied by clinical features that include glomerulonephritis, neuropathy, interstitial pneumonitis, and cardiomyopathy, ranging in their severity from mild to life threatening. The risk of developing non-Hodgkin lymphoma is also higher. The cumulative 10-year survival rate of CV patients is significantly lower than in the age- and sex-matched general population, with death typically caused by nephropathy, malignancies, liver involvement, and severe infections. Unfailing serological stigmata include both a cryoglobulin IgM fraction with rheumatoid factor activity and decreased complement C4 levels. On peripheral B cells, the expression of the CD81 B cell receptor is reduced while that of the CD19 receptor is increased. A monoclonal B cell lymphocytosis develops in almost one-third of patients. HCV-related proteins (but not HCV-RNA genomic sequences) can be detected on biopsy samples by immunofluorescence and immunohistochemistry and involve the vessel lumen, vessel walls, and the perivascular spaces of the skin, kidney, and peripheral nerves, supporting the pathogenetic role of HCV in the onset of a widespread microvasculitis. Based on the demonstration of HCV infection in the large majority of CV patients, a therapeutic regimen consisting of once-weekly pegylated interferon-α and the daily administration of ribavirin results in a sustained virologic response in ~50 % of patients. In those with refractory and relapsing disease, addition of the anti-CD20 monoclonal antibody rituximab has significantly increased the overall response rates. The extension to CV of latest-generation direct-acting antivirals, strikingly successful in non-CV HCV-positive patients, has yielded high complete response rates according to the few studies published thus far.

Immunoglobulin variable region heavy chain (IgVH ) somatic gene diversification is instrumental in the transformation process that characterizes hepatitis C virus (HCV)-related B cell lymphoproliferative disorders. However, the extent to which activation-induced cytidine deaminase (AID), an enzyme essential for IgV gene somatic hypermutation (SHM), is active in cryoglobulinaemic vasculitis (CV) remains unclear. AID mRNA expression in the peripheral blood of 102 chronically hepatitis C virus (HCV)-infected patients (58 with and 44 without CV) and 26 healthy subjects was investigated using real-time reverse transcription-polymerase chain reaction (RT-PCR). The features of activation-induced cytidine deaminase (AID) protein and mRNA transcripts were explored in liver tissue biopsies and portal tracts isolated using laser capture microdissection. In chronically HCV-infected patients, AID mRNA expression was almost threefold higher in those with than in those without CV and sevenfold higher than in healthy subjects (median-fold: 6.68 versus 2.54, P = 0.03 and versus 0.95, P = 0.0003). AID transcript levels were significantly higher in polyclonal than in clonally restricted B cell preparations in either CV or non-CV patients (median-fold, 15.0 versus 2.70, P = 0.009 and 3.46 versus 1.58, P = 0.02, respectively). AID gene expression was found to be related negatively to age and virological parameters. AID protein was found in portal tracts containing inflammatory cells that, in several instances, expressed AID mRNA transcripts. Our data indicate that the aberrant expression of AID may reflect continuous B cell activation and sustained survival signals in HCV-related CV patients., (© 2015 British Society for Immunology.)

Introduction: Hepatitis C virus (HCV) infection can be detected in virtually all patients with cryoglobulinemic vasculitis (CV). Among its many effects, the virus is able to stimulate the production of thymic stromal lymphopoietin (TSLP) by infected hepatocytes. In this study, we assessed the systemic levels and tissue distribution of TSLP in 60 chronically HCV-infected patients, 36 with and 24 without CV., Methods: Serum TSLP levels were measured by an enzyme-linked immunosorbent assay (ELISA) method. TSLP mRNA was assessed in patient samples by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). TSLP protein in liver and skin biopsy samples was revealed by indirect immunofluorescence. All other methods were carried out according to standardized procedures., Results: Serum TSLP levels were significantly higher in patients with than in those without CV and in healthy individuals. Higher TSLP levels paralleled specific mRNA expression and the up-regulation of TSLP protein in liver tissue. Compared with non-CV patients, higher TSLP levels in CV were accompanied by a higher frequency of circulating mono/oligoclonal B-cell expansions (8% vs. 92%, p<0.0001) and a higher number of peripheral CD20+ B-cells (10.3% vs. 15.5% p=0.04). In addition, TSLP mRNA expression in the liver of CV patients was lower than in their correspondent skin tissue and paralleled specific immune deposits of TSLP protein in keratinocytes., Conclusion: Overall, this study shows that TSLP secreted by hepatocytes and keratinocytes of HCV-infected patients with CV is involved in the pathogenesis of vasculitis and may possibly support the therapeutic use of TSLP-targeted monoclonal antibodies.

Introduction: In hepatitis C virus (HCV)-related mixed cryoglobulinemia (MCG), the nonenveloped HCV core protein (HCV-Cp) is a constituent of the characteristic cold-precipitating immune complexes (ICs). A possible correlation between HCV-Cp, virologic, laboratory, and clinical parameters in both untreated MCG patients and those undergoing specific treatment was explored., Methods: HCV-Cp was quantified by a fully automated immune assay. Correlations between HCV-Cp and HCV RNA, cryocrit, and virus genotype (gt) were investigated in 102 chronically HCV-infected MCG patients., Results: HCV-Cp concentrations strongly correlated with HCV RNA levels in baseline samples. An average ratio of 1,425 IU and 12,850 IU HCV RNA per picogram HCV-Cp was estimated in HCV gt-1 and gt-2 patients, respectively. This equation allowed us to estimate that, on average, HCV-Cp was associated with the viral genome in only 3.4% of the former and in 35% of the latter group of patients. The direct relation between HCV-Cp and the cryocrit level suggests that the protein directly influences the amount of cryoprecipitate. Although the therapy with rituximab (RTX) as a single agent resulted in the enhancement of HCV-Cp levels, in patients treated with RTX in combination with a specific antiviral therapy (pegylated interferon-α plus ribavirin), the prompt and effective clearance of HCV-Cp was documented., Conclusions: Our data provide evidence that HCV-Cp has a direct effect on the cold-precipitation process in a virus genotype-dependence in HCV-related MCG patients.

Objective: Single-nucleotide polymorphisms (SNP) in the interleukin 28B (IL-28B) gene region are strongly predictive of the response of infected patients to antiviral therapy for hepatitis C virus (HCV). We sought to determine the prevalence of SNP IL-28B rs12979860 C/C and non-C/C (C/T plus T/T) genotypes in HCV-related cryoglobulinemic vasculitis (CV), as compared with HCV-positive patients without CV. We also searched for their association with peculiar clinical manifestations of CV and potential influence on the complete response (virological, molecular, and immunological) to the therapy., Methods: The study cohort comprised 159 and 172 HCV-infected patients with and without CV, respectively, prospectively followed starting from 1990. SNP rs12979860 genotyping was performed by Taq-Man allelic discrimination. In 106 patients (66.6%) with CV, the profile of circulating B cell clonalities was determined as well. All patients with CV were treated with pegylated interferon-α/ribavirin-based antiviral therapy., Results: The T/T IL-28B genotype was more common in patients with CV than in those without (17% vs 8.1%, p = 0.02). In patients with CV, compared with non-C/C variants, the IL-28B C/C genotype was associated with a higher rate of complete response (52.6% vs 39.2%, p = 0.13), whereas a treatment response of 61.4% was demonstrated when solely virological response was considered (p = 0.008). A higher frequency of expanded B cell clonalities in the circulation (84.2% vs 55.9%; p = 0.005), kidney involvement (21% vs 2.9%; p = 0.003), and B cell non-Hodgkin lymphoma (17.5% vs 6.8%; p = 0.048), were also observed., Conclusion: In HCV-positive patients with CV, the IL-28B C/C genotype is distinguished biologically by a higher frequency of restriction of B cell response and clinically by a higher risk of cryoglobulinemic nephropathy and B cell malignancies, while acting as an independent predictor of a sustained virological response to antiviral therapy. In addition, we found that IL-28B T/T variant was more prevalent in patients with CV than in those without.

This study illustrates the use and efficacy of a combination of pegylated interferon-alpha (Peg-IFN-alpha) and ribavirin (RBV), with or without rituximab (RTX), in hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC). Twenty-two patients with HCV-related MC received Peg-IFN-alpha (2a: 180 mug or 2b: 1.5 mug/kg) weekly plus RBV (1000 or 1200 mg) daily for 48 weeks, and RTX (375 mg/m(2)) once a week for 1 month followed by two 5-monthly infusions (termed PIRR). Fifteen additional patients received Peg-IFN-alpha/RBV with the same modalities as the PIRR schedule. Complete response was achieved in 54.5% (12/22) and in 33.3% (5/15) of patients who received PIRR and Peg-IFN-alpha/RBV, respectively (P < .05). Clearance of HCV RNA and conversion of B-cell populations from oligoclonal to polyclonal in liver, bone marrow, and peripheral blood was maintained for up to 3 years in 10 of 12 (83.3%) and in 2 of 5 (40%) patients receiving PIRR and Peg-IFN-alpha/RBV, respectively (P < .01). Cryoproteins in 22.7% (5/22) of patients with PIRR and in 33.3% (5/15) with Peg-IFN-alpha/RBV persisted despite sustained HCV RNA clearance. No response occurred in remaining 5 patients of both groups. PIRR therapy is well tolerated and more effective than Peg-IFN-alpha/RBV combination in HCV-related MC. Its effect may last for more than 3 years.

Mixed cryoglobulinemia (MC) is a lymphoproliferative disorder observed in approximately 10 to 15% of hepatitis C virus (HCV)-infected patients. Circulating, nonenveloped HCV core protein, which has been detected in cryoprecipitable immune complexes, interacts with immunocytes through the receptor for the globular domain of C1q protein (gC1q-R). In this study, we have evaluated circulating gC1q-R levels in chronically HCV-infected patients, with and without MC. These levels were significantly higher in MC patients than in those without MC and in healthy controls and paralleled specific mRNA expression in PBL. Soluble gC1q-R circulates as a complexed form containing both C1q and HCV core proteins. Higher serum gC1q-R levels negatively correlated with circulating concentrations of the C4d fragment. The presence of sequestered C4d in the vascular bed of skin biopsies from MC patients was indicative of in situ complement activation. In vitro studies showed that release of soluble gC1q-R is regulated by HCV core-mediated inhibition of cell proliferation. Our results indicate that up-regulation of gC1q-R expression is a distinctive feature of MC, and that dysregulated shedding of C1q-R molecules contributes to vascular cryoglobulin-induced damage via the classic complement-mediated pathway.

Chemokine CXCL13, also known as BCA-1 (B cell-attracting chemokine-1) or BLC (B-lymphocyte chemoattractant), is a major regulator of B-cell trafficking. Hepatitis C virus (HCV) infection may be associated with B-cell dysfunction and lymphoproliferative disorders, including mixed cryoglobulinemia (MC). This study evaluates circulating levels of CXCL13 protein and specific mRNA expression in chronically HCV-infected patients with and without MC. Compared with healthy controls and HCV-infected patients without MC, CXCL13 serum levels were significantly higher in MC patients. The highest CXCL13 levels strongly correlated with active cutaneous vasculitis. CXCL13 gene expression in portal tracts, isolated from liver biopsy tissues with laser capture microdissection, showed enhanced levels of specific mRNA in MC patients with active cutaneous vasculitis. Specific CXCL13 gene mRNA expression was also up-regulated in skin tissue of these patients. These findings paralleled specific deposits of CXCL13 protein both in the liver and in the skin. Our results indicate that up-regulation of CXCL13 gene expression is a distinctive feature of HCV-infected patients. Higher levels of this chemokine in the liver as well as in the skin of patients with active MC vasculitis suggest a possible interrelation between these biologic compartments.

Hepatitis C virus (HCV) is the major cause of cryoglobulinemia. Skin lesions are frequent and can be cured from the removal of cryoglobulins by therapeutic apheresis. We describe a case of HCV-positive type I cryoglobulinemia with severe leg ulcers, not responsive to antiviral and immunosuppressive treatment. Thirty sessions of double filtration plasmapheresis were performed, over a period of 6 months, with no other associated treatment. Before and after each session an assessment of immunoglobulins, complement, cryocrit, and fibrinogen was made. HCV RNA levels were determined in serum cryoprecipitate, supernatant before and after each session, and in the collection bag. No differences in pre and postapheresis values were observed in the serum concentrations and the supernatant, whereas the postapheresis cryoprecipitate showed a significantly reduced viral load (P < 0.02) as compared with the preapheresis values. There was improvement in the condition of ulcers in the leg during apheresis and had completely regressed by the end of the cycle., (Copyright 2008 Wiley-Liss, Inc.)

Recognition of hepatitis C virus (HCV) as an etiological factor in mixed cryoglobulinemia (MC) has dramatically changed our point of view in its treatment. Emphasis is placed on abatement and clearance of viral load and deletion of clonal expansions of IgM molecules with rheumatoid factor activity-synthesising B cells. The purpose of this review is to discuss the underlying scientific rationale and results of clinical studies of new treatment approaches to MC, with a focus on cell-depleting therapies and chemokine blockade. Additional antiviral agents directed to several phases of HCV life cycle acting with different or alternate mechanisms are proposed with the goal to enhance response rates more broadly suitable for MC patients with vasculitis and peripheral neuropathies. The majority of the available data on these new treatment approaches stems from open-label studies, but controlled trials are under way. Therapy directed against chemokines and/or cytokines represents an interesting and promising future target.

Like other hepatotropic viruses, hepatitis C virus (HCV) exhibits the property of inducing hepatocellular damage, possibly through induction of immune mechanisms that lead to hepatocellular necrosis. The early activated mechanisms include production of non-specific and specific antibodies, which represent the first line of defence against invading foreign pathogens. As a consequence, circulating immune complexes are promptly formed and antigen uptake and processing by specialized cells are enhanced. A major fraction of circulating immunoglobulins (Igs) are part of the spectrum of so-called "natural" antibodies, which include anti-idiotypic antibodies and molecules with rheumatoid factor (RF) activity. They mainly belong to the IgM class, are polyclonal and have no intrinsic pathogenetic potential. In 20-30% of HCV-infected patients, RFs share characteristics of high affinity molecules, are monoclonal in nature, and result in the production of cold-precipitating immune complexes and mixed cryoglobulinemia (MC). The persistent production of monoclonal RF molecules implies the existence of a further mechanism capable of restricting the reactivity and reflects a distinct selection of a cell population that can be maintained throughout life because it is continuously exposed to antigen pressure. Either polyclonal or monoclonal profiles of B-cell expansion are demonstrable in the liver of the majority of HCV-infected patients. The frequent detection of oligoclonal B cell expansion may, indeed, represent a key pathobiologic feature which sustains non-malignant B-cell lymphoproliferation. The preferential expansion of one clone would in turn lead to a monoclonal pattern, which could favor stochastic oncogenic events. Thus, it can be postulated that HCV is the stimulus not only for the apparent benign lymphoproliferative process underlying a wide spectrum of clinical features, but also for the progression to frank lymphoid malignancy in a subgroup of patients.

The ability of the immune system to distinguish between self and non-self is critical to the functioning of the immune response. A breakdown in these mechanisms can lead to the onset of autoimmune disease. Clinical and molecular data suggest that shared immunogenetic mechanisms lead to the autoimmune process. The most studied part of the autoimmune process is the human leukocyte antigen (HLA) region. Recently, progress has been made in narrowing down HLA cluster classifications based on structural and functional features of HLA alleles. Using this approach we have investigated 175 patients with hepatitis C virus (HCV)-induced type II cryoglobulinemia (MC), and compared them to a control group of 14,923 bone marrow donors. Additionally, we investigated the frequency of HLA homozygosity in the same groups of subjects. Our results provide evidence of a role for DR5 and DQ3 HLA class II clusters and a higher frequency of HLA homozygous leading to the clinical outcome of type II mixed cryoglobulinemic autoimmune disease. The DR5 cluster is characterized by a Glu in beta 9 and its polymorphism is connected with preferred anchors at beta 9 of the binding peptide, while the DQ3 cluster is characterized by Glu B86 and Leu B87, which allows the binding of large hydrophobic amino acids at p1 of the binding peptide. The mechanisms by which variations in HLA lead to autoimmunity remain unknown, although they are likely to be mediated by continuous presentation of HCV epitopes to T cells and a genetic background that limits the effective clearance of HCV. The results presented in this paper have increased our knowledge of the mechanism of autoimmune disease and B-cell lymphoproliferation during HCV infection. The work was performed in accordance with the principles of the 1983 Declaration of Helsinki. There is no conflict of interest.

Hepatitis C virus (HCV) infection is the major cause of mixed cryoglobulinaemia (MC), an immune complex (IC)-mediated systemic vasculitis mainly involving the small blood vessels. The precise mechanism of cryoprotein production is currently unknown. HCV virions and non-enveloped core protein participate in the formation of cold-insoluble ICs. Cryoglobulinaemic patients represent a distinct HCV-infected population, in that significant HCV enrichment of lymphoid cells is accompanied by evidence of productive virus infection and increased frequency of B cells. Liver, the major target organ of HCV, is the site of accumulation of inflammatory infiltrates that shares many architectural features with lymphoid tissue and reflects a distorted homeostatic balance between factors that enhance cellular recruitment, proliferation and retention, and those that decrease cellularity (cell death and emigration). There is now overwhelming evidence of a direct contribution to B-cell growth and survival through production of a variety of cytokines and chemokines. Liver tissue over-expression and abnormal circulating levels of B-cell activating factor belonging to the TNF family can provide effective costimulatory mechanisms to sustain the B-cell clonal expansion, which constitutes molecular stigmata of MC. Indolent lymphoproliferation might act as the starting point of chronic, multistage lymphomagenesis. An innovative therapeutic strategy is directed to 'eradication of the virus' and deletion of B-cell clonalities.

The relationship between the occurrence of cryoglobulins and hepatitis C virus (HCV) productive infection in peripheral blood and bone marrow-derived lymphocytes was explored. HCV minus strand RNA, the viral replicative intermediate, was searched for by a polyA(+) tract strand-specific Tth-based reverse transcriptase-polymerase chain reaction (RT-PCR) in lymphoid cells of 46 patients with acute and chronic infection. The HCV minus strand was demonstrated in RNA extracted from six (13%) and five (11%) peripheral blood and bone marrow-derived lymphocytes, respectively. The HCV replicating form in lymphoid cells was associated strictly with mixed cryoglobulinaemia (MCG), in that it was found in six of 13 (46%) MCG patients, including two with B cell non-Hodgkin's lymphoma (NHL). No traces of HCV-negative strand RNA were found in four patients with acute hepatitis C, in 15 with chronic active hepatitis without extrahepatic disorders, in seven with monoclonal gammopathy of undetermined significance, and in seven with B-NHL without MCG. These results emphasize the direct role of the virus in the pathogenesis of MCG and support the contention that HCV is not specifically lymphotropic, its entry and replication in lymphoid cells being determined largely by selective interactions.

We demonstrate that in three cases of MC (two with immunocytoma), the IgM-RF+ component of their cryoprecipitated represents the circulating counterpart of the B-cell receptor (BCR) of the monoclonal overexpanded B-cell population. These IgMs were isolated and used to demonstrate a crossreactivity against both hepatitis C virus (HCV) NS3 antigen and the Fc portion of IgG. Epitopes were identified in a fraction of exemplary samples by using epitope excision approach (NS(31250-1334) and IgG Fc(345-355)). The same phenomenon of crossreactivity has been shown to occur in vivo after immunization of a mouse with the NS3(1251-1270) peptide. To verify if the same reaction was also present in MC samples characterized by an oligo/polyclonal B-cell proliferation, IgM crossreactivity was tested in 14 additional samples. Five out of the 14 were reactive against HCV NS3 and 11 out of 14 were reactive against IgG-Fc peptide. The data support the role of HCV NS3 antigen in a subset of patients with MC, whereas the high frequency of the IgG-Fc epitope suggests that these B cells originate from precursors strongly selected for auto-IgG specificity. We suggest that engagement of specific BCRs by NS3 (or NS3-immunocomplex) antigen could explain the prevalence of IgM cryoglobulins in these patients.

Objective: To identify and characterize rheumatoid factor (RF)-producing B-cells and cryoprecipitate immunoglobulin (Ig) M in hepatitis C virus (HCV)-positive patients., Methods: We purified and characterized, by peptide mass fingerprinting integrated with an NCBI IgBlast data bank search, the IgM component of cryoprecipitate and analysed the VDJ pattern of bone marrow B-cells by gene scan analysis of 17 HCV-positive patients with type II mixed-cryoglobulinaemia., Results: IgM purified from all of the patients presented an RF specificity. In three of these patients a high and predominant B-cell clone (>or=30%) was found in the bone marrow. B-cell-receptor sequences were determined and immunophenotyping of these clones was performed. Peptide masses originating after tryptic digestion of the B-cell-receptor combinatory regions and those originating by tryptic digestion of the cryoprecipitated IgM from the same patient were comparable. In the remaining patients an oligoclonal/polyclonality was found. However, in some of these patients we were able to find peptides that matched with the B-cell-receptor sequences of overexpanded B cells, indicating that, even in the absence of a clear monoclonal expansion, a fraction of total cryoprecipated IgM may derive from overexpanded B-cell clones found in patients' bone marrow., Conclusions: In the majority of mixed cryoglobulinaemia-HCV-positive patients, both in the serum and in B cells from the bone marrow, an oligoclonal pattern is the main molecular picture. When a monoclonal B-cell clone is found, its B-cell-receptor shows an antigen-binding fragment identical to that of cryoprecipitable RF-IgM. Phenotypically, B cells are CD20-positive but CD5-negative, suggesting that the B-1 B-cell subset is not likely to produce high-affinity IgM-RF molecules.

In clinical and pathological terms hepatitis C virus (HCV)-infected patients can be subdivided into two main groups with and without mixed cryoglobulinaemia (MC). Involvement of blood mononuclear cells by HCV has potentially important implications. To this end, HCV-RNA levels in peripheral blood lymphocytes (PBL) preparations of 20 chronically HCV-infected patients with MC were measured and compared with those found in a group of 20 patients without MC matched for age, serum HCV-RNA, infectious genotype, source and presumable duration of infection. Phenotypic abnormalities of PBL subsets in each group of patients were determined by cell surface marker expression and compared. Results showed a significant enrichment of HCV-RNA in PBL of MC patients compared with a non-MC group (P = 0.01). Different distribution of HCV-RNA was accompanied by evidence of an increased frequency of circulating B cells. These data indicate that MC patients are characterized distinctly by a higher quota of cell-associated viral load.

Several viruses are involved in the development of systemic vasculitides. Hepatitis C virus (HCV) has been shown to be closely related to mixed cryoglobulinaemia, an immune complex-mediated vasculitis. HCV particles and non-enveloped nucleocapsid protein participate in the formation of immune complexes. Once formed, immune complexes precipitate in many organs, including the skin, kidneys, and peripheral nerve fibres. Viral proteins confer peculiar physical and chemical properties on cryoimmunoglobulins. Since expansion of rheumatoid factor-synthesising B cells is the biological hallmark of mixed cryoglobulinaemia, it may be that the combination of rheumatoid factor activity and cryoprecipitability is responsible for the vasculitis. B-cell clonal expansion occurs primarily in the liver and correlates with a high intrahepatic viral load, pointing to a major role for HCV in the emergence and maintenance of B-cell clonalities. Recognition of HCV as an aetiological factor in most cryoglobulinaemic vasculitides has dramatically changed the approach to their treatment. Emphasis, in fact, is now placed on abatement of the viral load and deletion of B-cell clonalities.

Hepatitis C virus (HCV) infection has been detected in a large proportion of patients with mixed cryoglobulinaemia (MC). Circulating 'free' non-enveloped HCV core protein has been demonstrated in HCV-infected patients, and this suggests its possible involvement in the formation of cryoprecipitable immune complexes (ICs). Thirty-two anti-HCV, HCV RNA-positive patients with type II MC were evaluated. Non-enveloped HCV core protein, HCV RNA sequences, total IgM, rheumatoid factor (RF) activity, IgG and IgG subclasses, C3 and C4 fractions, C1q protein and C1q binding activity were assessed in both cryoprecipitates and supernatants. Non-enveloped HCV core protein was demonstrated in 30 of 32 (93.7%) type II MC patients. After separation of cold-precipitable material, the protein was removed completely from supernatant in 12 patients (40%), whereas it was enriched in the cryoprecipitates of the remaining 18. In addition, HCV RNA and IgM molecules with RF activity were concentrated selectively in the cryoprecipitates. Differential precipitation was found for both total IgG and IgG subclasses, as they were less represented in the cryoglobulins and no selective enrichment was noted. Immunological characterization of HCV core protein-containing cryoprecipitating ICs after chromatographic fractionation showed that the IgM monoclonal component had RF activity, whereas anti-HCV core reactivity was confined to the IgG fraction. C1q enrichment in addition to high avidity of ICs for C1q binding in the cryoprecipitates suggest that complement activation may occur through the C1q protein pathway. The present data demonstrate that non-enveloped HCV core protein is a constitutive component of cryoprecipitable ICs in type II MC patients.

A controlled study has been carried out to assess the efficacy of rituximab, a chimeric antibody that binds to the B-cell surface antigen CD20, in 20 patients with mixed cryoglobulinemia (MC) and hepatitis C virus (HCV)-positive chronic active liver disease, resistant to interferon alpha (IFN-alpha) therapy. They received an intravenous infusion of 375 mg/m(2) rituximab once a week for 4 consecutive weeks. Infusion of rituximab had a good safety profile and no severe side effects were reported. Sixteen patients (80%) showed a complete response (CR), characterized by rapid improvement of clinical signs (disappearance of purpura and weakness arthralgia and improvement of peripheral neuropathy), and decline of cryocrit. CR was associated with a significant reduction of rheumatoid factor (RF) activity and anti-HCV antibody titers. Decline of IgG anti-HCV titers in the cryoprecipitates was usually associated with a favorable response (r = 0.81; P <.005). No differences in the dynamics of B-cell depletion and recovery were found between responders and nonresponders. Molecular monitoring of the B-cell response revealed disappearance/deletion of peripheral clones in the responders and great stability in the nonresponders. Rituximab had a deep impact on hepatitis C viremia; HCV RNA increased approximately twice the baseline levels in the responders, whereas it remained much the same in the nonresponders. Twelve (75%) of 16 responders remained in remission throughout the follow-up. The results indicate that rituximab has clinical and biologic activity in patients with HCV(+) MC. However, in view of the increased viremia in the responders, additional modes of application and combination of rituximab with other agents need to be investigated.

Cryoglobulins are cold-precipitable immunoglobulins associated with a number of infectious, autoimmune and neoplastic disorders. Their appearance along with rheumatoid factor (RF) can be considered a normal event in the clearance of immune complexes and rarely produces any symptoms. The association between hepatitis C virus (HCV) and mixed cryoglobulinemia (MC) has been rendered evident since the recognition of serological markers of HCV infection. There is thus every reason to suppose that direct or indirect involvement of B cells on the part of the HCV results in their persistent stimulation, clonal expansion and release of molecules with RF activity. The formation of RF/IgG immune complexes is the key pathogenetic mechanism. The close correlation between HCV infection and MC also throws new light on the interpretation of autoimmune phenomena in the course of viral infection and on the close link between autoimmune diseases and lymphoproliferative disorders. The higher risk of non-Hodgkin's lymphoma (NHL) displayed by HCV positive subjects, especially in the Mediterranean basin, suggests that the HCV's chronic lymphoproliferative drive may progress towards frank lymphoid neoplasia. The presence of MC does not represent an in situ or 'occult' NHL, because recent evidences indicate that none of the clones interpreted as predominant displays the molecular features of a true neoplastic process. The cryoglobulinemic syndrome is probably the consequence of pathogenic noxae that act upon the immune system of a host in which regulation of the peripheral T cell response appears to be in some way altered.

Like other hepatotropic viruses, hepatitis C virus (HCV) shares the property of inducing hepatocellular damage, possibly through induction of immune mechanisms that lead to hepatocellular necrosis. After infection of hepatocytes, and possibly other cells, humoral and cellular responses occur aimed at prevention of virus dissemination and elimination of infected cells. The early activated mechanisms include production of nonspecific and specific antibodies that represent the first-line of defense against invading foreign pathogens. As a consequence, circulating immune complexes are promptly formed, and antigen uptake and processing by specialized cells are enhanced. A major fraction of circulating immunoglobulins (Igs) are part of the spectrum of the so-called natural antibodies, which include anti-idiotypic antibodies and molecules with rheumatoid factor (RF) activity. They mainly belong to the IgM class, are polyclonal, and have no intrinsic pathogenetic potential. In 20-30% of HCV-infected patients, RFs share characteristics of high affinity molecules, are monoclonal in nature, and result in the production of cold-precipitating immune complexes and mixed cryoglobulinemia. It has been shown that anti-idiotypic antibodies and polyclonal and monoclonal RF molecules have the same cross-reactive idiotype, called WA, suggesting that their production is highly restricted. This strongly indicates that they arise from stimulation with the same antigen, likely HCV. It has also been speculated that B-1 (CD5+) and B-2 (CD5-) B-cell subsets, which use a limited number of VH germline genes, underlie the production of low-affinity polyclonal and high-affinity monoclonal antibodies, respectively. The persistent production of monoclonal RF molecules implies the existence of a further mechanism capable of restricting the reactivity and reflects a distinct selection of a cell population that can be maintained throughout life because they are continuously exposed to antigen pressure. Either polyclonal or monoclonal profiles of B-cell expansion are demonstrable in the liver of most HCV-infected patients. The occurrence of B-cell clonal expansion is strictly related to intrahepatic production of RF molecules, and this suggests that liver is a microenvironment, other than lymphoid tissue, in which a germinal centerlike reaction is induced. The frequent detection of oligoclonal B-cell expansion may, indeed, represent a key pathobiologic feature that sustains nonmalignant B-cell lymphoproliferation. The preferential expansion of one clone would in turn lead to a monoclonal pattern that could favor stochastic oncogenic events. It can be postulated that HCV is the stimulus not only for the apparent benign lymphoproliferative process underlying a wide spectrum of clinical features, but also for the progression to frank lymphoid malignancy in a subgroup of patients. Current data indicate a higher prevalence of overt B-cell non-Hodgkin's lymphoma in HCV-infected patients, especially in some geographic areas.

Hepatitis C virus (HCV) is a single-stranded RNA agent which expresses its genetic informations in the form of a single, large polyprotein encoded by an open reading frame (ORF) that extends through most of its genomic RNA. Proteolytic cleavage of the ORF product is essential for the virogenesis and the production of viral progeny. HCV is responsible for chronic liver disease, cirrhosis and possibly hepatocellular carcinoma. Viral persistence is considered the greatest problem in the management of HCV infection. It may result from several mechanisms, two of which are established. In the first, the high rate of genetic variations during viral replication results in the production of mutants capable of escaping the immune attack. In the second, the virus infects cells of the immune system itself, which represent a privileged site that cannot be reached by virus-specific T cell response. Involvement of lymphoid cells in the early stages of HCV infection may provide insight into the pathobiologic patterns of extrahepatic dissemination (lymph nodes, major salivary glands, kidneys, blood vessels). Dissemination of HCV-infected lymphoid cells throughout the organism is likely to maintain a mobile and extensive reservoir of the virus. In this respect, extrahepatic sites may act as a source of continuous reinfection of hepatocytes. Studies of intrahepatic B lymphocytes indicate that they are infected with HCV, clonally expanded and activated to secrete IgM molecules with rheumatoid factor activity. This strongly suggests that HCV directly stimulates B cell expansion, which may result in an indolent stage of lymphoproliferation (i.e., mixed cryoglobulinemia) or in frank B cell non-Hodgkin's lymphoma (NHL). The frequency of NHL, however, is much lower than that of HCV infection, suggesting that HCV alone is not able to induce tumors and that cellular events, in addition to the presence of virus and virus-encoded products, are necessary in order to obtain a malignant B cell phenotype. The demonstration of HCV productive infection in bone marrow-recruited and circulating pluripotent hematopoietic CD34+ stem cells indicates that HCV replication occurs in the early differentiation stages of hematopoietic progenitors. These are stable cell populations and are likely to represent the initial site of infection and a continuous source of virus production.

Clonal rearrangements of Ig heavy chain (IgH) genes and hepatitis C virus (HCV) genomic sequences were assayed on intrahepatic B lymphocytes isolated from HCV chronically infected patients with and without type II mixed cryoglobulinemia (MC). Liver tissue samples from eight patients with and nine without MC were subjected to routine histologic studies, immunophenotyping, and genotypic analysis including IgH V-D-J region gene rearrangements by PCR. RT-PCR, signal amplification by branched DNA assay, and in situ hybridization technique were used to detect and quantitate HCV RNA genomic sequences in selected B cells purified from each tissue sample. Although HCV infection of intrahepatic B cells was shown in all patients both with and without MC, frank B cell monoclonal and oligoclonal patterns were found in only three and four patients with MC, respectively. No monoclonal profile was seen in the noncryoglobulinemic patients, whereas an oligoclonal profile was demonstrated in four of them. No clonalities were shown in HCV-unrelated patients matched for age and severity of liver disease. No obvious difference in HCV genotype distribution was found in relation to the clonal expansion profile. Noncryoglobulinemic patients showing clonal expansion in liver tissue had higher titers of serum rheumatoid factor (RF). Spontaneous production of RF was shown in cell cultures of intrahepatic B cells, suggesting their persistent stimulation in vivo. These data indicate that HCV infection of B cells and B cell clonal expansions occur in the liver microenvironment and preferentially involve RF-producing cells.

Leukocytoclastic vasculitis is the dominant lesion of mixed cryoglobulinemia (MC). The high prevalence of antibodies to hepatitis C virus (HCV) in association with the higher concentration of HCV RNA genomic sequences in the cryoglobulins suggests a close relationship between MC and HCV infection and strongly supports the view that this virus plays a key role in causing vascular damage. Analysis of the composition of immune complexes (ICs) provides evidence that cryoglobulins include virions mostly bound to IgG that is specifically reactive with HCV-related proteins, which in turn are crosslinked by monoclonal IgM with rheumatoid factor (RF) activity, frequently bearing the WA crossidiotype (XId). This structure is similar (if not identical) to that of circulating ICs from HCV-infected patients without cryoglobulins, suggesting that the virus may be directly responsible for the production of WA RF. Evidence for the role of circulating cryoproteins in the pathogenesis of cutaneous and renal vasculitis stems from the demonstration of HCV-related proteins and/or HCV RNA genomic sequences in the vessel wall of patients with MC. Our data indicate that endothelial cells are fully susceptible to infection by and replication of HCV, and support the contention that they serve as sufficient targets for the binding of HCV proteins expressed on the cell surface to serum immunoglobulins. The in situ demonstration of IgM RF WA XId adds further evidence that RF of the WA group participates in the development of vasculitis and probably stabilizes the binding of IgG antibodies. Lymphocytes may be crucial in the infection of endothelial cells by acting as a circulating viral reservoir. After encouraging initial results, controlled trials have defined the substantive efficacy of IFN-alpha in the treatment of MC. A response of IFN can be achieved in more than 50% of patients and includes improvement of cutaneous vasculitis and renal function. This clinical response is accompanied by a reduction in hepatitis C viremia, serum cryoglobulin concentration, and IgM RF synthesis. However, almost 80% of responders eventually have a clinical and biochemical relapse. Additional studies are required to improve the outcome and extension of this therapy, define the best candidates, and indicate the situations in which it is needed.

The role of hepatitis C virus (HCV) in the pathogenesis of type II mixed cryoglobulinemia (MC) has been strongly emphasized in the last few years. Although MC is a benign lymphoproliferative disorder, the risk of overt B-cell malignancy greatly increases during its course. The occurrence of HCV infection in 10% to 30% of patients with non-Hodgkin's lymphoma (NHL) suggests that this virus may have a role in the development of MC-associated B-cell malignancies. We identified 2 patients with hyperplastic reactive lymphadenopathy (HRL) and 12 with NHL in two series of MC patients chronically infected with HCV collected over a 5-year period. Structural and nonstructural HCV-related proteins were investigated in lymph node sections by immunohistochemistry and their location and distribution were correlated with clinical and histologic findings, viremic state, and HCV genotypes. In HRL, HCV proteins were found in the cytoplasm of lymphoid cells, mainly in interfollicular areas. However, occasional positive cells were found in the mantle zone and in the germinal centers of follicles. In addition, strong reactivity was found in the circulating mononuclear cells of capsular blood vessels. HCV immunodeposits were found in 3 of 12 (25%) NHL cases. Positive cells were frequently restricted to the cortex; if not, they were randomly diffused in the neoplastic tissue. Positivity was related to the low-grade type of NHL; in the 2 composite cases, HCV immunodetection was found in the small cells, whereas large anaplastic cells were regularly negative. Other viruses previously involved in lymphoproliferation, ie, human herpes virus-6 and Epstein-Barr virus, were absent in all tissues. These data emphasize that lymphoid organs may be a site of HCV infection. The demonstration of HCV-related proteins in a nonmalignant condition, namely HRL, indicates that HCV infection precedes the neoplastic transformation and possibly plays a major role in lymphomagenesis in MC.

Objectives: Lymph node biopsies from patients with mixed cryoglobulinemia (MC) were investigated for: a) the presence of the hepatitis C virus (HCV) peptides C22 and C100; b) the degree of angiogenesis and of inflammatory infiltrate (macrophages and neutrophils); c) the distribution of laminin and type IV collagen, the main components of the subendothelial basement membrane; and d) the ultrastructural characteristics of the microvessels., Methods: Cryostat sections were stained by immunoperoxidase to highlight HCV peptides, endothelial cells, their basement membrane components, and inflammatory cells. Planimetric methods were applied to count the microvessels and inflammatory cells, and to evaluate the distribution pattern of the basement membrane components. Microvessels were also analysed at the ultrastructural level., Results: HCV+ lymph nodes showed higher microvessel and inflammatory cell counts than the HCV- lymph nodes. Their laminin and type IV collagen distribution and ultrastructural findings also indicated immature capillaries., Conclusions: HCV could be introduced into the lymph nodes by infected mononuclear cells, and trigger inflammation and angiogenesis, thus contributing to the inflammatory process. Angiogenesis, in turn, could facilitate HCV dissemination and persistence in MC.

Purpose: The purpose of this study was to assess whether granulocytopenia observed in 3 of 38 patients with essential mixed cryoglobulinemia who were treated with low-dose interferon was due to the underlying disease or to synergistic toxicity of interferon with other drugs., Patients and Methods: Adverse effects of interferon therapy were monitored in 38 patients affected with type II essential mixed cryoglobulinemia. Patients were treated with 3 million units (MU), daily or on alternate days, of recombinant interferon-alpha 2a (35 patients) or with natural interferon-beta (3 patients). The duration of treatment ranged between 6 and 15 months; the total duration of follow-up, including after therapy, ranged between 8 and 93 months., Results: None of 35 patients treated with interferon alone developed significant hematologic alterations. In addition, none of 7 patients treated with angiotensin-converting enzyme (ACE) inhibitors alone showed hematologic toxicity. Three patients who were treated with a combination of interferon and ACE inhibitors developed severe granulocytopenia a few days after starting treatment. Granulocytopenia subsided within 1 to 2 weeks after suspending therapy. Resumption of treatment with this drug combination produced a granulocytopenia relapse in 1 patient. In these 3 patients, interferon treatment alone, or ACE inhibitor monotherapy, was not followed by granulocytopenia., Conclusion: Although severe hematologic toxicity rarely develops in patients treated with low-dose interferon, granulocytopenia occurred in all 3 of our patients with mixed cryoglobulinemia who were treated with a combination of low-dose interferon-alpha 2a and ACE inhibitors. Neither drug alone was toxic in any of our cryoglobulinemic patients, indicating a high risk of severe hematologic toxicity for this drug combination, at least in patients with this disease. Physicians should be aware of this danger when using interferon treatment in patients with this, or possibly other, disorder(s) that also require antihypertensive therapy.

Hepatitis C virus (HCV) has been implicated as the major etiologic factor sustaining B-cell clonal expansion in type II mixed cryoglobulinemia (MC). A putative pathogenetic role of HCV in the development of MC-associated B-cell malignancies has also been speculated. We report for the first time the localization of HCV within a parotid non-Hodgkin's lymphoma (NHL) lesion in the course of HCV-related type II essential MC, an important step to implicate any infectious agent in the lymphomagenesis. Plus and minus strand HCV RNA was first demonstrated by polymerase chain reaction on the whole RNA from the lesion. Further immunohistochemical studies localized HCV c22 proteins in the residual ductal or acinar parotid structures, which also abnormally expressed HLA-DR antigens. Weak c22 signals were inconstantly detected in cells strictly confined around the residual epithelium, while all the remaining infiltrating cells in the parotid lesion stained c-22-negative. Staining for c33 and c100 HCV antigens was negative. In situ hybridization (ISH) studies again identified the residual parotid epithelial cells as the site of HCV infection and replication in the NHL lesion. Sialotropic viruses previously involved in lymphoproliferation, ie, Epstein-Barr virus and human herpesvirus-6, were absent in the same tissue lesion. According to the current models of B-cell lymphomagenesis, a role of HCV as an exogenous antigenic stimulus should be considered for NHL development in the present case, whereas malignant B cells do not appear permissive of active HCV replication. Further efforts would be worthwhile to clarify a role of HCV infection in the development of some B-cell malignancies.

Skin and/or liver biopsy specimens were obtained from the following patients: 15 anti-hepatitis C virus (HCV), HCV RNA-positive patients and 3 anti-HCV, HCV RNA-negative patients with type II mixed cryoglobulinemia (MC); 7 anti-HCV, HCV RNA-positive patients with chronic active liver disease (CALD); 5 anti-HCV, HCV RNA-negative patients with noncryoglobulinemic vasculitis; and 7 anti-HCV, HCV RNA-negative patients with lichen ruber planus. A pool of murine monoclonal antibodies (MAbs) developed against c22-3, c33c, and c100-3 proteins was used to detect HCV-related antigens (Ags) by indirect immunohistochemistry. Acid electroelution (AEE) of tissue sections was performed to enhance the sensitivity of the immunohistochemical method. In anti-HCV-positive MC patients, specific HCV-related Ags were detected in the small vessels of the skin and in the cytoplasm of hepatocytes. Prior AEE of biopsy sections allowed detection of HCV Ags in 6 of 15 (40%) skin biopsy and in 9 of 14 (64.3%) liver biopsy specimens. HCV immunoreactive deposits in the skin displayed two immunohistochemical patterns: (1) coarse intraluminal material associated with dermal inflammatory infiltrates and intravascular deposition of eosinophilic hyaline material; and (2) reactivity confined to the vessel wall in the context of an apparently normal tissue. Immunoglobulin (Ig) G and IgM deposition in the skin showed immunohistochemical features comparable with those found for HCV Ag deposits.(ABSTRACT TRUNCATED AT 250 WORDS)

Type II mixed cryoglobulinemia (MC) is an often progressive vasculitis characterized by circulating cold-precipitable proteins that usually consists of polyclonal IgG and monoclonal IgM kappa with rheumatoid factor (RF) activity. Its etiology is unknown, although recent evidence strongly suggests that hepatitis C virus (HCV) plays a major role. Plasmapheresis, corticosteroids, and cytotoxic drugs have been used in the therapy of MC patients. Recently, favorable results with recombinant interferon-alpha (rIFN alpha) have been reported. To further assess its effectiveness, we studied the effects of natural human interferon-alpha (nIFN alpha), alone and in combination with 6-methyl-prednisolone (PDN), in a prospective, randomized, controlled trial in patients with symptomatic MC. Sixty-five patients were enrolled onto the trial, 52 (80%) of whom presented serum anti-HCV antibodies and specific genomic RNA sequences. Fifteen patients received nIFN alpha (3 MU) intramuscularly (IM) three times weekly, whereas 17 patients also received 16 mg/d of PDN orally on non-IFN days. Moreover, 18 patients received 16 mg/d of PDN only, and 15 were untreated. Treatment was discontinued after 1 year and patients were monitored for 8 to 17 months (mean, 13). A complete response was achieved in eight of 15 patients (53.3%) treated with nIFN alpha and nine of 17 (52.9%) treated with nIFN alpha plus PDN, as compared with three of 18 patients (16.7%) who received PDN only (P < .05) and one of 15 (6.7%) untreated controls (P < .01). Partial response occurred in two of 15 (13.3%) patients treated with nIFN alpha, three of 17 (17.6%) who received nIFN alpha plus PDN, one of 18 (5.5%) who received PDN only, and one of 15 (6.7%) controls. A complete response in six patients (66.7%) was achieved within 3 months in the group that received nIFN alpha plus PDN, as compared with two patients (25%) of those who received nIFN alpha alone (P < .02). In anti-HCV-positive patients, the clinical response occurred in step with reduced or undetectable levels of HCV RNA and transaminase normalization. Quantification of circulating HCV RNA represented a good predictive response marker. The probability of relapse within 3 months after treatment was 100% (three of three patients) and 75% (six of eight patients), respectively, in patients who received PDN alone or nIFN alpha alone as compared with none of those who received nIFN alpha plus PDN (P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

The high frequency of liver involvement in cryoglobulinemia is well established. Although both etiology and pathogenesis have remained so far undefined, recent studies suggest an association of mixed cryoglobulinemia with hepatitis C virus infection. To explore this hypothesis further, we assessed the prevalence of hepatitis C virus antibodies and RNA in a large group of patients, including: (1) 35 patients with cryoglobulinemia without clinical evidence of liver involvement (group 1), (2) 15 patients with symptomatic cryoglobulinemia associated with chronic liver disease (group 2) and (3) 12 patients with asymptomatic cryoglobulinemia associated with chronic liver disease (group 3). Anti-hepatitis C virus antibodies were detected by a second-generation enzyme-linked immunosorbent assay and third-generation immunoblot (SIA Prototype RIBA), whereas the polymerase chain reaction was used for the detection of viral RNA. Anti-hepatitis C virus antibodies, as detected by enzyme-linked immunosorbent assay, were demonstrated in 21 (60%) patients from group 1, 11 (73.3%) from group 2 and 10 (83.3%) from group 3. The immunoblot identified as positive 3 further patients in group 1 (giving a prevalence of 68.6%) and all patients in groups 2 and 3. Hepatitis C virus RNA was demonstrated in cryoprecipitates from 21 of 24 immunoblot-positives and from 6 of 11 immunoblot-negatives, indicating an actual active viral replication in 77.1% of group 1. This was also found in 13 (86.7%) and 10 (83.3%) cryoprecipitates of groups 2 and 3, respectively. Type II cryoglobulinemia was the prevalent form in group 1 (88.6%) and group 2 (73.3%), whereas type III was found in group 3 (58.3%) and in 26.7% of group 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Although essential mixed cryoglobulinaemia (EMC) is recognized to be frequently associated with chronic liver disease, aetiology and pathogenesis of liver damage remain unsolved questions. The purpose of this study was to assess the possible causative role of hepatitis C virus (HCV) in the liver impairment occurring in patients with EMC. Twenty-six consecutive EMC patients were evaluated. All patients underwent percutaneous liver biopsy. Anti-HCV antibodies were assayed by ELISA and supported by a recombinant immunoblotting assay (4-RIBA). The prevalence of anti-HCV antibodies in patients with and without chronic active liver disease (CALD) was compared. Anti-HCV antibodies were detected in 13 patients (50%) by ELISA and confirmed in 11 of them (42.3%) by 4-RIBA, the remaining two patients being indeterminate in the supportive assay. CALD correlated significantly with anti-HCV antibodies: indeed, 7/11 (63.6%) anti-HCV+ patients showed histological and clinical pictures of CALD, compared with 1/15 (6.6%) anti-HCV- patients (P less than 0.01). With the exception of the patient who was found to be HBsAg+, no liver tissue expressed hepatitis B virus-related antigens in the hepatocytes. Additional histological findings included discrete lymphoid aggregates in portal tracts, siderosis, fatty changes, hyperplasia of Kupffer cells. It can be concluded that chronic liver damage in EMC is frequently associated with anti-HCV antibodies. Although the cause of EMC remains unknown, this study has obvious implications for clarifying the etiology of associated CALD and further supports the therapeutic use of interferons in this disease.

Aims: Peripheral neuropathy (PN), the major neurological complication of chronic HCV infection, is frequently associated with mixed cryoglobulinaemia (MC) and small-vessel systemic vasculitis. While humoral and cellmediated immune mechanisms are suspected to act together in an aberrant immune response that results in peripheral nerve damage, the role of HCV remains largely speculative. The possible demonstration of HCV in peripheral nerve tissue would obviously assume important pathogenic implications. Methods: We studied sural nerve biopsies from 11 HCV-positive patients with neuropathic symptoms: five with and six without MC. In situ hybridization (ISH) and immunofluorescence studies were carried out to detect genomic and antigenomic HCV RNA sequences and HCV-encoded E2-glycoprotein, respectively. Results: Epineurial vascular deposits of E2- glycoprotein were found in four (80%) MC and in two (33.3%) non-MC patients, respectively. These findings were enhanced by the perivascular deposition of positive-, though not negative-strand replicative RNA, as also found in the nerve extracts of all patients. Mild inflammatory cell infiltrates with no deposits of immunoglobulins and/or complement proteins were revealed around small vessels, without distinct vasculitis changes between MC and non-MC patients. Conclusions: These results indicate that nerve vascular HCV RNA/E2 deposits associated to perivascular inflammatory infiltrates were similar in chronically HCV-infected patients, regardless of cryoglobulin occurrence. Given the failure to demonstrate HCV productive infection in the examined sural nerve biopsies, nerve damage is likely to result from virus-triggered immune-mediated mechanisms. [ABSTRACT FROM AUTHOR]