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Objectives The biomarkers of an immunological dysregulation due to a chronic HBV infection are indeed understudied. If untreated, this condition may evolve into liver impairment co-occurring with extrahepatic involvements. Here, we aim to identify a new panel of biomarkers [including immunoglobulin G (IgG) subclasses, RF, and Free Light Chains (FLCs)] that may be useful and reliable for clinical evaluation of HBV-related cryoglobulinemia. Methods We retrospectively analysed clinical data from 44 HBV-positive patients. The patients were stratified (according to the presence/absence of mixed cryoglobulinemia) into two groups: 22 with cryoglobulins (CGs) and 22 without CGs. Samples from 20 healthy blood donors (HDs) were used as negative controls. Serum samples were tested for IgG subclasses, RF (-IgM, -IgG, and -IgA type), and FLCs. Results We detected a strikingly different distribution of serum IgG subclasses between HDs and HBV-positive patients, together with different RF isotypes; in addition, FLCs were significantly increased in HBV-positive patients compared with HDs, while no significant difference was shown between HBV-positive patients with/without mixed cryoglobulinemia. Conclusion The immune-inflammatory response triggered by HBV may be monitored by a peculiar profile of biomarkers. Our results open a new perspective in the precision medicine era; in these challenging times, they could also be employed to monitor the clinical course of those COVID-19 patients who are at high risk of HBV reactivation due to liver impairment and/or immunosuppressive therapies.

BACKGROUND: Genetic variants of IFNL4 and PDCD1 genes have been shown to influence the spontaneous clearance of hepatitis C virus (HCV) infection. We investigated the IFNL4 rs12979860 and the PDCD1 polymorphisms in 734 HCV-positive patients, including 461 cases with liver disease of varying severity and 273 patients with lymphoproliferative disorders to determine the association of these genes with patient's outcome. METHODS: Expression levels of PDCD1 mRNA encoded by haplotypes were investigated by quantitative PCR in hepatocellular carcinoma (HCC) tissue and peripheral blood mononuclear cells. Flow cytometry was used to detect PD-1 and its ligand PD-L1. RESULTS: The frequency of IFNL4 rs12979860 C/T or T/T genotypes was significantly higher in patients with HCV-related diseases than blood donors (P

Hepatitis C virus (HCV) causes B-cell lymphoproliferative disorders (LPDs) expressing stereotyped B-cell receptors (BCRs) endowed with rheumatoid factor (RF) activity and putatively recognizing the HCV E2 protein. To further untangle the shaping and function of these BCRs, we analyzed immunoglobulin gene rearrangements of monoclonal B cells from 13 patients with HCV-associated LPDs and correlated their features with the clinical outcomes of antiviral therapy. While only two patients shared a stereotyped heavy-chain complementarity determining region 3 (CDR3) sequence, two kappa chain CDR3 stereotyped sequences accounted for 77% of BCRs. Light chains were enriched in sequences homologous to anti-HCV E2 antibodies compared with heavy chains (7/13 vs. 0/13; p = 0.005). Anti-HCV E2 homology was uniquely associated (7/7 vs. 0/6; p = 0.0006) with a stereotyped CDR3 sequence encoded by IGKV3-20/3D-20 gene(s) accounting for 54% of BCRs. An IGKV3-15/IGKJ1-encoded stereotyped sequence homologous to WA RF accounted for 23% of BCRs. LPDs expressing KCDR3s homologous to anti-HCV E2 antibodies responded more frequently to the eradication of HCV by antiviral therapy (6/6 vs. 1/6; p = 0.015). These findings, although limited by the small sample size, suggest that a stereotyped KCDR3 may predominantly shape anti-HCV specificity of BCRs, possibly providing a signature that may help identifying bona fide HCV-dependent LPDs.

Autoimmune systemic diseases (ASD) may show impaired immunogenicity to COVID-19 vaccines. Our prospective observational multicenter study aimed to evaluate the seroconversion after the vaccination cycle and at 6-12-month follow-up, as well the safety and efficacy of vaccines in preventing COVID-19. The study included 478 unselected ASD patients (mean age 59 ± 15 years), namely 101 rheumatoid arthritis (RA), 38 systemic lupus erythematosus (SLE), 265 systemic sclerosis (SSc), 61 cryoglobulinemic vasculitis (CV), and a miscellanea of 13 systemic vasculitis. The control group included 502 individuals from the general population (mean age 59 ± 14SD years). The immunogenicity of mRNA COVID-19 vaccines (BNT162b2 and mRNA-1273) was evaluated by measuring serum IgG-neutralizing antibody (NAb) (SARS-CoV-2 IgG II Quant antibody test kit; Abbott Laboratories, Chicago, IL) on samples obtained within 3 weeks after vaccination cycle. The short-term results of our prospective study revealed significantly lower NAb levels in ASD series compared to controls [286 (53–1203) vs 825 (451–1542) BAU/mL, p < 0.0001], as well as between single ASD subgroups and controls. More interestingly, higher percentage of non-responders to vaccine was recorded in ASD patients compared to controls [13.2% (63/478), vs 2.8% (14/502); p < 0.0001]. Increased prevalence of non-response to vaccine was also observed in different ASD subgroups, in patients with ASD-related interstitial lung disease (p = 0.009), and in those treated with glucocorticoids (p = 0.002), mycophenolate-mofetil (p < 0.0001), or rituximab (p < 0.0001). Comparable percentages of vaccine-related adverse effects were recorded among responder and non-responder ASD patients. Patients with weak/absent seroconversion, believed to be immune to SARS-CoV-2 infection, are at high risk to develop COVID-19. Early determination of serum NAb after vaccination cycle may allow to identify three main groups of ASD patients: responders, subjects with suboptimal response, non-responders. Patients with suboptimal response should be prioritized for a booster-dose of vaccine, while a different type of vaccine could be administered to non-responder individuals.

Studies on the safety and immunogenicity of SARS-CoV-2 vaccination in patients with inflammatory rheumatic diseases have so far not included mixed cryoglobulinaemia (MC) vasculitis.1–3 We report a prospective observational multicentre study on this disorder. Participants were followed at four tertiary referral centres and were instructed to promptly inform the attending physicians about unusual events felt as possibly related to vaccination. Seventy-one patients were recruited: they had infection-cured hepatitis C virus (HCV)-related MC, either uncomplicated (HCV-MC, n=50) or complicated by low-grade non-Hodgkin’s lymphoma (MC-NHL, n=8), or essential MC (EMC, n=13). The characteristics of the patients, exclusion criteria and definition of bona fide vaccination-related flare are described in online supplemental methods.### Supplementary data [annrheumdis-2021-221248supp001.pdf] Overall, 9 of 71 (12.7%) patients had postvaccination MC vasculitis flare. However, 8 of 71 patients had experienced within 12 months before vaccination spontaneous flares, where 7 cases required rituximab and 3 of them (37.5%) had postvaccination flare (see online supplemental information). Thus, to exclude the confounding effects of high proneness to spontaneous flare as the facilitator and of rituximab as the preventor, we further restricted the evaluation of postvaccination flare rate to 63 patients off-therapy and without spontaneous flares for 20–48 months before …

Background: New direct-acting antiviral drugs can eradicate hepatitis C virus (HCV) infection in over 90% of patients and can even reduce the risk of complications in advanced fibrosis/cirr...

INTRODUCTION Mixed cryoglobulinemia (MC) is a B-cell lymphoproliferative disorder largely attributable to Hepatitis C virus (HCV) infection. MC clinical manifestations are determined by systemic vasculitis of low/medium sized vessels (mixed cryoglobulinemia syndrome or cryoglobulinemic vasculitis [CV]) caused by the deposition of cryoglobulins in blood vessels. EVIDENCE ACQUISITION A systematic review was performed via the Medline and Scopus databases to evaluate studies concerning CV treatment with new direct antiviral agents (DAAs) and their effect on the syndrome. EVIDENCE SYNTHESIS The introduction of interferon-free protocols has led to more evident positive effects than those observed in the treatment of hepatitis C. In fact, IFN-free, DAA-based therapy minimized side effects permitting the treatment of previously contraindicated patients and led to a particularly high SVR rate and to a clinical/immunological response in the majority of patients, even if at different levels in different patients, from restitutio ad integrum to partial response. In view of the clearly positive evolution in CV management, the persistence of CV manifestations, in partial or non-responders continues to pose problems in the clinical approach to patients who represent a new condition that is still not completely known. CONCLUSIONS Results of DAA-based therapy strongly confirm the use of anti-HCV therapy as the first-line therapeutic option in CV patients. However, growing evidence of a possible persistence or late relapse of CV suggests the need for longer/more accurate post-DAA follow-ups as well as biomarkers that are capable of predicting the risk of clinical relapse/persistence to allow for the design of rational post-HCV eradication clinical flow-charts.

Background and Aims Direct‐acting antivirals (DAAs) usually lead to improvement/remission of cryoglobulinemic vasculitis (CV), although symptoms may persist/recur after a sustained virological response (SVR). We evaluated hematological and genetic markers in patients with HCV‐SVR vasculitis with and without persisting/recurring symptoms to early predict the CV outcome. Approach and Results Ninety‐eight patients with HCV‐CV were prospectively enrolled after a DAA‐induced SVR: Group A: 52 with complete clinical response; Group B: 46 with symptom maintenance/recurrence. Monoclonal B‐cell lymphocytosis, t(14;18) translocation, and abnormal free light chains κ/λ ratios were detected by flow cytometry or nested‐PCR or nephelometry in 4% Group A versus 17% Group B (P = 0.04) patients, 17% Group A versus 40% Group B patients (P = 0.02), and 17% Group A versus 47% Group B (P = 0.003) patients, respectively. At least 1 out of 3 clonality markers was altered/positive in 29% of Group A versus 70% of Group B patients (P

Hepatitis B virus (HBV) chronic infection causes progressive liver damage, although about 20% of patients develop extrahepatic manifestations such as cryoglobulinemic vasculitis (CV). Clinical manifestations range from mild to moderate (purpura, asthenia, arthralgia) to severe (leg ulcers, peripheral neuropathy, glomerulonephritis, non-Hodgkin lymphoma). A comprehensive review of therapeutic options for HBV-related CV is lacking. Nucleos(t)ide analogues (NA) suppress HBV replication in 90–100% of cases and induce clinical response in most patients with mild-to-moderate CV. Plasma exchange can be performed in patients with severe CV and should be considered in severe or life-threatening cases combined with high doses of corticosteroids and antiviral treatment. A cautious use of rituximab can be considered only in association with NA treatment in refractory cases. A review of the literature and an analysis of data collected by six centers of the Italian Group for the Study of Cryoglobulinemia on 18 HBV-CV nucleotide/nucleoside analogues (NAs)-treated patients were carried out.

Background: The Covid-19 pandemic may have a deleterious impact on patients with autoimmune systemic diseases (ASD) due to their deep immune-system alterations. Objective: This study aims to investigate the prevalence of symptomatic Covid-19 and its correlations with both organ involvement and ongoing treatments in a large series of Italian ASD patients during the first wave of pandemic. Methods: Our multicenter telephone 6-week survey included 3,029 unselected ASD patients enrolled at 36 tertiary referral centers of northern, central, and southern Italian macro-areas with different diffusion of the pandemic. Symptomatic SARS-CoV-2 infection was classified as definite Covid-19 (presence of symptoms plus positive oral/nasopharyngeal swabs) or highly suspected Covid-19 (highly suggestive symptoms, in the absence of a swab testing). Results: A significantly higher prevalence of definite plus highly suspected Covid-19 compared to the Italian general population was detected in the whole ASD series (p=.000), as well as in patients from the three macro-areas (p=.000 in all). Statistically higher prevalence of Covid-19 was also found in connective tissue diseases compared to chronic arthritis subgroup (p=.000) and in ASD patients with pre-existing interstitial lung involvement (p=.000). Patients treated with either conventional disease-modifying anti-rheumatic drugs (DMARDs) and/or biological DMARDs showed a significantly lower prevalence of Covid-19 (p=.000 in both). Finally, scleroderma patients undergoing low-dose aspirin showed a significantly lower rate of Covid-19 compared to those without (p=0.003). Conclusion: The higher prevalence of Covid-19 in ASD patients, along with the significant correlations with important clinical features and therapeutic regimens, suggests the need to develop targeted prevention/management strategies during the current pandemic wave.

Hepatitis C virus (HCV) is a major public health problem. HCV is a hepatotropic and lymphotropic virus that leads to hepatocellular carcinoma (HCC) and lymphoproliferative disorders such as cryoglobulinemic vasculitis (CV) and non-Hodgkin&rsquo, s lymphoma (NHL). The molecular mechanisms by which HCV induces these diseases are not fully understood. MicroRNAs (miRNAs) are small non-coding molecules that negatively regulate post-transcriptional gene expression by decreasing their target gene expression. We will attempt to summarize the current knowledge on the role of miRNAs in the HCV life cycle, HCV-related HCC, and lymphoproliferative disorders, focusing on both the functional effects of their deregulation as well as on their putative role as biomarkers, based on association analyses. We will also provide original new data regarding the miR 17-92 cluster in chronically infected HCV patients with and without lymphoproliferative disorders who underwent antiviral therapy. All of the cluster members were significantly upregulated in CV patients compared to patients without CV and significantly decreased in those who achieved vasculitis clinical remission after viral eradication. To conclude, miRNAs play an important role in HCV infection and related oncogenic processes, but their molecular pathways are not completely clear. In some cases, they may be potential therapeutic targets or non-invasive biomarkers of tumor progression.

Meanwhile, measurement of liver stiffness (LS) is the standard for fibrosis screening in patients with chronic hepatitis C virus (HCV) and chronic hepatitis B virus (HBV), namely for the diagnosis of severe fibrosis and cirrhosis (F3 or F4 according to the METAVIR scoring system, respectively) and for the exclusion of significant fibrosis. LS values can be influenced by the degree of necroinflammation in both HBV and HCV infection. In particular, the international guidelines suggest considering normal ALT versus elevated ALT (5 × ULN) in the measurement of LS in HBV-infected patients. Several studies were performed using transient elastography to evaluate its reliability in defining the presence of portal hypertension and esophageal varices. A wide range of cut-off levels have been reported to date. LS is also a strong pre-treatment predictor with regard to the response to antiviral treatment. The precise distinction between advanced fibrosis and cirrhosis is of fundamental importance in terms of both diagnostic and therapeutic approaches. Regression of fibrosis has been demonstrated in patients with HCV after virus elimination and in patients with HBV during antiviral treatment. In HCV patients, a sustained virological response was accompanied by a significant improvement in hepatic function and a decrease of LS. In HBV patients, pre-treatment and LS changes during nucleoside/nucleotide analogues can predict liver-related events. In conclusion, LS measurement has become essential for the clinical management of patients with HCV and HBV chronic infection.

Rituximab (RTX) represents a milestone in the treatment of mixed cryoglobulinemic vasculitis (MCV). Despite usually well-tolerated, RTX may induce different types of adverse drug reactions, including exacerbation of vasculitis. Recently, RTX biosimilar CT-P10 has been approved in Europe for the treatment of rheumatoid arthritis, but no data are available about effectiveness and safety of CT-P10 in the treatment of MCV. In this multicenter open-label study, we analyzed the safety of CT-P10 in patients with MCV treated in first-line or after a shift by RTX originator. Fifty-one consecutive MCV patients (females/males 35/16, median age 68 years, median disease duration 42 months, 51% HCV positive) were included in the study between July and December 2018 and were treated with CT-P10 (group 1). Safety and effectiveness of CT-P10 were compared with a retrospective group (group 2) including 75 consecutive patients treated with RTX originator between July 2017 and July 2018. Thirty-six patients were treated with CT-P10 for the first time, while the other 15 switched from RTX originator. RTX was administrated with high or dosage schemes (375 mg/m2 four times a week apart/1000 mg twice one week apart or 250 mg/m2 twice one week apart). During a month period after the last infusion, 13/51 adverse events (AE) were observed in group 1 and 17/75 in group 2 (p not significant). Among them, 7/13 and 6/17 (in group 1 and 2, respectively) could be considered immune-mediated AE (p not significant). At univariate analysis patients with IM-AE were more frequently males (p = 0.04) and with a lower disease duration (p = 0.03), but both the parameters were not significant at logistic regression. About clinical response after 6 months by the end of the treatment, no differences were observed between patients treated with originator and CT-P10 regarding the response to the therapy. No differences were observed in safety and effectiveness between patients naive at RTX or switching from originator. Despite the higher prevalence of immune-mediated AE among patients treated with CT-P10 than originator, we have observed no significant differences between the 2 groups. The use of a low-dosage regimen is more common in group 1 than in group 2, representing a possible bias of the study, possibly influencing the appearance of AE. Considering the cost/efficacy ratio of biosimilars, their use could be helpful to treat a large number of MCV patients with an effectiveness and safety comparable to originator. Multicenter studies including a large number of patients and the new RTX biosimilars could be useful to fully elucidate the possible risk of immune-mediated adverse events with biosimilar drugs. Considering the cost/efficacy ratio of CT-P10, its use could help to treat a large number of MCV patients with an effectiveness and safety comparable to originator.

Background and Aims: Polymorphisms in the immune response genes can contribute to clearance of hepatitis C virus (HCV) infection but also mediate liver inflammation and cancer pathogenesis. This study aimed to investigate the association of polymorphisms in PD-1 (PDCD1), IFNL3 (IL28B), and TLR2 immune related genes in chronic HCV patients with different hepatic and lymphoproliferative HCV-related diseases. Methods: Selected PDCD1, IFNL3, and TLR2 genes were tested by molecular approaches in 450 HCV-positive patients with increasing severity of underlying liver diseases [including chronic infection (CHC), cirrhosis and hepatocellular carcinoma (HCC)], in 238 HCV-positive patients with lymphoproliferative diseases [such as cryoglobulinemia and non-Hodgkin lymphoma (NHL)] and in 94 blood donors (BD). Results: While the rs12979860 IFNL3 T allele was found a good marker associated with HCV-outcome together with the rs111200466 TLR2 del variant, the rs10204525 PD-1.6 A allele was found to have an insignificant role in patients with HCV-related hepatic disorders. Though in Asian patients the combination of IFNL3 and PD-1.6 markers better define the HCV-related outcomes, in our series of Caucasian patients the PD-1.6 A-allele variant was observed very rarely. Conclusion: Differences in the incidence of HCV-related HCC and clinical response between Asians and Europeans may be partially due to the distribution of PD-1.6 genotype that we found divergent between these two populations. On the other hand, we confirmed in this study that the polymorphic variants within IFNL3 and TLR2 immune response genes are significantly associated with HCV-related disease progression in our cohort of Italian patients.

BACKGROUND & AIMS Hepatitis C virus (HCV)-related mixed cryoglobulinaemia vasculitis (MCV) is characterized by the expansion of rheumatoid factor-producing B-cell clones. The aim of this study was to assess whether B-cell clones may persist in these patients after the clearance of the virus with antiviral therapy, and whether their persistence influences clinical outcomes. METHODS Forty-five HCV-cured MCV patients were followed up for a median of 18.5 (range 9-38) months after the clearance of HCV. Circulating B-cell clones were detected using flow cytometry either by the skewing of kappa/lambda ratio or by the expression of a VH 1-69-encoded idiotype. RESULTS The clinical response of vasculitis was 78% complete, 18% partial and 4% null. However, cryoglobulins remained detectable in 42% of patients for more than 12 months. Circulating B-cell clones were detected in 18 of 45 patients, and in 17 of them persisted through the follow-up; nine of the latter patients cleared cryoglobulins and had complete response of vasculitis. Several months later, two of these patients had relapse of MCV. CONCLUSIONS B-cell clones persist in MCV patients long after HCV infection has been cleared but halt the production of pathogenic antibody. These 'dormant' cells may be reactivated by events that perturb B-cell homeostasis and can give rise to the relapse of cryoglobulinaemic vasculitis.

Notch receptors are single-pass transmembrane proteins that play a critical role in cell fate decisions and have been implicated in the regulation of many developmental processes. The human Notch family comprises of four receptors (Notch 1 to 4) and five ligands. Their signaling can regulate extremely basic cellular processes such as differentiation, proliferation and death. Notch is also involved in hematopoiesis and angiogenesis, and increasing evidence suggests that these genes are involved and frequently deregulated in several human malignancies, contributing to cell autonomous activities that may be either oncogenic or tumor suppressive. It was recently proposed that Notch signaling could play an active role in promoting and sustaining a broad spectrum of lymphoid malignancies as well as mutations in Notch family members that are present in several disorders of T- and B-cells, which could be responsible for altering the related signaling. Therefore, different Notch pathway molecules could be considered as potential therapeutic targets for hematological cancers. In this review, we will summarize and discuss compelling evidence pointing to Notch receptors as pleiotropic regulators of hematologic malignancies biology, first describing the physiological role of their signaling in T- and B-cell development and homeostasis, in order to fully understand the pathological alterations reported.

Hepatitis C virus (HCV)-associated mixed cryoglobulinemia (MC) vasculitis commonly regresses upon virus eradication, but conventional therapy with pegylated interferon and ribavirin yields approximately 40% sustained virologic responses (SVR). We prospectively evaluated the efficacy and safety of sofosbuvir-based direct-acting antiviral therapy, individually tailored according to the latest guidelines, in a cohort of 44 consecutive patients with HCV-associated MC. In two patients MC had evolved into an indolent lymphoma with monoclonal B-cell lymphocytosis. All patients had negative HCV viremia at week 12 (SVR12) and at week 24 (SVR24) posttreatment, at which time all had a clinical response of vasculitis. The mean (±standard deviation) Birmingham Vasculitis Activity Score decreased from 5.41 (±3.53) at baseline to 2.35 (±2.25) (P < 0.001) at week 4 on treatment to 1.39 (±1.48) (P < 0.001) at SVR12 and to 1.27 (±1.68) (P < 0.001) at SVR24. The mean cryocrit value fell from 7.2 (±15.4)% at baseline to 2.9 (±7.4)% (P < 0.01) at SVR12 and to 1.8 (±5.1)% (P < 0.001) at SVR24. Intriguingly, in the 2 patients with MC and lymphoma there was a partial clinical response of vasculitis and ∼50% decrease of cryocrit, although none experienced a significant decrease of monoclonal B-cell lymphocytosis. Adverse events occurred in 59% of patients and were generally mild, with the exception of 1 patient with ribavirin-related anemia requiring blood transfusion. Conclusion: Interferon-free, guideline-tailored therapy with direct-acting antivirals is highly effective and safe for HCV-associated MC patients; the overall 100% rate of clinical response of vasculitis, on an intention-to-treat basis, opens the perspective for curing the large majority of these so far difficult-to-treat patients. (Hepatology 2016;64:1473-1482)

Background HCV can lead to both chronic liver disease and B-cell lymphoproliferative disorders. A strong association exists between HCV and mixed cryoglobulinaemia (MC). Methods Anti-nuclear antibodies (ANA), rheumatoid factor Ig-G (RF-IgG), free light chain κ and λ (FLC-κ, FLC-λ) levels and κ/λ ratio were evaluated in 50/420 subjects unexpectedly resulted anti-HCV positive after routine screenings for non-hepathological procedures. Results Three/fifty patients had HCV-RNA undetectable in the serum and were excluded from the analysis. Thirty-nine/fifty patients had laboratory evidence of circulating cryoglobulins without liver disease and MC-related symptoms. Among them, 17 resulted ANA-positive. The mean cryocrit was higher in ANA-positive patients, while no other demographic/clinical differences were observed between the groups. Significantly higher levels of RF-IgG were observed in ANA-positive vs ANA-negative patients. κ and λ FLC were higher in ANA-positive patients. A ROC analysis, based on ANA-positivity vs ANA-negativity, confirmed a high sensitivity and specificity of RF-IgG test. Conclusions Published data concerning MC come mostly from symptomatic vasculitis. We analyzed HCV-patients without MC symptoms, founding cryoglobulins in the majority of them. The increased levels of FR-IgG and FLC in CGs-ANA-positive patients, suggest these test could be used to identify a state of silent autoimmune and/or lymphoproliferative condition before the transition to a frank disease in naive HCV-patients without symptoms of extrahepatic manifestations.

Background Reversible cryoprecipitability of proteins is observed as a concomitant feature of immune complex formation. Mixed cryoglobulinemia (MC) is systemic vasculitis, associated with mixed IgM and IgG cryoglobulins (CGs) showing rheumatoid factor (RF) activity. It is frequently associated with hepatitis C virus (HCV). This study investigates the presence of IgG RF and anti-nuclear antibodies (ANA) in cryoprecipitates of patients with type III and type II MC, to understand the biochemical patterns associated with different types of MC to a greater degree. Methods Sera from 70 HCV untreated patients with type III or type II MC were tested by immunofixation for IgG3 and through ELISA for IgG RF. Cryoprecipitates were analysed for ANA by indirect immunofluorescence to identify specific patterns. Results After stratification according to MC type, the ANA patterns between type II and type III MC were statistically different. IgG3 levels and IgG-RF positivity were significantly higher in type III cryoprecipitate. We observed a higher positivity of IgG3 and a significant difference between the liver fibrosis stage, ANA and IgG-RF in the cryoprecipitate. Conclusion Results show a combination of biochemical markers and autoantibodies associated to mixed cryoglobulinemia; these findings could be further investigated in order to ascertain their usefulness in assessing the risk for the development of mixed cryoglobulinemia.

BACKGROUND Cryoglobulinaemic vasculitis (CV) is a lymphoproliferative disorder related to hepatitis C virus (HCV) infection; anti-viral therapy is the first therapeutic option. CV can be incapacitating, compromising the patients' quality of life (QoL). In a controlled study, interferon-based therapy was associated with a lower virological response in vasculitic patients than in patients without vasculitis. Limited, uncontrolled data on direct-acting anti-virals are available. AIM To evaluate safety, clinical efficacy, virological response and the impact of interferon-free treatment on QoL in HCV patients with and without mixed cryoglobulinaemia (MC). METHODS We prospectively studied HCV patients with cryoglobulinaemia (with vasculitis-CV- and without vasculitis-MC-) and without cryoglobulinaemia (controls), treated with direct-acting anti-virals. Hepato-virological parameters, CV clinical response and impact on QoL were assessed. RESULTS One hundred and eighty-two HCV patients were recruited (85 with CV, 54 with MC and 43 controls). A sustained virological response at 12 weeks (SVR12) was achieved in 166 (91.2%) patients (77/85 CV, 48/54 MC, 41/43 controls). In CV SVR patients, cryocrit levels progressively decreased and clinical response progressively improved, reaching 96.7%, 24 weeks after treatment. QoL, baseline physical and mental component summaries were lower in the CV group compared to the other groups (P < 0.05). Scores improved in all groups, and significantly in CV patients after SVR. CONCLUSIONS No significant differences in SVR rates were recorded between cryoglobulinaemic patients and controls and a high clinical and immunological efficacy was confirmed in CV, supporting the role of interferon-free therapy as the first therapeutic option. Interestingly, CV patients had worse baseline QoL than other HCV-positive groups and interferon-free therapy was effective in significantly increasing QoL, suggesting the important role of direct-acting anti-viral-based therapy in improving CV's individual and social burden.

HCV chronic infection is characterized by possible development of both hepatic and extrahepatic manifestations. The infection by this both hepatotropic and lymphotropic virus is responsible for polyoligoclonal B-lymphocyte expansion, leading to several immune-mediated disorders. Mixed cryoglobulinemia syndrome that in some cases may evolve to frank B-cell non-Hodgkin's lymphoma is the prototype of HCV-driven autoimmune and lymphoproliferative disorders. The HCV oncogenic potential has been suggested by several clinicoepidemiological and laboratory studies; it includes hepatocellular carcinoma, B-cell non-Hodgkin's lymphoma and papillary thyroid cancer. The definition HCV syndrome refers to the complex of HCV-driven diseases; these latter are characterized by heterogeneous geographical distribution, suggesting a role of other important genetic and/or environmental cofactors. The natural history of HCV syndrome is the result of a multifactorial and multistep pathogenetic process, which may evolve from mild manifestations to systemic autoimmune disorders, and less frequently to malignant neoplasias. The present updated review analyzes the clinical and pathogenetic aspects of the main HCV-associated diseases.

// Laura Gragnani 1, * , Elisa Fognani 1, * , Valli De Re 2 , Massimo Libra 3 , Adriana Garozzo 3 , Patrizio Caini 1 , Guia Cerretelli 1 , Andrea Giovannelli 1 , Serena Lorini 1 , Monica Monti 1 , Silvia Bagnoli 4 , Irene Piaceri 4 and Anna Linda Zignego 1 1 Center for Systemic Manifestations of Hepatitis Viruses (MaSVE), Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 2 Centro di Riferimento oncologico, National Cancer Institute, Aviano, Italy 3 Department of Biomedical and Biotechnological Sciences, Section of Microbiology, University of Catania, Italy 4 Department of Neuroscience, Psychology, Drug Research and Children’s Health, University of Florence, Florence, Italy * These authors contributed equally to this work Correspondence to: Anna Linda Zignego, email: a.zignego@dmi.unifi.it Keywords: HCV, mixed cryoglobulinemia, NOTCH4, HLA-II, lymphoma Received: January 31, 2017 Accepted: March 29, 2017 Published: May 06, 2017 ABSTRACT Mixed cryoglobulinemia (MC), is a HCV-related, clinically benign, lymphoproliferative disorder (LPD) that may evolve into a non Hodgkin’s lymphoma (NHL). Significant associations were found between two single nucleotide polymorphisms near NOTCH4 (rs2071286) and the HLA class II (rs9461776) genes and HCV-related MC syndrome (MCS). We analyzed NOTCH4 rs2071286 and HLA-II rs9461776 in 3 HCV-related LPD groups (asymptomatic MC, MCS, NHL) with HCV infection without lymphoproliferative disorders. We found a positive relationship between NOTCH4 rs207186 T minor allele frequency (MAF) and patients with HCV-related LPDs at risk of NHL (Chi-square test for trend = 14.84 p = 0.0001), in accordance with an over-dominant model in the NHL group (CT vs CC + TT, OR=1.88, 95% CI 1.24–2.83, p = 0.0026). Regarding HLA II rs9461776, G MAF increased in patients with HCV-related LPDs at risk of NHL (Chi-square test for trend = 8.40 p = 0.0038), in accordance with a recessive genotypic model in the NHL group (G/G vs A/A + A/G, OR = 11.07, 95% CI 2.37–51.64, p = 0.0022). Both NOTCH4 rs2071286 and HLA-II rs9461776 were present on chromosome 6 and showed D’ and r values of linkage disequilibrium (LD) of about 0.5 values, thereby suggesting there is no extensive LD in the HCV+ population. This data shows that the previously demonstrated association between NOTCH4 rs2071286 and HLA-II rs9461776 polymorphisms and HCV-related MCS could be extended to overall patients with HCV-related LPDs. The significant relationship between rs2071286 and rs9461776 MAF and the increased risk for NHL, suggests their use as non-invasive markers to categorize patients at risk of lymphoma.

Background Mixed cryoglobulinaemia is strongly related to hepatitis C virus infection. Treatment with peg-interferon and ribavirin has been indicated as first-line therapy for mild/moderate hepatitis C virus-related mixed cryoglobulinaemia. Aim To evaluate the safety and efficacy of triple boceprevir-based antiviral therapy in patients with or without mixed cryoglobulinaemia previously treated with peg-interferon and ribavirin, and with advanced liver disease. Methods Thirty-five hepatitis C virus-positive patients (17 with asymptomatic mixed cryoglobulinaemia, 5 with symptomatic mixed cryoglobulinaemia, and 11 without mixed cryoglobulinaemia) were treated with triple boceprevir-based antiviral therapy. Results In 19/22 cryoglobulinaemic subjects (86%), the addition of boceprevir induced cryocrit disappearance. Cryocrit behaviour was related to virological response, with improvement of symptoms upon undetectable viraemia and reappearance after virological breakthrough. The rate of sustained virological response was lower in cryoglobulinaemic patients than in patients without mixed cryoglobulinaemia (23.8% vs 70% respectively, p = 0.01). Conclusion Boceprevir-based therapy was safe and effective in cryoglobulinaemic patients. The correlation between direct inhibition of hepatitis C virus replication and clinical improvement in mixed cryoglobulinaemic patients is definitive proof of the key pathogenetic role played by viral replication. Further studies are needed to confirm and clarify the reduced virological response in patients with mixed cryoglobulinaemia.

To explore the relationship between innate immunity and hepatitis C Virus (HCV) in determining the risk of cirrhosis (CIR), hepatocellular carcinoma (HCC), mixed cryoglobulinemia syndrome (MCS) and non-Hodgkin lymphoma (NHL), we investigated the impact of the toll-like receptor-2 (TLR2) and interleukin-28B (IL28B) genetic variants. TLR2 -174 del variant was associated with TLR2 expression and with specific downstream molecules that drive the expression of different interleukins; rs12979860 Il28B was important in response to interferon-treatment and in spontaneous clearance of HCV. The risk for liver and lymphoproliferative diseases in HCV progression was clarified by stratifying 862 HCV-positive patients into groups based on liver (CIR, HCC) and lymphoproliferative HCV-related diseases (MCS, NHL) and compared with chronic HCV (CHC) infection. Analysis of TLR2-IL28B haplotypes showed an association of wild type haplotype with the lymphoproliferative diseases (OR 1.77, p = 0.029) and a slight increase in HCV viral load (HR 1.38, p = 0.054). Wild type haplotype (TLR2 ins/ins- IL28B C/C) was also found associated with older age in patients with an hepatic diseases (in CIR and in HCC p = 0.038 and p = 0.020, respectively) supporting an effect of innate immunity in the liver disease progression. TLR2 and IL28B polymorphisms in combination showed a role in the control of HCV viral load and different HCV disease progression.

The possibility that HCV infects lymphoid cells has been widely discussed. Evidence in favor of HCV tropism for lymphoid cells derives from a series of data including: (1) the higher sensitivity of testing HCVRNA in PBMC than in serum or plasma samples, with possible detection of HCV RNA-positive PBMC in the absence of HCV viremia; (2) short-term cultures of PBMC which yield a significant increase in the amount of viral RNA on stimulation by mitogens; (3) results of "in situ" methods (i.e. in situ hybridization, immunofluorescence); (4) efficient infection of lymphoid cell lines or PBMC from normal individuals; (5) the persistence of HCV RNA in PBMC obtained from HCV-positive subjects and injected into SCID mice; (6) the long-term persistence of HCV RNA in PBMC in spite of HCV RNA negativity of serum and liver in sustained responder patients after therapy. The principal criticisms concerning effective HCV infection of lymphoid cells arise from technical difficulty in identifying HCV RNA replicative intermediate in these elements. Conflicting data may also result from differences in PBMC infection by different genotypes, samples taken at different stages in the disease process and differences in the sensitivity of detection methods, as well as low replication levels and/or proportion of infected PBMC. Interesting available data about HCV lymphotropism, which is possibly important in influencing the natural history of infection, include: (1) possible preferential viral tropism for specific PBMC subsets; (2) different lymphotropism of different viral strains; (3) selection of distinctive viral strains; (4) identification of putative HCV cell receptors; (5) association between determination of HCV lymphatic infection and t(14; 18) translocation. The clinical correlates of HCV lymphotropism are potentially very numerous, including, first, its role in determining HCV-related lymphoproliferative disorders.

Mixed Cryoglobulinemia (MC) is the most frequent extrahepatic manifestation of Hepatitis C virus (HCV) infection. MC is an autoimmune /B-cell lymphoproliferative disorder characterized by circulating immune-complexes, named cryoglobulins. MC patients exhibit symptoms due to a systemic vasculitis of small/medium size vessels (mixed cryoglobulinemia syndrome, MCS) in a percentage going from 5 to 30%. The first-line therapeutic option in MCS patients is the etiologic treatment and, in the past fifteen years, antiviral therapy with Pegylated-Interferon (Peg-IFN) plus Ribavirin (RBV) represented the standard of care. Lately, the arrival of direct acting antivirals (DAAs) significantly modified the cure of HCV infection, consenting the use of IFN-free regimens. Here we report a review of the literature about the role of antiviral treatment, following its evolution, in treating HCVrelated MC. Furthermore, we report the results, after 8 weeks of treatment, of a preliminary pilot prospective study, counting 17 patients with HCV-related MC with or without MCS, treated with new generation DAAs in IFN-free regimens. After 8 weeks of DAA administration, all the subjects were HCV RNA negative. Moreover, in 6/17 (35%) patients cryoglobulins disappeared and, on the whole, in all patients a decrease of the cryocrit values was observed (plt;0.05). Furthermore, three MCS-HCV patients (30%) resulted to be complete clinical responders and 5 subjects (50%) partial clinical responders. Therefore, IFN-free anti-HCV treatment appears to be safe and effective in MC patients from virological and clinical points of view, thus supporting the importance of HCV eradication in leading MC remission.

Objective To evaluate whether rituximab at a low dose of 250 mg/m 2 × 2 may be as effective as at higher dosages, most commonly 375 mg/m 2 × 4, used in previous studies on the treatment of patients with refractory mixed cryoglobulinemia (MC) vasculitis associated with hepatitis C virus (HCV) infection. Methods We conducted a phase 2, single-arm two-stage trial (EUDRACT n. 2008-000086-38) of low-dose rituximab in 52 patients with HCV-associated MC who were ineligible/intolerant or non-responder to antiviral therapy. The primary outcomes were response of vasculitis evaluated by the Birmingham Vasculitis Activity Score (BVAS) at months 3, 6 and 12, rate of relapses and time to relapse, and rate of adverse events. Our data were compared with those reported in 19 published studies selected among 291 reviewed in a literature search. Results The cumulative response rate (complete and partial) at month 3 was 81% in our patients, and 86% in 208 patients from studies using high-dose rituximab. The relapse rate and median time to relapse were, respectively, 41% and 6 months in our study, and 32% and 7 months in high-dose studies. Treatment-related adverse events were 11.5% in our study and 19.9% in high-dose studies. None of these differences was statistically significant. Conclusion Rituximab at a low dosage of 250 mg/m 2 × 2 is as effective as at higher dosages for treating MC vasculitis. This low-dose regimen may improve the cost/benefit profile of rituximab therapy for MC.

Background and aim: Hepatocellular carcinoma is one of the major causes of death due to cancer worldwide, and its association with hepatitis C virus infection has been definitively established. Hepatitis C virus is also involved in the pathogenesis of non-Hodgkin’s lymphoma. This is the only virus infecting humans that is able to induce two different malignancies. We analyzed the expression levels of a panel of microRNA in peripheral blood mononuclear cells of patients with hepatitis C virus-related malignancies in order to find a disease-associated deregulation and identify specific biomarkers. Methods: We tested peripheral blood mononuclear cells isolated from patients with hepatocellular carcinoma, non-Hodgkin’s lymphoma, hepatitis C virus without malignancies and healthy subjects for a panel of microRNA selected on the basis of previous studies. MicroRNA expression was evaluated by real-time PCR. Results: Our results showed an upregulation of miRNA-21 and downregulation of miRNA-26b in hepatocellular carcinoma and non-Hodgkin’s lymphoma patients compared to controls (p

Mixed cryoglobulinemia (MC) is an autoimmune/B-cell lymphoproliferative disorder associated with Hepatitis C Virus (HCV) infection, manifesting as a systemic vasculitis. In the last decade, antiviral treatment (AT) with pegylated interferon (Peg-IFN) plus ribavirin (RBV) was considered the first therapeutic option for HCV-MC. In MC patients ineligible or not responsive to antivirals, the anti-CD20 monoclonal antibody rituximab (RTX) is effective. A combined AT plus RTX was also suggested. Since the introduction of direct acting antivirals (DAAs), few data were published about MC and no data about a combined schedule. Here, we report a complete remission of MC after a sustained virological response following a combined RTX/Peg-IFN+RBV+DAA (boceprevir) treatment and review the literature about the combined RTX/AT.

s / Digestive and Liver Disease 48S (2016) e1–e17 e5 eGFR was ≥60mL/min/1.73m2 in 92 patients (93.8%) and between45and59mL/min/1.73m2 in6patients (6.1%).Glomerular involvement was found in 19 patients (19.4%), tubular involvement in 31 (31.6%) and they co-occurred in 10 patients (p=0.034). Subjects with glomerular or tubular involvement, or both, showed significantly lower eGFRvalues (p=0.005). AROC curvewasdrafted and a cut point of 90ml/min predicted renal involvement (RI) (sensitivity 63%, specificity 75%), although it was unable to distinguish tubular vs glomerular involvement (p=0.914). Patients with RI were older, had higher ACR and 1MCR levels and exhibited a more severe KDIGO stage. No association was found between RI and: HCV-RNA levels, liver stiffness and liver function tests. L-FABP andKIM-1 levelswere significantlyhigher inpatientswithRI. Tubular involvement was significantly associated with increased levels of L-FABPandKIM-1,while glomerular involvementwas associated only with high L-FABP level. Conclusion: Tubular and/or glomerular involvement are quite frequent in HCV cirrhotic patients. The occurrence of eGFR

The host genetic basis of mixed cryoglobulin vasculitis is not well understood and has not been studied in large cohorts. A genome-wide association study was conducted among 356 hepatitis C virus (HCV) RNA-positive individuals with cryoglobulin-related vasculitis and 447 ethnically matched, HCV RNA-positive controls. All cases had both serum cryoglobulins and a vasculitis syndrome. A total of 899 641 markers from the Illumina HumanOmni1-Quad chip were analyzed using logistic regression adjusted for sex, as well as genetically determined ancestry. Replication of select single-nucleotide polymorphisms (SNPs) was conducted using 91 cases and 180 controls, adjusting for sex and country of origin. The most significant associations were identified on chromosome 6 near the NOTCH4 and MHC class II genes. A genome-wide significant association was detected on chromosome 6 at SNP rs9461776 (odds ratio=2.16, P=1.16E-07) between HLA-DRB1 and DQA1: this association was further replicated in additional independent samples (meta-analysis P=7.1 × 10(-9)). A genome-wide significant association with cryoglobulin-related vasculitis was identified with SNPs near NOTCH4 and MHC Class II genes. The two regions are correlated and it is difficult to disentangle which gene is responsible for the association with mixed cryoglobulinemia vasculitis in this extended major histocompatibility complex region.

Limited data are available about the efficacy of antiviral treatment in hepatitis C virus (HCV)–associated mixed cryoglobulinemia (MC), especially concerning the long-term effects of HCV eradication. The aim of this study was to evaluate the influence of MC on the virological response and the long-term effects of viral eradication on MC. We prospectively enrolled 424 HCV+ patients belonging to the following groups: MC syndrome (MCS)-HCV (121 patients with symptomatic MC), MC-HCV (132 patients with asymptomatic MC), and HCV (158 patients without MC). Pegylated interferon plus ribavirin treatment was administered according to standard protocols. Posttreatment follow-up ranged from 35 to 124 months (mean 92.5 months). A significant difference was observed in the rate of sustained virological response between the HCV group and both the MC-HCV (P = 0.009) and MC-HCV+MCS-HCV (P = 0.014) groups. Multivariate logistic regression analysis identified cryoglobulinemia as an independent prognostic factor of nonresponse. The clinical–immunological response in MCS-HCV correlated with the virological one. All patients with sustained virological response also experienced a sustained clinical response, either complete or partial. In the majority of sustained virological response patients all MCS symptoms persistently disappeared (36 patients, 57%); in only two (3%) did definite MCS persist. All virological nonresponders were also clinical nonresponders, in spite of a transient improvement in some cases. No evolution to lymphoma was observed. For the first time we have evaluated both the effects of interferon-based therapy on HCV patients with and without MC and with and without symptoms, as well as the long-term effects of viral eradication on MC. Conclusion: MC is a negative prognostic factor of virological response. Clearance of HCV led to persistent resolution or improvement of MCS, strongly suggesting the need for a next generation of highly effective antiviral drugs. (Hepatology 2015;61:1145-1153)

Introduction Mixed cryoglobulinemia (MC) is a HCV-related lymphoproliferative disorder generally associated with advanced liver disease. Liver stiffness has been significantly correlated with histopathological stage of fibrosis. Moreover, it was influenced by necroinflammatory activity. Rituximab (RTX) is a chimeric anti-CD20 monoclonal antibody inducing transient B lymphocytes depletion that was shown to be useful and safe in the majority of HCV MC patients, leading also to improvement of cirrhotic syndrome. Aim of this study was to evaluate the modifications of liver stiffness following RTX treatment in HCV-related MC patients. Materials and methods Fourteen consecutive patients (10 F, 4 M; mean age 60.43 ± 43) with HCV-related chronic hepatitis (n = 10) or cirrhosis (n = 4) and MC, eligible for RTX treatment, were prospectively enrolled. Intravenous injection of 1 g of RTX was performed at day 0 and at day 15. Assessment of stiffness was carried out by Fibroscan® (Echosens, Paris-France) at baseline, 15 days after the first infusion, and at month 1, 3 and 6 after therapy. Results MC symptoms significantly improved during the study, especially during the first 3 months. Liver stiffness observed 3 months after treatment was significantly reduced when compared with pre-treatment values (p = 0.01). This difference disappeared after 6 months of follow-up. Cytofluorimetric analysis showed a decrease of CD19+ peripheral blood cells, with the nadir at month 3 after therapy and B cell compartment reconstitution after 6 months. Conclusion This study, for the first time showed that RTX-treatment in HCV-related MC induces a reduction of liver stiffness that is strictly associated with the B-cell depletion.

SummaryChronic hepatitis C is the main cause of morbidity and mortality in adult haemophilic patients who received non-virally inactivated plasma-derived clotting factor concentrates. Overall, spontaneous viral clearance rate is 10–25% and the only approach that can halt disease progression is hepatitis C virus (HCV) eradication by means of antiviral therapy. In non-haemophilic patients a single nucleotide polymorphism located upstream the gene of interferon lambda 3 (IFNλ3) has been associated with both spontaneous viral clearance and sustained virological response after antiviral treatment. The aim of this study was to assess whether the rs12979860 polymorphism was a predictor of spontaneous viral clearance and of sustained virological response after antiviral therapy in a large cohort of haemophilic patients with HCV infection. The rs12979860 polymorphism, defined as CC genotype or T allele, was tested in a cohort of 342 haemophilic patients and evaluated as predictor of spontaneous clearance or response to antiviral therapy. By multivariate regression analysis the IFNλ3 CC genotype was an independent predictor of spontaneous viral clearance (odds ratio: 3.7, 95% confidence interval: 2.0–6.8). Sustained virological response rates were doubled in patients with the CC genotype than in those with the T allele (78% vs 44%; p

With the aim of investigating whether interleukin 28B gene (IL28B) rs1297860 polymorphism is associated with different hepatitis C (HCV) infection statuses, we compared IL28B allelic distribution in an Italian case series of 1050 patients with chronic infection and different outcomes, 47 individuals who spontaneously cleared HCV, and 178 blood donors. Furthermore, we compared IL28B variants among 3882 Caucasian patients with chronic infection, 397 with spontaneous clearance, and 1366 blood donors reported in PubMed. Overall data confirmed a relation between IL28B C allele and HCV spontaneous clearance. Furthermore, we found that IL28B T allele had a weak relation with chronic HCV progression to hepatocellular carcinoma. Study findings are in accordance with the hepatocellular carcinogenic model where IL28B TT genotype, by promoting a persistent chronic hepatitis which leads to both hepatocyte injury and chronic inflammation, could facilitate HCC development. Conversely, patients with lymphoproliferative disorders had not any significantly different IL28B rs1297860 allelic distribution than those with chronic HCV, but, like all chronic HCV-related diseases, they showed a lower CC frequency than patients who spontaneously cleared HCV. Study results confirmed the model of persistent HCV infection as a risk factor for the pathogenesis of both liver and lymphoproliferative disorders.

Hepatitis C Virus (HCV) is a major health problem, infecting about 3 % of people worldwide and leading to liver as well as extrahepatic diseases. This justifies the definition of HCV infection as a systemic disease. Based on available data, the link between the virus and some of these extrahepatic disorders is certain, whereas for some others needs further confirmation. HCV-related lymphoproliferative disorders, ranging from benign, but pre-lymphomatous conditions, like mixed cryoglobulinemia, to frank lymphomas, represent the extrahepatic manifestations most closely related to HCV. The primary involvement of the liver and lymphatic system corresponds to the double viral tropism, being HCV able to infect both hepatic and lymphatic cells. Other HCV-associated disorders include renal, endocrine, dermatological, cardiovascular, rheumatologic and central nervous system diseases. On the whole, the HCV disease appears a very important, mainly hidden, public health problem leading to heavy direct and indirect costs. The possibility that HCV may be eradicated following antiviral therapy is important for both the therapeutic and preventive points of view, making the HCV disease an ideal model for pathogenetic studies.

HCV-related mixed cryoglobulinemia (MC) is characterized by clonal expansion of B cells producing a polyreactive natural antibody (rheumatoid factor) and interferon (IFN)-based therapy is the first therapeutic option in mild-moderate MC. Single nucleotide polymorphisms (SNPs) proximal to genes involved in the innate response (IL28B/IFN-λ gene family) are strongly associated with spontaneous and IFN-induced viral clearance in hepatitis C, but no data exist about their role in HCV-positive MC. A large cohort of patients with HCV and MC was studied to evaluate the influence of IL28B genotype on the response to treatment and/or the evolution to MC of HCV infection. The rs12979860/rs8099917 IL28B polymorphisms were analysed in 481 consecutive HCV-positive subjects (250 with MC and 231 without MC, as controls) using real-time PCR and direct sequencing. Hundred and fifteen HCV patients with MC received standard anti-HCV therapy, and the results were evaluated according to the IL28B SNP distribution. Similar IL28B SNPs allele frequencies were recorded for patients and controls. IL28B major allele homozygosis (for both SNPs tested) was tightly correlated with virological and clinical response (P = 0.002). A statistically significant association was limited to 'difficult-to-treat' (G1/4) HCV genotypes. The IL28B genotype was a strong independent predictor of response (P = 0.007, OR 6.06; CI 1.65-22.22). The IL28B genotype was confirmed to be a useful predictor of response to IFN-based therapy in patients with HCV and MC. The very close correlation between IL28B SNP distribution, virological and clinical response definitively confirmed the key role played by HCV in MC. Conversely, the IL28B genotype does not seem to influence the evolution to MC.

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

Objective An uncommon manifestation of hepatitis C virus (HCV) infection is systemic vasculitis associated with type II cryoglobulinemia (cryoglobulinemic vasculitis), a proliferative B cell disorder that transforms into B cell malignancy in 5–10% of patients. The monoclonal rheumatoid factors (mRF) that bear the WA cross-idiotype (Xid) are responsible for most cases of cryoglobulinemic vasculitis in patients with HCV infection. The purpose of this study was to determine whether WA B cells can be detected in asymptomatic patients with HCV infection, using sequence analysis of B cell clonal expansions (BCEs) to identify the WA Xid. Methods Asymptomatic patients with HCV infection and those without HCV infection as well as respective control patients with cryoglobulinemic vasculitis, whose serum was either negative or positive for WA mRF, were studied. BCEs were isolated in the patients' blood, and WA BCEs were identified by sequencing analysis. Results BCEs were detected in all control patients with cryoglobulinemic vasculitis, but only control patients with HCV infection had WA BCEs. None of the 33 asymptomatic patients without HCV infection had a BCE. WA BCEs were detected in 4 (7.4%) of 55 asymptomatic patients with HCV infection, in none of 14 patients with HCV infection and type III cryoglobulinemia, and in 5 (13.5%) of 37 patients with HCV infection and serum RF positivity. One patient with a WA BCE had splenic lymphoma markers and villous lymphocytes, and the villous lymphocytes were found to be WA B cells. Conclusion By identification of the WA Xid, WA B cells can be detected in asymptomatic HCV-infected patients. WA B cells in asymptomatic patients with HCV infection may be a marker for the development of cryoglobulinemic vasculitis and associated B cell malignancies. The results of this study provide a basis for the development of the first practical clinical application of cross-idiotype analysis.

To the editor: Mixed cryoglobulinemia (MC) is a benign but prelymphomatous condition whose clinical manifestations are secondary to systemic immune complex–related vasculitis, which is the final step in a complex process initiated by unregulated B-cell expansion. MC is strongly associated with

Background: Platelet-activating factor (PAF), a powerful phospholipid mediator of inflammation, is degraded by plasma PAF-acetyl-hydxolase (pPAF-AH), an enzyme which circulates in serum mainly in a complex with lipoproteins that confer its biological activity. Hepatitis C virus (HCV) is linked to lipoproteins in serum too. Reduced pPAF-AH activity was observed in several diseases, including systemic vasculitis. Aim: To evaluate if chronic HCV infection could alter pPAF-AH physiological functions. Subjects: 145 subjects were studied: 56 HCV- and 52 HBV-infected patients (pathologic controls); 37 healthy subjects (healthy controls). Methods: pPAF-AH activity, PAF and Apo B100 titers were determined in plasma; enzyme expression levels were evaluated in monocyte-derived macrophages. HCV-RNA was detected in plasma, peripheral blood mononuclear cells and liver samples. Results: HCV-infected patients showed an increase of PAF levels following a significant decrease of pPAF-AH activity. A recovery of pPAF-AH activity occurs only in patients who clear HCV after the antiviral treatment. Expression levels of pPAF-AH mRNA and Apo B100 titers were not modified in HCV patients in comparison to controls. Conclusion: In light of these results, it is tempting to hypothesize that during chronic HCV infection, the PAF/pPAF-AH system may be altered and this condition may contribute to HCV-related vascular damage.

Hepatitis C virus (HCV) chronically infects about 200 million individuals worldwide and leads to severe liver and lymphatic diseases. HCV circulates in the serum, associated with apoB-containing lipoproteins. Platelet-activating factor (PAF), a pro-inflammatory mediator, is mainly modulated by plasma PAF-acetylhydrolase (pPAF-AH), associated with ApoB100-containing low-density lipoproteins (LDL). The aim of the study was to evaluate the potential effects of chronic HCV infection on the PAF/pPAF-AH system. HCV-RNA was detected in plasma, peripheral blood mononuclear cells (PBMC) and liver samples. Plasma PAF levels, pPAF-AH activity, ApoB100 serum titres and pPAF-AH mRNA levels in cultured macrophages were determined. Plasma PAF levels were significantly higher and pPAF-AH activity was significantly lower in HCV patients than in controls. No significant modifications of pPAF-AH mRNA in macrophages or in ApoB100 values were observed in HCV patients compared with controls. Patients who cleared HCV after antiviral treatment showed a complete restoration of pPAF-AH activity and significant decrease of PAF levels during the follow-up. No data exist about the PAF/pPAF-AH system behaviour during HCV infection. This study shows that in HCV patients modifications of pPAF-AH activity/PAF levels take place and that HCV clearance restored pPAF-AH activity. This suggests that circulating viral particles play a role in PAF/pPAF-AH system modifications and such an alteration could be involved in HCV-related damage.

s / Digestive and Liver Disease 47S (2015) e1–e18 e13 escape the demonstration of arterial hypervascularity, CEUS must be performed immediately after conventional US to contrast the malignant fate of small lesions arising in cirrhotics. http://dx.doi.org/10.1016/j.dld.2015.01.030

Background & Aims Mixed cryoglobulinaemia (MC) is an HCV-related lymphoproliferative disorder characterized by the presence of circulating immune complexes called cryoglobulins. Treatment with anti-CD20 monoclonal antibody rituximab is proved to be very useful, especially in patients ineligible to interferon-based antiviral therapy. Recently, free light chain (FLC) κ/λ ratio and FLC patterns were associated with MC. The aim of this study was to evaluate changes in FLC-κ, FCL-λ, FLC ratio following rituximab treatment in patients with HCV-related MC and to correlate FLC-κ, FCL-λ and FLC ratio values with therapy response. Patients and Methods We retrospectively enrolled 46 patients with HCV infection (26 females, 20 males), including 10 patients without signs/symptoms of MC-related vasculitis, 36 with MC vasculitis. Clinical and biological data were recorded at baseline and 6 months after RTX treatment. Nephelometric measurement of serum FLCs was taken. Results The mean serum FLC-κ level and FLC ratio were significantly higher in patients with MC, compared to HCV patients without MC and to blood donors. An abnormal FLC ratio at baseline correlated with the presence of cryoglobulins, C4 consumption, higher RF level and higher vasculitis rate. To evaluate the predictive value of FLCs, patients with MC were divided into two groups according to RTX therapy outcome (responders and no/partial responders). Abnormal baseline FLC ratio was significantly associated with no/partial response. Conclusions RTX treatment in HCV-related MC induces a reduction in FLC-κ and RF levels. Moreover, pretreatment FLC ratio, which can be easily assessed by a routine test, may be useful to predict response to this expensive treatment for patients with HCV-related MC ineligible to IFN-based therapy.

Not only is chronic hepatitis C virus (HCV) infection a major public health problem, but also it can cause hepatocellular carcinoma and, more rarely, non-Hodgkin's lymphoma. These characteristics mean that HCV is the only virus infecting humans that is able to cause two different cancers. The fine pathogenetic and molecular mechanisms by which HCV induces these two malignancies are not completely clear. In the last decade, it has been shown that microRNAs (miRNAs), a class of 21-23-nucleotide molecules modulating post-transcriptional gene expression, make an important contribution to the pathogenesis of several cancers and are also considered highly promising biomarkers. Here, we briefly describe the current knowledge about microRNAs' involvement in HCV-related molecular oncogenesis. We decided to focus our attention on studies fully conducted on ex vivo samples with this specific etiology, and on cultured cell lines partially or completely expressing the HCV genome. Some of the results reported in this review are controversial, possibly because of methodological issues, differences in sampling size and features, and ethnicity of patients. What is certain is that miRNAs play a remarkable role in regulating gene expression during oncogenetic processes and in viral infection. A clear understanding of their effects is fundamental to elucidating the mechanisms underlying virus-induced malignancies.