Safety and efficacy of rituximab in patients with hepatitis C virus-related mixed cryoglobulinemia and severe liver disease.

The effectiveness of rituximab in hepatitis C virus (HCV)-related mixed cryoglobulinemia (MC) has been shown. However, the risk of an increase in viral replication limits its use in cirrhosis, a condition frequently observed in patients with MC. In this prospective study, 19 HCV-positive patients with MC and advanced liver disease, who were excluded from antiviral therapy, were treated with rituximab and followed for 6 months. MC symptoms included purpura, arthralgias, weakness, sensory-motor polyneuropathy, nephropathy, and leg ulcers. Liver cirrhosis was observed in 15 of 19 patients, with ascitic decompensation in 6 cases. A consistent improvement in MC syndrome was evident at the end-of-treatment (EOT) and end-of-follow-up (EOF-U). Variable modifications in both mean viral titers and alanine aminotransferase values were observed at admission, EOT, third month of follow-up, and EOF-U (2.62 x 10(6), 4.28 x 10(6), 4.82 x 10(6), and 2.02 x 10(6) IU/mL and 63.6, 49.1, 56.6, and 51.4 IU/L, respectively). Improvement in liver protidosynthetic activity and ascites degree was observed at EOT and EOF-U, especially in more advanced cases. This study shows the effectiveness and safety of rituximab in MC syndrome with advanced liver disease. Moreover, the depletion of CD20(+) B cells was also followed by cirrhosis syndrome improvement despite the possibility of transient increases of viremia titers.

Genetic determinants in hepatitis C virus-associated mixed cryoglobulinemia: role of polymorphic variants of BAFF promoter and Fcγ receptors.

OBJECTIVE: Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related immune complex disorder. Only some HCV-infected patients develop MC, which suggests that the genetic background of the host plays a key role. This study was undertaken to evaluate the contribution of host genetic factors in the pathogenesis of HCV-associated MC (HCV-MC) by analyzing allelic variants of low-affinity Fcγ receptor (FcγR) genes and BAFF promoter. METHODS: FcγR polymorphisms (FCGR2A 131 R/H, FCGR2B 232 I/T, FCGR3A 176 V/F, and FCGR3B NA1/NA2) and BAFF promoter polymorphism -871 C/T were analyzed in 102 patients with HCV-MC and 108 patients with HCV without MC, using polymerase chain reaction-based techniques. RESULTS: A higher prevalence of -871 T/T homozygosity (31% versus 16%; P = 0.001) and a greater frequency of T alleles of the BAFF promoter (80% versus 57%; P = 0.004) were found in the HCV-MC group than in the HCV group. A significant increase in serum BAFF concentration was significantly associated with the higher frequency of the T allele in HCV-MC (mean ± SD 4.12 ± 1.29 versus 2.09 ± 0.81 ng/ml; P < 0.0005). The distribution of the FcγR genotypes was not significantly different. In the 21 HCV-MC patients treated with rituximab, the response was strictly related to F allele homozygosity (significantly reduced in 5 of 5 patients with the FCGR3A F/F genotype versus 4 of 16 with V/V or V/F; P < 0.0005). CONCLUSION: These results indicate the importance of host genetic background in the development of HCV-MC, suggesting that mechanisms enhancing Ig production and B cell survival may play a relevant role. Genetic FcγR variants seem to be crucial to the effectiveness of rituximab therapy.

Involvement of PI3K in HCV-related lymphoproliferative disorders.

Hepatitis C virus (HCV) core protein has been shown to deregulate cell growth and programmed cell death in hepatoma cells, but only minimal informations are available about its possible role on B-lymphoproliferative disorders (LPDs). The aim of our work was to analyze the biological activity of HCV core protein on B-cell proliferation. We established Wil2-ns and Ramos B-cell lines that stably expressed the HCV core protein. Growth curve, thymidine incorporation analysis, as well as the expression of PCNA and activated-ERKs demonstrated that HCV core protein induced an increased growth in both cell lines. Interestingly, the HCV core protein expression determined, in our model, a downregulation of DNp73 and an upregulation of DNp63, which was essential for the maintenance of viral-dependent effects on cell growth. Finally, we have identified phosphoinositide 3-kinase (PI3K) as mediator of HCV core-dependent transcriptional increase of DNp63, which in turn correlated with the increasing of lymphocyte proliferation. In primary B-lymphocytes, derived from HCV-related low-grade non-Hodgkin's lymphoma patients, consistent results were obtained. These findings provide evidence for a possible pathogenetic role played by HCV core protein in HCV-related lymphomagenesis; it could occur through the deregulation of PI3K activity, consequent activation of Akt and overexpression of DNp63.

Notch4 and mhc class II polymorphisms are associated with hcv-related benign and malignant lymphoproliferative diseases.

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.

Prevalence of bcl-2 rearrangement in patients with hepatitis C virus-related mixed cryoglobulinemia with or without B-cell lymphomas.

BACKGROUND: Hepatitis C virus (HCV) infection is strictly associated with mixed cryoglobulinemia, a benign B-cell lymphoproliferative disorder that may evolve to lymphoma. An increased prevalence of bcl-2 rearrangement (the t(14;18) translocation) has been shown in patients infected with HCV. OBJECTIVE: To evaluate the prevalence of bcl-2 rearrangement in patients with HCV-related mixed cryoglobulinemia and patients with chronic hepatitis but no cryoglobulinemia. DESIGN: Prospective study. SETTING: Two university hospitals. PATIENTS: 37 consecutively recruited patients with HCV-related mixed cryoglobulinemia and 101 patients with chronic HCV infection but without mixed cryoglobulinemia. MEASUREMENTS: Clinical and serologic characteristics; liver biopsy; bcl-2 rearrangement, Bcl-2 expression, and the ratio of Bcl-2 to Bax in total peripheral blood mononuclear cells and cell subgroups; and sequence analysis of the junction of bcl-2 and IgH joining segments in positive samples. RESULTS: Rearrangement of bcl-2 was observed in 28 of 37 (75.7%) patients with mixed cryoglobulinemia (65% of those with type III disease and 85% of those with type II disease, including 3 of 4 patients with lymphoma) and in 38 of 101 (37.6%) patients with chronic HCV infection but not mixed cryoglobulinemia (P < 0.001). Overexpression of Bcl-2 protein and a high ratio of Bcl-2 to Bax were observed in samples from patients with bcl-2 rearrangement. In 2 patients followed over time, peripheral blood cells bearing the t(14;18) translocation disappeared after antiviral therapy. CONCLUSIONS: Rearrangement of bcl-2 was found with increased frequency in patients with chronic HCV infection and mixed cryoglobulinemia. The frequency was greatest in patients with type II mixed cryoglobulinemia. The high ratio of Bcl-2 to Bax in patients with bcl-2 rearrangement and disappearance of the rearrangement with antiviral therapy suggest that the translocation is associated with the antiapoptotic function of Bcl-2 and that HCV infection is linked to inhibition of B-cell apoptosis.

EEG correlates of social interaction at distance.

This study investigated EEG correlates of social interaction at distance between twenty-five pairs of participants who were not connected by any traditional channels of communication. Each session involved the application of 128 stimulations separated by intervals of random duration ranging from 4 to 6 seconds. One of the pair received a one-second stimulation from a light signal produced by an arrangement of red LEDs, and a simultaneous 500 Hz sinusoidal audio signal of the same length. The other member of the pair sat in an isolated sound-proof room, such that any sensory interaction between the pair was impossible. An analysis of the Event-Related Potentials associated with sensory stimulation using traditional averaging methods showed a distinct peak at approximately 300 ms, but only in the EEG activity of subjects who were directly stimulated. However, when a new algorithm was applied to the EEG activity based on the correlation between signals from all active electrodes, a weak but robust response was also detected in the EEG activity of the passive member of the pair, particularly within 9 - 10 Hz in the Alpha range. Using the Bootstrap method and the Monte Carlo emulation, this signal was found to be statistically significant.

Triple antiviral therapy in hepatitis C virus infection with or without mixed cryoglobulinaemia: a prospective, controlled pilot study.

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.

Role of microRNA profile modifications in hepatitis C virus-related mixed cryoglobulinemia.

Hepatitis C virus infection is closely related to lymphoproliferative disorders (LPDs), including mixed cryoglobulinemia (MC) and some lymphomas. Modification of the expression of specific microRNAs (miRNAs) has been associated with different autoimmune diseases and/or LPDs. No data exist about the modifications in miRNA expression in HCV-associated LPDs. The aim of this study was to analyze the expression levels of a panel of miRNAs previously associated with autoimmune/LPDs in a large population of HCV patients with and without MC or non-Hodgkin's lymphoma (NHL), to identify potential markers of evolution of HCV infection. PBMC expression of miR-Let-7d, miR-16, miR-21, miR-26b, miR-146a and miR-155 was evaluated by real-time PCR in 167 HCV patients (75 with MC [MC-HCV], 11 with HCV-associated NHL [NHL-HCV], 81 without LPD [HCV]) and in 35 healthy subjects (HS). A significant increase in miR-21 (p<0.001), miR-16 (p<0.01) and miR-155 (p<0.01) expression was detected in PBMCs from only NHL patients whereas a significant decrease in miR-26b was detected in both MC and NHL subjects (p<0.01) when compared to HS and HCV groups. A restoration of miR-26b levels was observed in the post-treatment PBMCs of 35 HCV-MC patients experiencing complete virological and clinical response following antiviral therapy. This study, for the first time, shows that specific microRNAs in PBMC from HCV patients who developed MC and/or NHL are modulated differently. The specific, reversible downregulation of miR-26b strongly suggests the key role it plays in the pathogenesis of HCV-related LPDs and its usefulness as a biomarker of the evolution of HCV infection to these disorders.

Elevated plasma levels of urotensin II do not correlate with systemic haemodynamics in patients with cirrhosis.

BACKGROUND: The hyperdynamic circulation of hepatic cirrhosis is related to decreased systemic vascular resistance due to arterial vasodilation. Urotensin II plasma levels are increased in cirrhotic patients, and have been suggested to play a role in the pathogenesis of systemic haemodynamic alterations. AIM: To evaluate the relationships between systemic haemodynamics and urotensin II plasma levels. METHODS: Thirty-six consecutive in-patients with cirrhosis and no alteration of plasma creatinine, and 20 age- and gender-matched healthy volunteers underwent noninvasive assessment of systemic haemodynamics and measurement of urotensin II plasma levels. RESULTS: In comparison to healthy controls, cirrhotic patients had signs of hyperdynamic circulation and higher plasma urotensin II levels. Plasma urotensin II was neither significantly different amongst patients with different severity of cirrhosis nor between patients with or without ascites. Both in controls and cirrhotic patients no significant correlations were found between parameters of systemic haemodynamics and plasma urotensin II levels. CONCLUSIONS: In patients with cirrhosis and hyperdynamic circulation, but with normal serum creatinine, urotensin II is higher than in healthy subjects. However, no correlation with cardiac index or other haemodynamic parameters was observed, indicating that other mechanisms prevail.

Improvement in liver cirrhosis after treatment of HCV-related mixed cryoglobulinemia with rituximab.

Mixed cryoglobulinemia (MC) is the most strictly virus-related extrahepatic HCV disease. Antiviral therapy is considered the first therapeutic option; however, MC patients are frequently excluded from treatment due to contraindications. The effectiveness of B-cell depletion by anti-CD20 monoclonal antibody (rituximab) has recently been described, but the possibility of an immunodepression- related increase in viral replication and aminotransferase values limits its use in patients with advanced liver disease. Unfortunately, MC patients frequently also have cirrhosis. To our knowledge, no data are available regarding the effect of rituximab therapy in patients with decompensated cirrhosis. We report the successful treatment with rituximab (4 weekly infusions of 375 mg/m 2) of two patients (a 58-year-old man, and a 65-year-old woman) with HCV-related MC syndrome and decompensated liver cirrhosis. These patients underwent at least 6 months of post-treatment follow-up. In both cases a consistent improvement of MC syndrome was evident after treatment. In addition, improvement of liver protidosynthetic activity, increased prothrombin time, impressive reduction or disappearance of ascites and encephalopathy were also observed, in spite of some increase in viral titers or in ALT values. The Child-Pugh score improved from B8 to A6 and from Cll to B7, respectively. Pre- and post-treatment transjugular liver biopsies were available in 1 patient, showing disappearance of lymphocytic infiltration after treatment. These case reports show the effectiveness and safety of rituximab in patients with HCV-related MC and advanced cirrhosis, and strongly suggest that the depletion of CD20+ B-cells induced by rituximab treatment may be responsible for liver function improvement. The mechanisms involved are unknown. Interesting working hypotheses may implicate a role played by B-cell infiltrates in conditioning liver damage. The improvement of Kupffer cell function due to the cryocrit value reduction might also play a role.

Effect of chronic hepatitis C virus infection on inflammatory lipid mediators.

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 core protein expression in human B-cell lines does not significantly modify main proliferative and apoptosis pathways.

Hepatitis C virus (HCV) chronic infection has been associated with many lymphoproliferative disorders. Several studies performed on hepatoma and fibroblast cell lines suggest a role of the HCV core protein in activation of cellular transduction pathways that lead to cell proliferation and inhibition of apoptosis. However, no data are available concerning the effects of HCV core expression on B-lymphocyte proliferation and apoptosis. B-lymphocyte cell lines permanently expressing full-length HCV 1b core sequences isolated from chronically infected patients were established using B-cell lines at different degrees of differentiation. Clones and pools of clones permanently expressing the HCV core were selected and characterized for protein expression by Western blot and FACS. Expression of HCV core proteins did not significantly enhance cell proliferation rates under normal culture conditions or under mitogenic stimulation. Analysis of NF-kappa B, CRE, TRE and SRE pathways by luciferase reporter genes did not show a significant influence of HCV core expression on these signal transduction cascades in B-lymphocytes. The effects of HCV core on anti-IgM and anti-FAS-induced apoptosis in B-cell lines was also analysed. In this experimental model, HCV core expression did not significantly modify the apoptotic profile of the B-lymphocyte cell lines tested. These data underline a cell type-specific effect of HCV core expression. In fact, it was not possible to show a significant contribution of the HCV core protein in activation of the major B-cell signal transduction pathways involved in the regulation of proliferation and programmed cell death, which is in contrast with the results reported in hepatoma cell lines.

Effect of antiviral treatment in patients with chronic HCV infection and t(14;18) translocation.

Hepatitis C virus (HCV) may be associated with the mixed cryoglobulinemia syndrome and other B-cell lymphoproliferative disorders (LPDs). The t(14;18) translocation may play a pathogenetic role. Limited data are available regarding the effects of antiviral therapy on rearranged B-cell clones. We evaluated the effects of interferon and ribavirin on serum, B-lymphocyte HCV RNA, and t(14; 18) in 30 HCV+, t(14;18)+ patients without either mixed cryoglobulinemia syndrome or other LPDs. The t(14;18) translocation was analyzed by both bcl-2/JH polymerase chain reaction and bcl-2/JH junction sequencing in peripheral blood mononuclear cells in all patients. Fifteen untreated patients with comparable characteristics served as controls. Throughout the study, the presence or absence of both t(14;18) and HCV RNA sequences were, in most cases, associated in the same cell samples. At the end of treatment, t(14;18) was no longer detected in 15 patients (50%) with complete or partial virologic response, whereas it was persistently detected in nonresponders (P <.05), as well as in 14 of 15 control patients. In 4 responder patients, t(14;18) and HCV RNA sequences were no longer detected in blood cells after treatment, but were again detected after viral relapse; the same B-cell clones were involved in the pretreatment and posttreatment periods. In conclusion, this study suggests that antiviral therapy may induce regression of t(14;18)-bearing B-cell clones in HCV+ patients and that this phenomenon may be related, at least in part, to the antiviral effect of therapy. This in turn suggests that antiviral treatment may help prevent or treat HCV-related LPDs.