Pascale Ghillani

Extrahepatic manifestations of chronic hepatitis C

Objective To assess the prevalence of clinical and biologic extrahepatic manifestations of hepatitis C virus (HCV) infection and to identify associations between clinical and biologic manifestations. Methods To analyze the natural history of extrahepatic manifestations of HCV infection, we reviewed only the data recorded prospectively during the first visit of 1,614 patients with chronic HCV infection, coming from a single monocenter cohort. Exclusion criteria were positivity for hepatitis B surface antigen or human immunodeficiency virus. The prevalence of dermatologic, rheumatologic, neurologic, and nephrologic manifestations; diabetes; arterial hypertension; autoantibodies; and cryoglobulins were assessed. Then, using multivariate analysis, we identified demographic, biochemical, immunologic, virologic, and liver histologic factors associated with the presence of extrahepatic manifestations. Results At least 1 clinical extrahepatic manifestation was observed in each of 1,202 patients (74%). Five manifestations had a prevalence >10%: arthralgia (23%), paresthesia (17%), myalgia (15%), pruritus (15%), and sicca syndrome (11%). Four biologic abnormalities had a prevalence >5%: cryoglobulins (40%), antinuclear antibodies (10%), low thyroxine level (10%), and anti–smooth muscle antibodies (7%). Only vasculitis, arterial hypertension, purpura, lichen planus, arthralgia, and low thyroxine level were associated with cryoglobulin positivity. By univariate and multivariate analyses, the most frequent risk factors for the presence of clinical and biologic extrahepatic manifestations were age, female sex, and extensive liver fibrosis. Conclusion Extrahepatic clinical manifestations are frequently observed in HCV patients and involve primarily the joints, muscles, and skin. The most frequent immunologic abnormalities include mixed cryoglobulins, antinuclear antibodies, and anti–smooth muscle antibodies. The most frequent risk factors for the presence of clinical and biologic extrahepatic manifestations are advanced age, female sex, and extensive liver fibrosis.

Impact of treatment on extra hepatic manifestations in patients with chronic hepatitis C

BACKGROUND/AIMS Fatigue and other extra hepatic manifestations of hepatitis C have never been studied prospectively in a large cohort. The aim was to assess the prevalence of these symptoms prior to any treatment, and on prolonged follow-up in treated and untreated patients. METHODS A single-center cohort of consecutive patients with chronic hepatitis C was investigated prior to any treatment. A questionnaire was completed every 6 months for 18 months of follow-up. RESULTS Of 1614 patients, 431 met the inclusion criteria (56% male; age 49 years; 60% with significant fibrosis or cirrhosis; 46% with cryoglobulinemia). Seventy-six were untreated; of the treated patients, 83 were sustained responders, 47 relapsers and 225 non-responders. At baseline, fatigue and other extrahepatic manifestations were present in 254 (59%) and 225 (52%) patients. Fatigue was improved in 29 of 83 (35%) responders versus 75 of 348 (22%) patients with detectable hepatitis C virus (HCV)-RNA (P=0.01). The impact of virologic response on fatigue persisted after adjusting for age, gender, fibrosis stage, and depression (odds ratio: 0.34, P<0.001). A cryoglobulin was detectable in two of 34 (6%) responders versus 38 of 115 (33%) patients with detectable HCV-RNA (P<0.001). CONCLUSIONS In hepatitis C, a sustained virologic response is associated with a reduction in fatigue and cryoglobulin, but fatigue frequently persists despite a virologic response.

Efficacy and Tolerability of Rituximab With or Without PEGylated Interferon Alfa-2b Plus Ribavirin in Severe Hepatitis C Virus-Related Vasculitis A Long-Term Followup Study of Thirty-Two Patients

OBJECTIVE To report on the long-term followup of a cohort of patients with hepatitis C virus (HCV)-related vasculitis treated with rituximab with or without PEGylated interferon alfa-2b (PEG-IFN alfa-2b) plus ribavirin. METHODS The study group comprised 32 HCV RNA-positive patients with HCV-related vasculitis: 20 patients were treated with rituximab and PEG-IFN alfa-2b (9 of whom had not previously received antiviral treatment and 11 of whom had experienced disease resistance to or relapse with antiviral treatment), and 12 antiviral-intolerant patients were treated with rituximab alone. RESULTS Treatment with rituximab and PEG-IFN alfa-2b plus ribavirin induced a complete clinical response and a partial clinical response in 80% and 15% of patients, respectively, a complete immunologic response and a partial immunologic response in 67% and 33% of patients, respectively, and a sustained virologic response in 55% of patients. Treatment with rituximab alone induced a complete clinical response and a partial clinical response in 58% and 9% of patients, respectively, and a complete immunologic response and a partial immunologic response in 46% and 36% of patients, respectively. B cell depletion was achieved in 96% of patients, and B cell recovery began after a median delay of 12 months. After a mean+/-SD followup period of 23+/-12 months, 22% of patients experienced a clinical relapse, and 34% of patients experienced an immunologic relapse. All relapses were associated with the absence of virologic control, and 78% of relapses were associated with B cell recovery. Six patients were re-treated with rituximab. All 6 of these patients had a complete clinical response, 50% had a complete immunologic response, and 50% had a partial immunologic response. Rituximab was well tolerated overall. CONCLUSION Rituximab is an effective treatment of severe and/or refractory HCV-related vasculitis. Relapses were consistently associated with the absence of virologic control. The clinical and immunologic efficacy of rituximab after repeated infusion appeared to be the same as that observed after induction therapy.

Influence of HLA-DR phenotype on the risk of hepatitis C virus-associated mixed cryoglobulinemia.

OBJECTIVE Circumstances predisposing hepatitis C virus (HCV)-infected patients to develop mixed cryoglobulinemia (MC), which may manifest as a small-vessel systemic vasculitis (MC vasculitis), remain unclear. Previous studies have failed to demonstrate a clear role of either viral factors (genotype, viral load) or host factors (lymphocytes or immunoglobulin subsets). This study was undertaken to examine a possible role of HLA class II alleles in HCV-associated MC. METHODS One hundred fifty-eight HCV-infected patients, of whom 76 had MC (56 with type II MC and 20 with type III MC) and 82 did not have MC, were studied prospectively. MC vasculitis was noted in 35 HCV-infected patients with type II IgMkappa-containing cryoglobulins. HLA-DRB1 and HLA-DQB1 polymorphism was analyzed by hybridization using allele-specific oligonucleotides, after gene amplification. The odds ratio (OR) was calculated with Woolfs method. Then, using multivariate analysis, demographic, biologic, immunologic, virologic, and liver histologic factors associated with the presence of MC and MC vasculitis were investigated. RESULTS HLA-DR11 was significantly more frequent in patients with type II MC than in those without MC (41.1% versus 17.1%; OR 3.4, corrected P [Pcorr] = 0.017), regardless of the presence of vasculitis accompanying the MC (37.1% of those with MC vasculitis, 34.1% of those with MC but no vasculitis). HLA-DR7 was less frequent in HCV-infected patients with MC than in those without MC (13.2% versus 30.5%; OR 0.34, P = 0.012, Pcorr not significant), with a particularly lower frequency in those with type II MC and those with MC vasculitis (12.5% and 8.6%, respectively). There was no significant difference in HLA-DQB1 distribution between the different patient groups. By univariate and multivariate analysis, HLA-DR11 was the only positive predictive factor, besides female sex and advanced age, for the presence of MC and HCV-associated MC vasculitis (OR 2.58). CONCLUSION Our results indicate that the presence of the DR11 phenotype is associated with a significantly increased risk for the development of type II MC in patients with chronic HCV infection. In contrast, HLA-DR7 appears to protect against the production of type II MC. These results suggest that the hosts immune response genes may play a role in the pathogenesis of HCV-associated MC.

Construction and clinical validation of a sensitive and specific assay for serum mature calcitonin using monoclonal anti-peptide antibodies.

Using calcitonin (CT) as an hapten, we have generated a library of monoclonal antibodies. Six monoclonal antibodies were developed and analyzed with respect to affinity and specificity for epitopes on CT by enzyme linked immunosorbent assay and radioimmunoassay. These antibodies were used in the construction and the optimization of a two-site monoclonal immunoradiometric assay (m-IRMA) for CT. We used a combination of two monoclonal antibodies to develop an assay which is rapid (overnight incubation), sensitive (10 pg/ml) and strictly specific for the mature form of calcitonin, ie the 32 amino acid-long polypeptide bearing a prolineamide as the C-terminal residue. This assay was utilized to demonstrate that mature calcitonin circulates as heterogeneous molecular species resulting from polymerization of calcitonin by disulphide linkage and/or by aggregation on irrelevant proteins. The clinical relevance of this assay was studied on a series of patients with medullary carcinoma of the thyroid (MCT) and the characteristics of the assay was compared with those of a conventional polyclonal radioimmunoassay. The m-IRMA for CT proved to be useful for both the diagnosis and the follow-up of MCT.

Anti-endothelial cell auto-antibodies in hepatitis C virus mixed cryoglobulinemia

BACKGROUND/AIM Hepatitis C virus (HCV) infection is often associated with mixed cryoglobulins (MC) and may manifest as small-vessel vasculitis. It has been suggested that antibody (Ab) or sensitized T cells to HCV-containing endothelial cells may initiate the vasculitis process. Anti-endothelial cell antibodies (AECA) have been found in various connective tissue disorders, with a high prevalence in systemic vasculitis. The aim of the study was to determine the prevalence of AECA in HCV patients with or without MC-associated vasculitis, and to identify associations with clinical, immunological, virological and liver characteristics. METHODS Sixty-nine HCV patients (Group 1), 46 of whom had MC (type II=30, type III=16), and 23 without MC, were prospectively studied. HCV-MC-associated vasculitis was noted in 25 patients who had at least one of the following clinical features: peripheral neuropathy, glomerulonephritis, skin purpura, cerebral vasculitis. Group 2 included 20 patients with non-HCV viral diseases: HHV8 (10), miscellaneous (10). Group 3 included 25 patients with biopsy-proven non-HCV chronic liver diseases: hepatitis B virus (10), miscellaneous (15). Controls were 100 blood donors (Group 4). Sera were adsorbed onto a pellet of A549/8 epithelial cells before being evaluated. AECA were then searched using a cellular ELISA, with a permanent cell line (EA.hy 926) as the substrate. All sera were also examined for the presence of cryoglobulin, antinuclear Ab, anticardiolipin Ab, and rheumatoid factor. RESULTS AECA were more frequently found in HCV patients than in blood donors (41% vs 5%, p=0.0001). The prevalence of AECA was lower in non-HCV than in group 1 patients [group 2=15%, p=0.03; group 3=16%, p=0.01]. There was no significant difference in AECA prevalence between groups 2, 3 and 4. In HCV patients, AECA were associated with age (p<0.001), the presence of MC (p=0.008), cryoglobulin level (p=0.016), HCV-associated vasculitis (p=0.04), genotype 1b (p=0.005) and severity of liver histologic damage. AECA isotypes were not different in the 4 groups. AECA were not associated with antinuclear Ab, anticardiolipin Ab, rheumatoid factor or interferon alpha treatment. CONCLUSION AECA are a common finding in HCV patients (41%), but not in other viral diseases or in non-HCV chronic liver diseases. In HCV patients, AECA are associated with MC-vasculitis, suggesting that AECA may be a marker for HCV-induced vasculitis.

Frequent joining of Bcl-2 to a JH6 gene in hepatitis C virus-associated t(14;18).

The t(14;18) chromosomal translocation, which joins the Bcl-2 proto-oncogene to an Ig JH gene, has increased prevalence in patients chronically infected with hepatitis C virus (HCV). We now establish a link between the molecular structure and clinical occurrence of HCV-associated t(14;18). A t(14;18) was detected by PCR in leukocytes from 22 of 46 HCV-infected patients (48%) and 11 of 54 healthy controls (20%) (p = 0.0053). Nucleotide sequence analysis of the Bcl-2/JH joins found a JH6 gene in 18 of 22 (82%) t(14;18) from HCV+ patients, and 3 of 8 (38%) from controls (p = 0.031). The t(14;18) rarely contained JH gene mutations, or an intervening region sequence suggestive of D gene rearrangement or templated nucleotide insertion. Analysis of published t(14;18) nucleotide sequences established that the JH6 prevalence in t(14;18) from normal/nonneoplastic controls (48%) was significantly lower than in t(14;18) from our HCV+ patients (p = 0.004) or from non-Hodgkin’s lymphomas (66%, p = 0.003). We conclude that the increased prevalence of t(14;18) in HCV+ patients occurs with a strong bias for Bcl-2/JH6 joins. In this regard, HCV-associated t(14;18) more closely resemble t(14;18) in lymphomas than t(14;18) from normal subjects.

Évaluation des performances analytiques de l’Optilite ® (The Binding Site) : nouvel automate spécialisé pour l’analyse des protéines spécifiques

We checked analytical performances of Optilite® analyser for immunoglobulins G, A, M, subclasses of IgG, free light chains of Ig (Freelite®) and complements fractions using Binding Site reagents. CVs for repeteability and reproducibility showed very good results, respectively <3% and <10% for all tested parameters, in agreement with Ricos and SFBC recommendations. Comparisons with results obtained on BN™II (Siemens) or SPAPLUS ® (Binding site) analysers showed a good agreement (>83%) according to Bland and Altman analysis. Sample throughput with either a batch of Freelite® only or Freelite® and immunoglobulins showed a gain of total realisation time on Optilite® versus BN™II. Optilite® analyser performed automatic dilutions until result and antigen excess determination for parameters as Freelite® or IgG4. In terms of practicability, traceability and maintenance, Optilite® turbidimeter alone or connected to LIS via DataSite software is well adapted to specialized laboratory for proteins determinations.

Manifestations extrahépatiques associées au virus de l'hépatite C : étude prospective d'une cohorte de 1 614 patients

Dix ans apres sa decouverte grâce aux progres des techniques de biologie moleculaire, le virus de l’hepatite C (VHC) a pris une place tres importante, non seulement dans le monde hepato-gastro-enterologique, puisqu’il est a l’origine de la plupart des hepatopathies chroniques dans les pays developpes, mais aussi dans l’ensemble des specialites medicales car de tres nombreuses atteintes extrahepatiques ont ete associees a l’infection chronique par le VHC. Toutefois, malgre le nombre tres important de publications rapportant des manifestations extrahepatiques tres diverses chez les patients infectes chroniquement par le VHC [1, 2], les liens de causalite entre une atteinte extrahepatique et le VHC sont loin d’etre toujours etablis. C’est pourquoi dans cet article nous avons volontairement scinde ces atteintes en quatre groupes : – les atteintes extrahepatiques liees avec certitude au VHC ; – les atteintes extrahepatiques possiblement associees au VHC, mais pour lesquelles des etudes complementaires sont necessaires pour affirmer definitivement ce lien ; – les manifestations extrahepatiques qui semblent associees fortuitement au VHC ; – enfin, les manifestations extrahepatiques pouvant apparaitre sous, ou etre declenchees par, le traitement de l’infection VHC, essentiellement l’interferon alpha.