Christoph Sarrazin

Summary. Chronic hepatitis C virus (HCV) infection leads to mixed cryoglobulinaemia (MC) and B-cell non-Hodgkin lymphoma (B-NHL). Aberrant somatic hypermutation and deregulation of the oncogene BCL-6 is associated with lymphomagenesis. Recently, HCV was shown to induce BCL-6 mutations in vitro. The BCL-6 gene (area B) was cloned and sequenced from peripheral blood mononuclear cells (PBMC) of 21 chronically HCV-infected patients with or without MC and B-NHL, and six healthy controls. Mutational frequencies, genetic complexity and diversity were calculated. BCL-6 mRNA from PBMC was quantified by real-time polymerase chain reaction, and additional sustained virologic responders to antiviral therapy and HBV patients served as controls. The overall/recurrent mutational frequencies tended to be lower in MC and B-NHL patients when compared with controls (P = 0.15 and 0.06, respectively). Genetic complexity was significantly lower in MC and B-NHL patients (P = 0.025). BCL-6 mRNA concentration was decreased in all HCV patients when compared with healthy controls, sustained virologic responder and HBV patients (P = 0.005). Although HCV can induce BCL-6 mutations in vitro, lower mutational frequencies and decreased BCL-6 mRNA expression in vivo suggest no major role of aberrant somatic hypermutation in HCV-associated MC and B-NHL.

SUMMARY: Chronic hepatitis C virus (HCV) infection affects more than 170 million persons worldwide and is responsible for the development of liver cirrhosis in many cases. Standard treatment with pegylated alpha interferon (IFN-α) in combination with the nucleoside analogue ribavirin leads to a sustained virologic response in approximately half of the patients. IFN-α is classified as an indirect treatment, as it interacts with the host's immune response. The mechanism of action of ribavirin is still unknown. The benefit of triple therapy by adding other antiviral agents, e.g., amantadine, is controversial. Currently, new direct antiviral drugs (HCV protease/polymerase inhibitors) are being evaluated in phase 1/phase 2 trials. Phenotypic resistance to antiviral therapy has been attributed to amino acid variations within distinct regions of the HCV polyprotein. While sensitivity to IFN-α-based antiviral therapy in vivo is clearly correlated with the number of mutations within the HCV NS5A protein, the underlying functional mechanisms for this association are unknown. In turn, in vitro, several mechanisms to circumvent the host immune defense or to block treatment-induced antiviral activities have been described for different HCV proteins. By the introduction of direct antiviral drugs, hepatitis C therapy now is entering a new era in which the development of resistance may become the most important parameter for treatment success or failure.

Background. Hereditary aceruloplasminemia is an adult-onset autosomal recessive disease characterized by increased iron overload in the liver, pancreas, retina, and central nervous system. So far, 45 families with cases of aceruloplasminemia have been reported world-wide and mainly missense and nonsense mutations in the ceruloplasmin gene were detected. Material and methods. Here, we report the identification, clinical characterization, and in silico analysis of a novel compound heterozygosity in the ceruloplasmin gene of a 31-year-old man with iron overload. Results. Increased serum ferritin levels, elevated iron saturation, as well as results of iron quantification in the liver and magnetic resonance imaging-based measurement of T2 relaxation times of the substantia nigra consistently suggested iron overload. By sequencing the ceruloplasmin gene, so far unknown nucleotide replacements G229C, and C2131A were detected in exons 2 and 12, respectively. In silico analyses showed that the resulting amino acid changes Asp58His and Gln692Lys are located at highly conserved positions. The Asp58His mutation is located on the surface of the protein, alters polarity, and may interfere with copper incorporation or ceruloplasmin trafficking. The Gln692Lys mutation is mapped to a β-strand of domain 4 and may lead to conformational change of the cupredoxin fold. Conclusions. As causative for aceruloplasminemia, a formerly unknown compound heterozygosity in the ceruloplasmin gene was identified. In silico characterization suggests an impact on ceruloplasmin conformation and function.

CD81 is a hepatitis C virus (HCV) coreceptor with important functions in lymphocytes. During treatment, CD81 expression may be changed directly by the antiviral therapy or indirectly by reduction of the HCV serum level. The regulation of CD81 on lymphocyte subtypes has not been investigated so far and may be relevant for the control of viral infection and treatment response. CD81 was analyzed by flow cytometry in CD8(+), CD4(+), CD19(+), and CD56(+) lymphocyte subtypes from 20 patients with chronic hepatitis C before, during, and after antiviral treatment with pegylated interferon-α (IFN-α) and ribavirin. A sustained virologic response (SVR) was achieved in 11 patients. Dynamics of CD81 were investigated in correlation with HCV-RNA dynamics and the outcome of therapy. During treatment, the following typical patterns of CD81 regulation were observed: down-regulation on CD8(+) T cells (P=0.022) and most significantly, on CD56(+) natural killer cells (P<0.001), transient up-regulation on CD19(+) B cells (P=0.006), and weak and late down-regulation on CD4(+) T cells (P=0.028). During treatment, CD81 expression was not associated with the HCV-RNA serum level on all lymphocyte subtypes. After end of treatment, CD81 increased again in CD8(+) and CD56(+) cells (P=0.001, P=0.002). On CD8(+) T cells post-treatment, CD81 remained lower in patients who achieved a SVR compared with patients who failed to eliminate HCV after treatment (P=0.033). Lymphocyte subsets show different patterns of CD81 response before and during antiviral treatment, which are associated with administration of IFN-α and antiviral response.

The key parameter for diagnosis and management of hepatitis C virus (HCV) infection is HCV RNA. Standardization of HCV RNA assays to IU is mainly based on genotype 1 panels. Little is known about the variability of commercially available HCV RNA assays for quantification of different genotypes. Two real-time reverse transcription (RT)-PCR assays (COBAS TaqMan HCV Test for use with the High-Pure System [HPS/CTM] and COBAS Ampliprep/COBAS TaqMan HCV Test [CAP/CTM]), one standard RT-PCR assay (COBAS Amplicor HCV Monitor 2.0 [CAM]), and one signal amplification assay (Versant Quantitative 3.0 [branched DNA {bDNA}]) were compared for quantification of genotypes 1 to 5 (n= 108). Using CAM as a reference assay for genotype 1-infected patients, the mean interassay differences compared with CAP/CTM, HPS/CTM, and bDNA were 0.16, −0.13, and −0.48 log₁₀IU/ml HCV RNA, respectively. Comparison of CAM with CAP/CTM, HPS/CTM, and bDNA for the remaining genotypes showed the following results, respectively: 2a/c, −0.24, −0.78, and −0.49; 2b, −0.21, −0.18, and −0.64; 3a, 0.13, −1.04, and −0.55; 4, −0.52, −1.51, and −0.05; and 5, −0.28, −1.00, and −0.24 log IU/ml HCV RNA. A correct decision for treatment discontinuation in genotype 1 patients at week 12 was possible only when the same assay was used at baseline and week 12. Comparison of CAM with the CAP/CTM assay showed equal quantifications of genotype 1, 2, 3, and 5 samples, while genotype 4 samples were slightly underestimated. For the HPS/CTM assay, a significant underestimation of the HCV RNA concentrations of genotypes 2a/c, 3, 4, and 5 was observed. For the bDNA assay, a constant lower quantification of genotypes 1 to 3 was detected.

BackgroundMutations in nonstructural (NS) hepatitis C virus (HCV) proteins enhance replication in HCV-1a/b replicons. The prevalence of such mutation

Pegylated interferon alfa in combination with ribavirin has been established as standard therapy for chronic hepatitis C virus (HCV) infection with sustained virologic response rates of 54–63%. The duration of therapy depends on the HCV genotype with currently 48 weeks for genotype 1 and 24 weeks for genotypes 2 and 3.