Large-scale survey of naturally occurring HBV polymerase mutations associated with anti-HBV drug resistance in untreated patients with chronic hepatitis B.

Drug resistance is a major limitation for the long-term efficacy of antiviral therapy with nucleos(t)ide analogues (NAs) in chronic hepatitis B (CHB). Antiviral resistance mutations may pre-exist in the overall viral population of untreated patients. We aimed to assess the prevalence of such hepatitis B virus (HBV) variants in a large cohort of NAs-naïve patients with CHB and to explore possible association with viral and host variables. Serum samples from 286 NAs-naïve consecutive patients with CHB were tested for serum HBV-DNA, and 255 of them having HBV-DNA > 1000 IU/mL were further analysed for drug resistance mutations by INNO-LiPA HBV DRv2/v3. NAs-naïve patients analysed were mainly men (73%), Caucasians (85%), hepatitis B e Antigen (HBeAg) negative (79%) and genotype D (69%), with a mean age of 43.2 ± 13.4 years. HBV mutations associated with antiviral drug resistance were detected in 13 (5%) patients: three patients infected with HBV genotype C had the rtM204V + rtL180M mutations associated with lamivudine (LMV) resistance. Four patients had the rtI233V mutation that may reduce sensitivity to adefovir, and three patients had the rtM250L/V mutation typical of entecavir resistance. LMV compensatory mutations rtL80V and rtV173L were seen in two and one patients, respectively. No relationship was seen between presence of resistant or compensatory mutations and HBV-DNA levels, HBeAg/anti-HBe status or previous IFN therapy. These results confirm that HBV mutations, which confer resistance against currently available anti-HBV NAs, may already exist in patients who have never received the drug.

Hyperinsulinaemia reduces the 24-h virological response to PEG-interferon therapy in patients with chronic hepatitis C and insulin resistance.

Insulin resistance (IR) reduces response to pegylated-interferon (PEG-IFN)/ribavirin in chronic hepatitis C (CHC), but the mechanisms are still undefined. We examined the relationship between baseline insulin levels, the main component affecting homeostasis model of assessment - insulin resistance (HOMA-IR) for assessment of IR in non-diabetic patients, and the 'acute' virological response to PEG-IFN measured 24 h after the first injection and taken as correlate of intracellular interferon signalling. In 62 patients treated with PEG-IFN/Ribavirin, serum insulin and HOMA-IR were assessed at baseline, while hepatitis C virus (HCV)-RNA was measured at baseline and 24 h, 1, 2, 4 and 12 weeks after treatment initiation. Sustained virological response was examined 24 weeks after therapy discontinuation. Mean baseline insulin was 11.52 +/- 8.51 U/L and mean HOMA-IR was 2.65 +/- 2.01 both being significantly higher with advanced liver fibrosis. Hepatitis C virus-RNA decay observed 24 h after the first injection of PEG-IFN was significantly lower (0.7 +/- 0.8 log) in patients with HOMA > or =3 compared with those with HOMA <3 (1.7 +/- 0.8, P = 0.001). A highly significant (r = -0.42) inverse correlation was observed between baseline insulin levels and the 24-h HCV-RNA decay. The difference in early viral kinetics between patients with HOMA > or =3 or <3 resulted in a significant difference in the percentage of patients achieving rapid (week 4) and sustained virological response. Multivariate analysis, inclusive of patient age, HCV genotype and fibrosis stage, identified baseline insulin levels as the main independent variable affecting the 24-h response to PEG-IFN. Hyperinsulinaemia reduces the cellular response to Pegylated-interferon in CHC with IR. Strategies to reduce insulin levels before initiation of treatment should be pursued to improve efficacy of anti-viral treatment.

Detailed analysis of the E2-IgM complex in hepatitis C-related type II mixed cryoglobulinaemia.

Hepatitis C virus (HCV) plays a major role in the induction of type II mixed cryoglobulinaemia (MCII). The role of HCV proteins and virus-host interaction in the pathogenesis of MC remains to be defined. To address this issue, we have characterized, in detail, the monoclonal IgM and the viral component of circulating immune complexes in eight patients with HCV-associated MCII. The proportion of HCV-RNA compartmentalized in the cryoprecipitate (CP) varied greatly (10-80% of total HCV-RNA). The complementary determining region (CDR)3 sequences of monoclonal immunoglobulin M (IgM) VH and VK genes were highly homologous to rheumatoid factor and to antibodies against HCV-E2. Furthermore, the CDR3 sequences in some of our MCII patients were highly similar to those described in HCV-positive patients with non-Hodgkin's lymphoma (NHL). From these results, it appears that, as in the case of NHL, the IgM-rheumatoid factor (RF) production in MCII patients is antigen driven, namely by E2. However, the limited number of mutations in VH and VK genes with respect to the germline and their distribution showed that the B-cell response in these cases was prevented from undergoing affinity maturation. Furthermore, in patients with monoclonal IgM and definite compartmentalization of HCV in either CP or supernatant, a highly homogeneous E2-hypervariable region (HVR)1 sequence distribution was found (90-100% identical clones), a feature of the quasispecies frequently associated with an impaired humoral immune response to HCV. These findings suggest that in patients with HCV-associated MCII, maturation of monoclonal B lymphocytes may be blocked in a primitive stage preventing serious damaging effects because of the auto-reactivity of their secreted immunoglobulins.

Characterization of human skeletal muscle Ankrd2.

Human Ankrd2 transcript encodes a 37-kDa protein that is similar to mouse Ankrd2 recently shown to be involved in hypertrophy of skeletal muscle. These novel ankyrin-rich proteins are related to C-193/CARP/MARP, a cardiac protein involved in the control of cardiac hypertrophy. A human genomic region of 14,300 bp was sequenced revealing a gene organization similar to mouse Ankrd2 with nine exons, four of which encode ankyrin repeats. The intracellular localization of Ankrd2 was unknown since no protein studies had been reported. In this paper we studied the intracellular localization of the protein and its expression on differentiation using polyclonal and monoclonal antibodies produced to human Ankrd2. In adult skeletal muscle Ankrd2 is found in slow fibers; however, not all of the slow fibers express Ankrd2 at the same level. This is particularly evident in dystrophic muscles, where the expression of Ankrd2 in slow fibers seems to be severely reduced.

FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z-disc of skeletal muscle.

We report the identification and characterization of a novel 32-kDa protein expressed in skeletal muscle and located in the Z-disc of the sarcomere. We found that this protein binds to three other Z-disc proteins; therefore, we have named it FATZ, gamma-filamin/ABP-L, alpha-actinin and telethonin binding protein of the Z-disc. From yeast two-hybrid experiments we are able to show that the SR3-SR4 domains of alpha-actinin 2 are required to bind the COOH-terminal region of the FATZ as does gamma-filamin/ABP-L. Furthermore, by using a glutathione S-transferase overlay assay we find that FATZ also binds telethonin. The level of FATZ protein in muscle cells increases during differentiation, being clearly detectable before the onset of myosin. Although FATZ has no known interaction domains, it would appear to be involved in a complex network of interactions with other Z-band components. On the basis of the information known about its binding partners, we could envisage a central role for FATZ in the myofibrillogenesis. After screening our muscle expressed sequence tag data base and the public expressed sequence tag data bases, we were able to assemble two other muscle transcripts that show a high level of identity with FATZ in two different domains. Therefore, FATZ may be the first member of a small family of novel muscle proteins.

ZASP: a new Z-band alternatively spliced PDZ-motif protein.

PDZ motifs are modular protein-protein interaction domains, consisting of 80-120 amino acid residues, whose function appears to be the direction of intracellular proteins to multiprotein complexes. In skeletal muscle, there are a few known PDZ-domain proteins, which include neuronal nitric oxide synthase and syntrophin, both of which are components of the dystrophin complex, and actinin-associated LIM protein, which binds to the spectrin-like repeats of alpha-actinin-2. Here, we report the identification and characterization of a new skeletal muscle protein containing a PDZ domain that binds to the COOH-terminal region of alpha-actinin-2. This novel 31-kD protein is specifically expressed in heart and skeletal muscle. Using antibodies produced to a fragment of the protein, we can show its location in the sarcomere at the level of the Z-band by immunoelectron microscopy. At least two proteins, 32 kD and 78 kD, can be detected by Western blot analysis of both heart and skeletal muscle, suggesting the existence of alternative forms of the protein. In fact, several forms were found that appear to be the result of alternative splicing. The transcript coding for this Z-band alternatively spliced PDZ motif (ZASP) protein maps on chromosome 10q22.3-10q23.2, near the locus for infantile-onset spinocerebellar ataxia.