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SUMMARYLittle is known about hepatitis C virus (HCV) breakthrough during antiviral therapy, although it would help in understanding HCV resistance to current antiviral treatments. To analyse the implication of virological factors and the vigour of humoral immune responses in this phenomenon, we studied 9 chronic hepatitis C patients with a viral breakthrough during IFN/ribavirin combination therapy, as well as 5 responders and 5 non responders. The IRES and regions coding for the capsid protein, the PePHD domain of envelope glycoprotein E2 and the NS5A and 5B proteins were amplified by RT-PCR before treatment, before and during breakthrough, and after treatment. The major variant sequence was obtained by direct sequencing. The heterogeneity of quasispecies was studied by SSCP in all patients and sequencing after cloning in seven genotype 1b-infected patients. Humoral responses against HCV epitopes were also analysed. The major sequences of IRES, PePHD and NS5B remained stable during treatment, regardless of the treatment response. However, the capsid protein and the regions flanking PePHD showed sequence variations in breakthrough patients, although no specific mutation was identified. The variable V3 region of NS5A, but not the PKR-binding domain and the ISDR, seemed to be associated with differences in response to treatment. The analysis of HCV quasispecies revealed no characteristic pattern during treatment in breakthrough patients, whose HCV genome profiles looked most similar to that of non responders. The humoral response was similar between groups. In conclusion, viral breakthrough does not seem to be due to selection of resistant strains with signature mutations.

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Concanavalin A (Con A) induces an immune-mediated liver cell injury analogous to that which occurs in several human diseases.We have recently shown that activation of CAR, a potent transcription factor in the nuclear receptor family, reduces liver cell apoptosis and injury in a murine model, following administration of Jo2 (a Fas agonistic antibody). However, whether Con A liver injury is reduced by CAR activation remains unknown. Thus, our aim was to ascertain if the CAR agonist ligand TCPOBOP attenuates acute and chronic Con A-induced liver injury. Methods: Acute liver injury: C57/BL6 CAR wild type (CAR ϩ/ϩ ) and knock out mice (CAR -/-) were injected i.p. (3mg/kg) daily with the CAR agonist TCPOBOP or vehicle (corn oil) for 3 days. Next, mice were injected with Con A 25 mg/kg i.v. and liver injury (H&E, and serum ALT determinations) was assessed 15 hrs later. Apoptosis was determined by the TUNEL assay and immunofluorescence for activated caspase 3/7. Chronic liver injury: Mice were injected with TCPOBOP or vehicle (corn oil) i.p. weekly followed by weekly Con A i.v. injections 8 mg/kg or vehicle for 6 weeks. Liver fibrosis was assessed in sirius red stained liver sections and ␣SMA mRNA expression by RT-PCR, respectively. Results: Acute injury: Following Con A administration, serum ALT values were elevated 120-fold in CAR ϩ/ϩ vehicle and CAR -/animals vs. TCPOBOP treated CAR ϩ/ϩ animals. TUNEL positive cells and caspase 3/7 immunofluorescence was also elevated in CAR ϩ/ϩ vehicle and CAR -/animals, but not TCPOBOP-treated mice after Con A administration. Chronic injury: ␣SMA mRNA expression was decreased by approximately 50% in TCPOBOP treated CAR ϩ/ ϩanimals compared to vehicle. Sirius red staining showed marked fibrosis in liver sections from animals without TCPOBOP pretreatment. In Conclusion, these original findings support a prominent role for CAR cytoprotection against Con A mediated acute and chronic liver injury. CAR agonists may represent a novel approach for reducing liver injury and fibrosis in immune mediated liver diseases. Disclosures:The following authors have indicated they have no relationships to disclose:We have previously shown that Bid, a pro-apoptotic Bcl-2 family protein, plays an important role in TNF-␣ and anti-Fas antibody induced apoptosis in primary cultured murine hepatocytes. However, the bid-deficient hepatocytes eventually died after 24hrs although the process delayed by 6hrs compared to wild type cells after TNF-␣ treatment. This suggests that a Bid-independent pathway may contribute to the cell death after TNF-␣ treatment. In the present study, we found that the reactive oxygen species (ROS) level was lower at the early time point but increased significantly at later time points after TNF-␣ treatment in bid-deficient cells. Moreover, MnTBAP, a superoxide dismutase mimetic, significantly inhibited the cell death in both wild type and biddeficient cells, suggesting that ROS may be involved in the Bidindependent pathway. Bax, another pro-apoptotic Bcl-2 family protein, can translo...