For the detection of persisting HCV-NS3 Ag in the liver, liver tissue samples isolated 21 days post-infection were homogenized in RIPA C buffer (50 mM Tris pH 7.5, 1% Triton X-100, 300 mM NaCl, 5 mM ethylenediaminetetraacetic

acid, 0.02% NaN3) to make 2% (w/v) extract and used for immune precipitation/western blot assay. Liver tissue extracts were incubated with protein-G sepharose beads for 30 min at 4°C to remove non-specifically bound proteins. After centrifugation, supernatants were incubated with anti-Flag-M2 antibody Selleckchem Temozolomide (Sigma-Aldrich) coupled protein-G sepharose beads for 2 h at 4°C. After centrifugation, HCV-NS3-3xFlag fusion protein bound to the beads were dissolved in sample buffer and separated on 10% sodium dodecylsulfate polyacrylamide gel electrophoresis gels (Mini PROTEAN TGX gel; Bio-Rad, Hercules, CA, USA) for immunoblot analysis using anti-Flag-M2 antibody and

goat antimouse Ig horseradish peroxidase (KPL, Gaithersburg, MD, USA). Electrochemiluminescence Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK) was used for chemiluminescent detection. Mann–Whitney U-tests were used to evaluate the significance of the differences. Correlations between parameters were tested for statistical http://www.selleckchem.com/products/PD-0332991.html significance by Pearson correlation. TO DETERMINE THE effect of the amount of virus dose, we evaluated hepatic inflammation and compared the magnitude of HCV-NS3-specific CD8 T-cell responses and their effector function in the liver of mice infected with 2 × 107, 1 × 109 and 1 × 1010 PFU Ad-HCV-NS3. In histological studies, we observed Ad-infection-mediated hepatic inflammation in mice injected with MCE公司 1 × 109 and 1 × 1010 PFU. Especially, infection with 1 × 1010 PFU caused drastic infiltrations

of inflammatory cells (Fig. 1a). We also observed that CD8 lymphocytes infiltrated into the lobular areas of the infected liver in mice injected with 1 × 1010 PFU (Fig. 1b). At 7 days post-infection, we found by flow cytometric assay that the numbers and the frequencies of CD8 T cells in the liver were markedly increased after infection with 1 × 109 PFU and 1 × 1010 PFU, and the increased CD8 T cells decreased with time (Fig. 1c). We did not find significant differences between the number of CD8 T cells of core (+) and core (−) at each time point and infectious dose. In addition, we evaluated core protein expression in the liver in each infectious dose at 7 and 14 days post-infection; there was no significant difference in core protein expression between Ad-infected and non-infected livers (Fig. 1e). Using major histocompatibility complex (MHC) class I tetramer complexed with the H2-Db-binding HCV-NS3 GAVQNEVTL epitope, we found that i.v. infection with 2 × 107 PFU generally elicited only a weak expansion of HCV-NS3 tet+ CD8+ IHL (Fig. 2a,b) and IFN-γ+ HCV-NS3 tet+ CD8+ IHL (Fig. 2a,c).