Human serum samples, containing high titers of genotype C HBV DNA (5.3 × 106 copies/mL), were obtained from patients with chronic hepatitis who provided written informed consent. Individual serum samples Ku 0059436 were divided into aliquots and stored in liquid nitrogen. Six weeks after hepatocyte transplantation,

chimeric mice were injected intravenously with 50 μL of HBV-positive human serum. DNA was extracted using SMITEST (Genome Science Laboratories, Tokyo, Japan) and dissolved in 20 μL of H2O. HBV DNA was measured by real-time polymerase chain reaction (PCR) using a light cycler (Roche, Mannheim, Germany). Primers used for amplification were 5′-TTTGGGCATGGACATTGAC-3′ and 5′-GGTGAACAATGTTCCGGAGAC-3′. Amplification conditions included initial denaturation at 95°C for 10 minutes, followed by 45 cycles of denaturation

at 95°C for 15 seconds, annealing IWR-1 molecular weight at 58°C for 5 seconds, and extension at 72°C for 6 seconds. The lower detection limit of this assay was 300 copies. PBMCs were isolated from healthy blood donors with HLA-A0201 and successfully vaccinated with recombinant yeast-derived hepatitis B surface antigen (HBsAg) vaccine (Bimmugen; Chemo-Sero Therapeutic Institute, Kumamoto, Japan) using Ficoll-Hypaque density gradient centrifugation. Neither monocytes nor macrophages were observed in the isolated PBMCs (Supporting Fig. 1). PBMCs isolated from 3 healthy, unvaccinated blood donors were also transplanted. Eight weeks after HBV inoculation, human PBMCs were transplanted into human hepatocyte chimeric mice. To deplete mouse NK cells and prevent

the elimination of human PBMCs from human hepatocyte chimeric mice, 200 μL of phosphate-buffered saline, containing 120 μL of anti–ganglio-N-tetraosylceramide (asialo GM1) antibody (Wako, Osaka, Japan), were administered this website intraperitoneally (IP) 1 day before (day 0; Fig. 1) the initial IP transplantation (day 1) of human PBMC. Then, 10 μL/g of liposome-encapsulated clodronate (Sigma-Aldrich, St. Louis, MO) were also administered 4 days before PBMC transplantation (day −2) to deplete mouse macrophages and DC cells. The second PBMC administration (4 × 107 cells/mouse) was performed 2 days after the initial administration (day 3). To assess the effect of the depletion of human DC, NK, or CD8-positive CTL cells from administered PBMCs on hepatitis formation, the BD IMag separation system (BD Biosciences, Franklin Lakes, NJ) was used. Alternatively, mice were treated with an IP administration of clodronate, as described above, 1 day before PBMC transplantation. To analyze the effect of inhibition of the Fas/FasL system, IFN-γ, IFN-α, antihuman FasL monoclonal antibody (mAb) (1.5 mg/mouse; R&D Systems, Minneapolis, MN), antihuman IFN-γ mAb (1.5 mg/mouse; R&D Systems), and antihuman IFN-α mAb (1.