PBS. Tumors were measured using a caliper, and tumor weight was calculated with the equation: tumor weight (in mg) = length (in mm) width (in mm2)/2 (22). On post-inoculation Day 0 and Day 14, blood was collected by retro-orbital sampling. On Day 28, the mice were sacrificed and blood was collected directly from the heart. Serum HBsAg and HBV DNA were analyzed as described subsequently. Determination of Serum Alanine Aminotransferase (ALT) and Serum HBsAg A blood sample was withdrawn from tumor-bearing ani- mals at the indicated times (Days 0, 14 and 28), and liver function was evaluated based on the levels of ALT. ALT activity was determined with the DTSC II Module detection system (Johnson-Johnson Co., USA) using the Vitros ALT/SGPT kit (Johnson-Johnson) as per the user manual. HBsAg levels in mouse serum were deter- mined semi-quantitatively by enzyme-linked immunosor- bent assay (ELISA; General Biologicals Corp., Taiwan, R.O.C.) according to the user manual. The cutoff value was calculated by the formula: cutoff value = ODnagative control + 0.025. An OD450 value higher than the cutoff value implies an HBV-positive result. Determination of HBV DNA by the Quantitative Real-time Polymerase Chain Reaction (PCR) Blood (500 ml) was withdrawn from test animals at the indicated times (Days 13 and 24). Blood was allowed to clot for 30 min and then centrifuged at 800g for 15 min at room temperature. Serum was collected and stored at 70 C prior to analysis of HBsAg and HBV DNA. Serum HBV DNA was extracted by a QIAamp DNA Blood Mini kit (Qiagen, USA). HBV DNA was quan- titated with the ABI 7500 Sequence Detection System using the HBV RealQuant PCR kit (General Biologicals Corp.). Briefly, PCR was performed with initial denatur- ing steps at 50 C for 2 min and 95 C for 10 min, followed by 45 cycles at 95 C for 15 s and annealing/extending at 58 C for 1 min. Validation of the Efficacy of BNE in HBV-viremia Animals For drug treatments, SCID mice were divided into five groups of eight mice each. Each animal was inoculated with 1 107 cells of HepG2 2.2.15 in 100 ml PBS on the flank subcutaneously; animals in the sham group received 100 ml PBS (Fig. 1 shows the experimental protocol). Tumors were allowed to grow, and blood was withdrawn on Day 13 prior to drug treatments. For drug treatments, 3TC (13 mg kg 1 day 1) was given by gavages, and BNE (195 mg kg 1 day 1) was administrated to animals via intraperitoneal and oral routes starting on Day 14 for eCAM 2010;7(2) 191 Figure 1. Scheme of the drug treatment protocol used in the HBV- viremia animal experiments. 10 consecutive days (for both 3TC and BNE). Tumor- bearing animals that were fed distilled water constituted the vehicle control group. All animals were sacrificed on Day 24, and the spleen was taken for NK cell activity analysis and blood was withdrawn to assess ALT, HBsAg and HBV DNA as described as earlier. All animals received human care, and the study protocol followed the guidelines of the Institutional Animal Care and Use Committees of the Development Center for Biotechnology. Assessment of the NK Cell Activity The spleen was taken from each drug-treated HBV- viremia mouse and then homogenized into a single cell suspension in Hank’s Balanced Salt Solution buffer (Gibco, USA) by passing through a 40 mm diameter nylon cell strainer (Becton Dickinson, USA). NK cell activity was measured using the LIVE/DEAD Cell- Mediated Cytotoxicity kit (Molecular Probes, USA). In brief, 1 106 YAC-1 cells were pre-labeled with 5 ml 3 mM DiOC18 fluorescent dye for 2 h, and excess dye was removed by washing with PBS. Then 1 106 of labeled YAC-1 cells were co-incubated with 2 106 splenocytes for 3 h, and 100 ml of 3.75 mM propidium iodine dye was added to the reaction at 4 C for 20 min avoiding the light. NK cell activity was analyzed by flow cytometry and was calculated with the equation: NK activity (%) = viability of treated group (%) – viability of blank group (%). Statistical Analysis Statistical analysis was performed using one-way ANOVA to analyze variances, and significant differences were assessed by Dunnett’s test for multiple comparisons. The paired t-test was used to assess significant differences before and after treatment in the same individuals. A P-value of <0.05 was considered statistically significant.