In this way, measurements of the Cr/C and D/V diameters were made. The transducer was then rotated 90 degrees and the procedure repeated. The second set of measurements included the L/R and a repeat of the D/V diameters. For calculating the mean tumor diameter, the PD173955? two separate measurements of the D/V diameter were averaged, followed by averaging the mean D/V diameter and the measurements of the Cr/C and L/R diameters. Tumor size also was determined by direct caliper measurements during laparotomy at week 0 and during necropsy at the end of the study. From woodchucks that were euthanized because of seizures, tumor measurements were also available during subsequent necropsy. The Cr/C, D/V, and L/R diameters were measured and averaged to calculate the mean tumor diameter.

Tumor size also was confirmed by CT prior to the start of the study, whenever possible between 6 and 10 weeks posttreatment, and at the end of the study at weeks 23 to 24. The tumor volume was calculated based on the diameters obtained by US using the formula V = ��abc/6, where a is the mean D/V diameter, b is the Cr/C diameter, and c is the L/R diameter. This formula was chosen because most large liver tumors (i.e., ��1.5 cm in diameter) in woodchucks are irregular and closely approximate an ellipsoid form. Analysis of WHV serum markers. Blood samples were obtained for WHV DNA analysis and serological testing prior to the start of the study; at week 0 prior to the administration of SFV-enhIL-12 or placebo; and then at 2, 4, 6, 10, 14, 18, and 23 to 24 weeks posttreatment.

WHV DNA in serum was determined quantitatively by dot blot hybridization (assay sensitivity, ��1.0 �� 107 WHV genome equivalents per ml (28). WHsAg in serum was determined qualitatively by enzyme-linked immunosorbent assay using dilutions of serum. (12) The cutoff value of this assay was defined as ��0.05 optical density unit. Analysis of T-cell responses. For determining T-cell responses against WHV, in vitro stimulators were used at concentrations previously determined as optimal for cultures of woodchuck peripheral blood mononuclear cells (PBMCs) (31, 32). Viral antigen stimulators consisted of native 22-nm WHsAg (2 ��g/ml) and synthetic peptides of WHsAg and WHcAg. WHs peptide S18 (10 ��g/ml; Sigma-Genosys, The Woodlands, TX) corresponded to amino acids 341 to 360 of WHsAg starting at the N terminus of the large surface protein (32).

WHc peptide C100-119 (10 ��g/ml; Sigma-Genosys) corresponded to amino acids 100 to 119 of WHcAg starting at the N terminus of WHcAg (31). Culture medium and a peptide unrelated to WHV (10 ��g/ml; Sigma-Genosys) were used as negative controls. Recombinant proteins that present tumor antigens in woodchuck liver were not available for Entinostat determining antitumoral T-cell responses.