Fig. 3

Analysis of toxicity and transgene expression following repeated dosage of first generation and helper-dependent adenoviral vectors. (A) Increased hepatotoxicity following repeated doses of first-generation but not helper-dependent adenoviral vectors, as measured by liver enzyme levels. The experimental protocol for Figure 3 experiments is outlined below the graphs. Mouse genotypes along with their corresponding symbols and vector treatments are listed. Arrows indicate the time of vector delivery. The vectors delivered are shown below the arrows. Liver enzyme (ALT, AP, and AST) analysis for serum drawn at different times for each group of experimental animals. ALT = alanine aminotransferase; AP = alkaline phosphatase; AST = aspartate aminotransferase. Times of harvest of serum for liver enzyme analysis (always 1 day prior and 7 days subsequent to each vector injection indicated by arrows). The vectors used for each injection are indicated below the large arrows. All first generation vectors (AdJM17ΔE1, AdhAATΔE1, and AdβgalΔE1) were given by tail vein injection at 1 × 10⁹ PFU/mouse. AdSTK109 was administered by tail vein injection at 2 × 10¹⁰ particles/mouse. Significant increases in enzyme levels above pretreatment values for each group of animals are indicated by asterisks (*). The statistical method used was a one-way analysis of variance with the Bonferroni method to compare post-treatment values with pretreatment values. The Kruskal-Wallis one-way analysis of variance on ranks procedure was used when normality or equal variance tests failed, in which case, values were compared with pretreatment values using the Dunn method. (B) Levels of hAAT for groups of mice receiving first-generation vectors. The data are from the same mice shown in Figure 3A. Day 0 in panel 3B corresponds to day 68 in panel 3A, which corresponds to the day of administration of the vector AdhAATΔE1. Symbols shown correspond to mice and treatments as in panel 3A. The asterisk indicates a significant difference from the control group receiving only one dose of vector. Statistical analysis was as in Figure 2B. The percent expression on day 7, compared with day 3 is shown in parenthesis, as described in Figure 2B. (C) Evaluation of hepatocyte proliferation, as determined by Ki-67 staining (top panel), and hepatocyte hypertrophy, as determined by counting nuclei within a given area (bottom panel; as the degree of hypertrophy increases, the number of hepatocytes per field decreases). The mice are the same animals as described in Figure 3A, except for a new group of naive μMT mice receiving one dose of AdβgalΔE1 (seen in the fourth bar in the graphs). The mouse strains and the vectors used for each group are given below the columns. The number of times the animals received each vector is indicated by “X”. The number of Ki-67 positive hepatocytes was determined by counting Ki-67 positive nuclei in 10 high power (40 × objective) fields. The number of hepatocytes per field was determined by counting nuclei in seven fields of a reticle that has an area of 0.0156 mm². All mice were sacrificed 3 days after the final vector dose indicated in Figure 3A. Horizontal bars with an asterisk indicate values that were significantly different using one-way analysis of variance (p = 0.5) and the Student-Newman-Keuls method for multiple comparisons. (D) Levels of hAAT for μMT mice injected with AdSTK109. Mice and treatments are the same as described in Figure 3A for the group receiving AdSTK109. Arrows indicate time of vector administration. Error bars indicate standard deviation for all graphs in Figure 3. (E) Photomicrographs of heptocytes showing cell proliferation and hypertrophy after treatment with first-generation, but not helper-dependent vector. Panels 1–4 are hematoxylin and eosin stained sections (H & E) and panels 5–8 are stained for the cell proliferation marker Ki-67. Mouse genotypes and vector treatments are indicated below the panels. The group symbol corresponds to the groups as described in Figure 3A. All pictures are the same magnification and were originally taken at 400×. Examples of Ki-67 positive hepatocytes are indicated by small arrows in panels 6 and 7. Proliferating hepatocytes are not present in panels 5 and 8. The other positive cells seen in the Ki-67 stain are proliferating immune cells.