HD-Ad5/35 vector genome distribution and inflammatory reaction in hCD46tg mice. (A) HD-Ad–GFP (4 × 10¹⁰ vp) was IV injected into mobilized hCD46tg mice. Animals received dexamethasone (10 mg/kg) IP 16 hours and 2 hours before virus injection. Three days later, genomic DNA from tissue samples was analyzed for HD-Ad vector genomes using quantitative PCR with GFP-specific primers. Shown are vector copies per cell. N = 3. (B) Immunofluorescence analysis of liver sections at day 3 after vector injection into mobilized mice. Mice received either HD-Ad–GFP or first-generation Ad5-GFP at a dose of 4 × 10¹⁰ vp per animal. GFP appears in green, murine CD45 in red, and DAPI-stained nuclei in blue. The arrows in the middle panel indicate transduced blood cells present in a liver blood vessel. The scale bar is 50 μm. No specific feature within images shown in panel B was enhanced, obscured, moved, removed, or introduced. Previously, we and others have shown that Ad5 entry into hepatocytes is mediated by Ad5 hexon protein interaction with coagulation FX^26,27  and that this pathway is inefficient if Ad5 vectors contain the short Ad35 fibers (such as in the Ad5/35⁺⁺ vectors used here), most likely due to a steric block of the FX-interacting domains within the Ad5 hexon.²⁸  (C) Levels of serum alanine transaminase and aspartate transaminase at day 3 after Ad injection. N = 3. *P < .05, ****P < .0001 after one-way ANOVA with Bonferroni posttesting. (D) Serum levels of different cytokines, 6 hours after vector injection in mobilized animals. Animals had been mobilized as before and a total of 8 × 10¹⁰ vp HD-Ad–GFP was IV injected. Shown are mean ± SD. Dotted lines represent the detection limits of the different cytokines. Serum of unmobilized, uninjected animals was used as a control (untreated). N = 5. CTRL, control; FX, factor X; IFN, interferon; MCP, membrane cofactor protein; TNF, tumor necrosis factor.