Fig. 4. Ad5-3M virus escapes sequestration in liver macrophages and fails to trigger hepatotoxicity and inflammatory cytokine activation after systemic delivery.

(A) Integrated density of virus-positive staining within CD68- and F4/80-positive cells for indicated viruses on liver sections at 1 h post virus injection, normalized to the median integrated density of Ad5-WT virus staining (n = 8-12). (B) Comparison of virus DNA copies in Kupffer cells by qPCR analysis for indicated viruses 30 min post intravenous virus injection (n = 3). (C) Graphical representation and (D) immunofluorescent staining of liver sections showing in vivo necrosis (PI permeability, red) of CD68-positive Kupffer cells (green) after administration of mice with indicated viruses. DAPI staining of the nuclei is in blue. Arrows point to the PI-positive necrotic Kupffer cells (n = 2-3). (E) Accumulation of viral DNA in livers of mice 1 hour after intravenous virus administration, when compared to the total injected dose for indicated viruses (n = 3-6). (F) Activity of virus-encoded nano-luciferase in liver lysates at the indicated time points (n = 3). Liver function was evaluated by measuring alanine aminotransferase (ALT) (G), and aspartate aminotransferase (AST) (H) in mouse serum 48 h post virus injection (n = 4-6). (I) Comparison of cytokine and chemokine concentration in the spleens of mice (pg or ng per 130mg of spleen tissue) at 1 h post intravenous virus injection (n = 4-6). (J) Amounts of human TNF-α and IL-6 released from primary human macrophages 72 h after their incubation with indicated viruses (n = 4). Bars show mean ± SD. The p-values of one-way ANOVA with multiple comparison tests are shown above the bars, the color of the p-value number indicates the comparison partner. The methodology for individual settings are as described in Table S1 and Methods.