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liv12571-sup-0001-FigS1.tifimage/tif2607KFig. S1. Depletion of Kupffer cells by clodronate liposomes. Balb/c mice were treated or not with intravenous clodronate liposomes (2 μl/g of animal weight) and sacrificed 24 h later. Immunohistochemical detection of Kupffer cells in liver sections using anti-F4/80 antibody. Microphotographs show representative fields at 200× (left) and 400× (right) magnification.
liv12571-sup-0002-FigS2.tifimage/tif1034KFig. S2. Long-term stabilization of hepatic transgene expression by clodronate liposomes. Mice received an intravenous injection of Ad-CMV-Luc (109 iu/mouse) at day 0 of the experiment. One subset of animals (n = 5) was treated with clodronate liposomes 24 h before infection (Clod group), whereas other was left untreated (Cont. group). Evolution of transgene expression analysed by BLI. The graph shows relative light emission for each group (average fold of photons/s), using as a reference the initial (day 1) luminescence in the control group. Differences between groups were significant (P = 0.006).
liv12571-sup-0003-FigS3.tifimage/tif1516KFig. S3. Lack of anti-Ad humoral response in µMT mice. Wild type C57BL/6J mice or µMT knock-out mice lacking B cells (n = 6) received an intravenous injection of Ad-CMV-Luc (108 iu/mouse) at day 0 of the experiment. (A) Evolution of transgene expression analysed by BLI. The graph shows relative light emission for each group, using as a reference the initial (day 1) luminescence in the wild type group (in this case, 1.87 × 107 photons/s). (B) At the end of the observation period, all mice received an intravenous injection of the Ad.hIL12 virus, and the presence of hIL-12 was detected in serum 48 h later by ELISA. The graph represents the relative concentration of the cytokine, using an age-matched group of wild type or mutant naïve animals as a reference. (C) Presence of anti-Ad NAbs in the serum of the indicated groups before the second viral administration.
liv12571-sup-0004-FigS4.tifimage/tif1010KFig. S4. Transient CD4+ T-cell depletion stabilizes vector genome copy numbers in the liver. Balb/c mice received an intravenous injection of Ad-CMV-Luc (109 iu/mouse) at day 0 of the experiment. One group of animals (n = 10) was assigned to the CD4-depleted group, whereas other group received the same total dose of unspecific rat IgG (Cont). The complete depletion protocol consisted on intraperitoneal injections of 100 µg anti-CD4 mAb at days −6, −2, 5, 12, 19 and 27. A subset of mice from each group (n = 5) was sacrificed 5 or 30 days after vector administration, and viral copy numbers were quantified in liver samples by qPCR. The graph represents copy numbers per milligram of liver tissue.
liv12571-sup-0005-FigS5.tifimage/tif1134KFig. S5. Transient CD4+ T-cell depletion does not increase adenovirus-mediated liver damage. Balb/c mice received an intravenous injection of Ad-CMV-Luc (109 iu/mouse) at day 0 of the experiment. Different subsets of animals (n = 7) were treated with 100 µg of anti-CD4 or anti-CD8 mAbs by intraperitoneal injection at days −6, −2, 5, 12, 19 and 27. Other group received the same total dose of unspecific rat IgG (Cont). Blood collection was performed at the indicated days for determination of liver enzymes aspartate and alanine aminotransferases (AST and ALT, panels A and B respectively). Average normal values for each parameter are indicated with a dotted line.
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