Siu-Wah Chung, Xiao-Ying Huang, Jianlin Song, Rebecca Thomas and Peter MC Wong

Department of Pathology & Laboratory Medicine, Fels Institute for Cancer Research & Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140

FIGURES

Figure 1. Ad-RanT/n and Ad-RanC/d vector distribution and routes of inoculation. The vectors were inoculated into mice, i.v. or i.p., with a titer of 1x10¹¹ pfu per mouse. Two hours after vector administration, we extracted DNA from peritoneal cells (PM), bone marrow (BM), liver (LIV), peripheral blood (PB) and spleen (SPL) and performed PCR amplification using Ran-specific primers as described in Materials and Methods. The upper band, with a size of 158bp, indicates the viral Ran transgene (with 39nt HA epitope sequence inserted within either T/n or C/d sequence); the lower band, with a size of 119bp, shows the endogenous Ran sequence. The chart represents cumulative data from 6 mice and % viral band intensity is the intensity of the viral band versus intensity of both viral and endogenous bands in each lane. Standard deviation of the mean is shown on top of each bar as well.

Figure 2. Early difference in tissue distribution of vectors after intraperitoneal inoculation. Ad-RanT/n and Ad-RanC/d were injected i.p. into each mouse in groups of 4 mice, at 10¹¹ pfu/mouse. Genomice DNA from Peripheral blood (PB), peritoneal macrophages (PM), bone marrow (BM), livers (LIV) and spleens (SPL) were extracted at various indicated time after virus inoculation. About 0.2ug of extracted genomic DNA were subjected to competitive PCR analysis as previously described (11). Ten microliters of each PCR product was loaded onto 2% agarose gel. The upper band represents amplified viral Ran-HA DNA fragment with a size of 158bp; the lower band represents amplified endogenous Ran DNA fragment with a size of 119bp.

Figure 3. Histochemical analysis on liver biopsy. The hepatocytes around this central vein (Zone 3) show dropout with replacement by a mixed inflammatory cell infiltrate. There is congestion of the central vein and some damage to the endothelial cells. Different panels represent histological slides of livers of (a) untreated mice, (b) Ad-RanT/n-treated mice, (c) Ad-RanC/d-treated mice. Hematoxylin and eosin stain, magnification: 200x for all slides.

Figure 4. Plasma cytokine levels after vector administration. Groups of 6 mice were inoculated i.p. with PBS, Ad-LacZ, Ad-RanT/n or Ad-RanC/d at 1011 pfu/mouse. Peripheral blood was collected 1 and 8 hours after inoculation. Various cytokines were measured by standard ELISA assay as we described previously (11). Black squares = PBS controls; Black circles = Ad-LacZ; red triangles = Ad-RanT/n; blue triangles = Ad-RanC/d.

Figure 5. Prophylaxis against adenoviral inflammation and endotoxin challenge. Acclimatized mice were inoculated i.p. with Ad-RanT/n (open circles), Ad-RanC/d (open squares), or with PBS alone (closed triangles). Each vector was given at a titer of 5x10¹⁰pfu/mouse. Four days later, each mouse was challenged with 450ug of E. coli LPS (Sigma). The survival rate was monitored daily and experiment was terminated after 30 days. The number of mice in each experimental group (T/n and C/d mice) was n= 25; for control mice (PBS+LPS), n= 20. The dotted line at 100%-mark represents groups of 10 mice inoculated with Ad-RanT/n alone or Ad-RanC/d alone. None died.

Figure 6. Level of plasma pro-inflammatory cytokines. Groups of 20 mice were each given either Ad-RanT/n, Ad-RanC/d, i.p., at 5x10¹⁰ pfu/mouse, or with PBS. Four days later, all mice were challenged with 450ug of E coli LPS. At various time points afterwards, peripheral blood were collected from 4 mice per group and level of TNFa and IL6 in each mouse was measured by ELISA. Solid black triangles are values from mice with no vector but with LPS; open red circles, mice with Ad-RanT/n and LPS; open blue squares, mice with Ad-RanC/d and LPS. For plasma TNFa level at one-hour time point, the difference between Ad-RanT/n mice and Ad-RanC/d mice has a P value of <0.0016. For serum IL-6 at 2-hr time point, the difference between Ad-RanT/n mice and Ad-RanC/d mice has a P value of <0.008, and at 8-hr time point, the difference between mice with no virus and T/n mice has a P value of <0.0016.

Figure 7. Survival of mice after endotoxin challenge. Different shipments of CD-1 (filled square) and Balb/c mice (empty square) from two different vendors (Jackson Laboratory, Charles River Laboratory: labeled as C ) were challenged with different doses of LPS. Survival rates were monitored daily for a month. Each point represented at least 20 mice (ranged 20-25). Experimental points connected with dotted lines represented mice from the same shipment but tested with different doses of endotoxin in different experiments done within 10 days.

Figure 8. Plasma endotoxin levels in two different strains of mice from different shipments. Shortly upon arrival, blood samples were taken randomly from 10 mice (except for CD1-2, only 6 mice were tested). Endotoxin level was determined using LAL assay following manufacturer's protocol. Each data point represented one mouse entry shown with average in each group (horizontal line).