Fig. 6.

Evidence for effects on adenosine on EPEC in vivo in ligated rabbit intestinal loops. Rabbits were subjected to laparatomy and 10 cm loops of ileum were ligated as described in Materials and Methods, then the loops were infected with human EPEC E2348/69 or rabbit EPEC strain E22. 19 to 20 h after infection, the rabbit was re-anesthetized, loop fluid was collected and the volume measured and bacterial cell counts were performed by dilution and plate counts on indicator agar. Because strain E22 is more virulent in the rabbit, the inoculum was reduced to 10⁷ cfu/loop for most experiments, or even lower on occasion (Fig. 6D). Human EPEC strain was inoculated at 10⁸ cfu/loop or occasionally even higher (Fig. 6D). Gene expression in vivo was also analyzed by quantitative RT-PCR. Panels A and B, effect of exogenous adenosine deaminase (ADA, 35 U/ml) on outcome of infection with REPEC E22 in vivo. Panel A, effect of ADA on fluid secretion, measured as the volume-to-length ratio; †, p not significant. Panel B, effect of ADA on the number of REPEC bacteria recovered from loop fluid; *, p < 0.001. Panel C, effect of ADA on the gross appearance of the intestinal fluids after low-speed centrifugation, showing bloodier fluids in the presence of ADA. Panels D–H, effect of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) on infection with E2348 and E22. Panel D, effect of EHNA on fluid secretion into the ligated rabbit ileal loops. Panel E, effect of EHNA on E22 bacterial CFUs recovered from loop fluid, * p < 0.03. Panels F and G, effect of EHNA on expression of espA and espB in loop fluid, normalized to that in the input inoculum (E22 grown in LB broth medium). *, significantly increased compared to in vivo without EHNA, by Student’s t-test.