Ali, N.A.; Mahida, Y.R.
April 2003
Gut;Apr2003 Supplement 1, Vol. 52, pA50
Academic Journal
Introduction: Clinical presentation after C difficile infection can range from asymptomatic carriage to pseudomembranous colitis. C difficile induces diarrhoea and colonic inflammation via secreted toxins A and B. To induce disease, the toxins have to first interact with colonic epithelial cells. We have investigated the binding of C difficile toxin A to membrane preparations of primary human colonic epithelial cells. Methods: Primary colonic epithelial cells from different individuals (A to D) were isolated by treatment with EDTA and used for membrane preparations (confirmed by electron microscopy). Caco2 cell membrane preparations were also obtained (and shown to be enriched for sucrase-isomaltase and alkaline phosphatase). Purified C difficile toxin A was label[ed with tritium [³H] using the Bolten-Hunter reagent N-succinimidy] [2,3-³H] propionate. Binding studies were carried out using purified epithelial membrane preparations and [³H]-toxin A. Specific binding was calculated by subtracting non-specific binding in the presence of 1000-fold excess unlabelled toxin A. Results: Biological activity of [³H]-toxin A was confirmed using Vero cells. In studies using membrane preparations from different individuals, binding of [³H]-toxin A varied markedly (binding per mcg protein, expressed as % of binding to Caco2 membrane preps: A 25.7%, B-32.8%, C-184.9% and D-0.8%). Using pooled samples of membrane preparations, specific binding of [³H]-toxin A occurred at 4°C, 19 °C, and 37 °C. The binding achieved saturation in the presence of increasing concentrations of [³H]-toxin A, and reached equilibrium within 60 min at 4°C. Conclusions: The specificity of [³H]-toxin A binding to membrane preparations of colonic epithelial cells and the ability to saturate this binding suggests involvement of receptor-ligand interaction. Variation in the binding of [³H]-toxin A to membrane preparations from different...


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