Permeability to [3H]mannitol increased 16-fold after exposure to 32 nM of toxin A and to 3 nM of toxin B when compared with controls (P < 0.05). Light and scanning electron microscopy after exposure to either toxin revealed patchy damage and exfoliation of superficial epithelial cells, while crypt epithelium remained intact. Fluorescent microscopy of phalloidin-stained sections showed that both toxins caused disruption and condensation of cellular F-actin. Our results demonstrate that the human colon is -10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important than toxin A in the pathogenesis of C. difficUe colitis in man. (J.
Commensal and enteroinvasive microbes in the human gut release bacterial flagellin, a specific microbial ligand of Toll-like receptor 5 (TLR5). However, the pathophysiological role of bacterial flagellin in gastrointestinal inflammation has not been determined. Here we evaluated the role of bacterial flagellin using native human colonic mucosa and the mouse colitis model of dextran sulfate sodium (DSS). We demonstrate that, in intact human colonic mucosa, the flagellin͞TLR5 response occurs only after exposure to the basolateral, not the apical, surface, implying a basolaterally polarized TLR5 response in human colonic mucosa. In this context, flagellin exposure to injured colonic mucosa due to DSS administration in mice resulted in a TLR5-associated response evaluated by in vivo activation of mitogen-activated protein kinase͞extracellu-lar signal-related kinase 1͞2 (MEK1͞2) and elevated IL-6, TNF-␣, and keratinocyte-derived chemokine production, whereas intact colonic mucosa did not respond to flagellin. Moreover, flagellin exposure to injured mouse colon in vivo, but not to intact colon, also significantly aggravated colonic inflammation, increased mouse mortality, and enhanced histopathological damage in the colonic mucosa. However, the TLR2-specific agonist, peptidoglycan or lipoteichoic acid, did not cause an inflammatory response in intact or DSS-injured mouse colon. Furthermore, intracolonic flagellin administration in mice causes severe apoptosis in colonic epithelium disrupted by DSS administration. These data suggest that intracolonic flagellin via TLR5 engagement is able to elicit inflammatory responses in disrupted colon, whereas the normal colon is not responsive to bacterial flagellin. These results demonstrate that bacterial flagellin plays an important role in the development and progress of colitis.innate immunity ͉ colitis ͉ commensal bacteria
Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibitsC. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardiion toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects ofS. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii onC. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.
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