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Infection and Immunity, January 2006, p. 81-87, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.81-87.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Caspase and Bid Involvement in Clostridium difficile Toxin A-Induced Apoptosis and Modulation of Toxin A Effects by Glutamine and Alanyl-Glutamine In Vivo and In Vitro

Benedito A. Carneiro,^1, Jun Fujii,² Gerly A. C. Brito,³ Cirle Alcantara,¹ Reinaldo B. Oriá,^1,3 Aldo A. M. Lima,⁴ Tom Obrig,² and Richard L. Guerrant^1*

Center for Global Health,¹ Division of Nephrology, University of Virginia, Charlottesville, Virginia,² Department of Morphology,³ Institute of Biomedicine and Clinical Research Unit, University Hospital, Federal University of Ceará, Fortaleza, CE, Brazil⁴

Received 10 October 2004/ Returned for modification 26 November 2004/ Accepted 22 September 2005

Clostridium difficile is the leading cause of nosocomial bacterial diarrhea. Glutamine and its stable and highly soluble derivative alanyl-glutamine, have been beneficial in models of intestinal injury. In this study, we extend our work on the mechanisms of Clostridium difficile toxin A (TxA)-induced apoptosis in human intestinal epithelial T84 cells and evaluate the effects of glutamine and alanyl-glutamine on TxA-induced apoptosis in vitro and disruption of ileal mucosa in vivo. T84 cells were incubated with TxA (100 ng/ml) in medium with or without glutamine or alanyl-glutamine (3 to 100 mM). Apoptosis was evaluated by DNA fragmentation in vitro and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method in vivo. Caspase and Bid involvement were investigated by Western blotting. Ligated rabbit ileal loops were used for the evaluation of intestinal secretion, mucosal disruption, and apoptosis. TxA induced caspases 6, 8, and 9 prior to caspase 3 activation in T84 cells and induced Bid cleavage by a caspase-independent mechanism. Glutamine or alanyl-glutamine significantly reduced TxA-induced apoptosis of T84 cells by 47% and inhibited activation of caspase 8. Both glutamine and alanyl-glutamine reduced TxA-induced ileal mucosal disruption and secretion. Altogether, we further delineated the apoptosis-signaling cascade induced by TxA in T84 cells and demonstrated the protective effects of glutamine and alanyl-glutamine. Glutamine and alanyl-glutamine inhibited the apoptosis of T84 cells by preventing caspase 8 activation and reduced TxA-induced intestinal secretion and disruption.

Editor: D. L. Burns

Present address: Evanston Northwestern Healthcare, Evanston, Ill.

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