title: Clostridium difficile toxin A induces intestinal epithelial cell apoptosis and damage: role of Gln and Ala-Gln in toxin A effects.
abstract: The aim of this study was to investigate the effect of Clostridium difficile toxin A (TxA) on intestinal epithelial cell migration, apoptosis, and transepithelial resistance and to evaluate the effect of glutamine (Gln) and its stable derivative, alanyl-glutamine (Ala-Gln), on TxA-induced damage. Migration was measured in rat intestinal epithelial cells (IEC-6) 6 and 24 hr after a razor scrape of the cell monolayer. Cell proliferation was indirectly measured utilizing the tetrazolium salt WST-1. The cells were incubated with TxA (1-100 ng/ml) in medium without Gln or medium containing Gln or Ala-Gln (1-30 mM). Apoptosis was quantified in IEC-6 cells using annexin V assay. Transepithelial resistance was measured using an epithelial voltohmmeter across T84 cells seeded on a transwell filter. TxA-induced a dose-dependent reduction of migration and also caused dose and time-dependent apoptosis in IEC-6 cells. Gln and Aln-Gln significantly enhanced IEC-6 cell migration and proliferation. Gln and Ala-Gln also prevented the inhibition of migration, apoptosis, and the initial drop in transepithelial resistance induced by TxA. In conclusion, both peptides reduced toxin-induced epithelial damage and thus might play an adjunctive role in C. difficile-induced colitis therapy.
authors.affiliation: Center for Global Health, Department of Intemal Medicine, School of Medicine, University of Virginia, Charlottesville, Virginia, USA.
authors.forename: Gerly A C
authors.initials: GA
authors.lastname: Brito
authors.forename: Benedito
authors.initials: B
authors.lastname: Carneiro-Filho
authors.forename: Reinaldo B
authors.initials: RB
authors.lastname: Oriá
authors.forename: Raul V
authors.initials: RV
authors.lastname: Destura
authors.forename: Aldo A M
authors.initials: AA
authors.lastname: Lima
authors.forename: Richard L
authors.initials: RL
authors.lastname: Guerrant
mesh_terms: D000818:Animals; D017209:Apoptosis; D001427:Bacterial Toxins / Q000008:administration & dosage / Q000494:pharmacology*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004151:Dipeptides / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004768:Enterotoxins / Q000008:administration & dosage / Q000494:pharmacology*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005973:Glutamine / Q000378:metabolism* / Q000494:pharmacology; D007422:Intestines / Q000187:drug effects / Q000473:pathology* / Q000503:physiopathology*; D009336:Necrosis; D051381:Rats; D013997:Time Factors
chemical_list: D001427:Bacterial Toxins; D004151:Dipeptides; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D005973:Glutamine; C054122:alanylglutamine