This study is an investigation into the mechanism of Clostridium difficile toxin A-induced apoptosis in human intestinal epithelial cells. Toxin A induced apoptosis of T84 cells in a dose- and time-dependent fashion. Toxin A-induced apoptosis was completely inhibited by blocking toxin enzymatic activity on Rho GTPases with uridine 5'-diphosphate-2',3'-dialdehyde by a nonspecific caspase inhibitor and was partially inhibited by caspase-1, -3, -6, -8, and -9 inhibitors. Caspases 3, 6, 8, and 9 and Bid activation were detected. Toxin A also induced changes in mitochondrial membrane potential and cytochrome c release at 18-24 h, a time course similar to caspase-9 activation. In conclusion, toxin A induces apoptosis by a mechanism dependent on inactivation of Rho, activation of caspases 3, 6, 8, and 9 and Bid, and mitochondrial damage followed by cytochrome c release. Toxin A proapoptotic activity may contribute to the mucosal disruption seen in toxin A-induced enteritis.

Polymorphisms in the apolipoprotein E (APOE) have constituted the major rationale to identify potential risk groups for developing late-onset Alzheimer's disease and help to predict recovery of cognitive function after brain injury. However, the APOE impact on cognitive development in children living in poor areas of the developing world, where we have discovered profound significant associations of early childhood diarrhea (at 0-2 y) with lasting impairments of growth, cognition, and school performance, is not known. Therefore, we conducted APOE genotyping in 72 Brazilian shantytown children under active surveillance since birth, using purified DNA extracted from buccal cell samples. We found a high frequency of APOE4 alleles (18% versus 9-11% expected) in children with lower diarrhea burdens. When we examined the children who experienced the heavier diarrhea burdens (greater than or equal to the median of seven illnesses in the first 2 y of life), those with APOE4 did significantly better in the coding subtest (p=0.01) when compared with APOE4-negative children with similar diarrhea burdens. Positive correlations between the APOE4 occurrence and coding scores remained, even after adjusting for family income, maternal education, and breast-feeding. Moreover, the APOE4-positive group, under heavy burdens of diarrhea, had preserved semantic fluency and the mean difference in fluency scores, p=0.025, a standardized coefficient for disproportional verbal fluency impairment. Our findings show that APOE4 is relatively common in favela children and suggest a protective role of the APOE4 allele in children with a history of heavy burdens of diarrhea in their first 2 y of life.

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.

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The aim of this study was to evaluate the protective effect of hydrogen sulfide (H 2 S) on ethanol-induced gastric lesions in mice and the influence of ATP-sensitive potassium (K ATP ) channels, capsaicin-sensitive sensory afferent neurons, and transient receptor potential vanilloid (TRPV) 1 receptors on such an effect. Saline and L-cysteine alone or with propargylglycine, sodium hydrogen sulfide (NaHS), or Lawesson's reagent were administrated for testing purposes. For other experiments, mice were pretreated with glibenclamide, neurotoxic doses of capsaicin, or capsazepine. Afterward, mice received L-cysteine, NaHS, or Lawesson's reagent. After 30 min, 50% ethanol was administrated by gavage. After 1 h, mice were sacrificed, and gastric damage was evaluated by macroscopic and microscopic analyses. L-Cysteine, NaHS, and Lawesson's reagent treatment prevented ethanol-induced macroscopic and microscopic gastric damage in a dose-dependent manner. Administration of propargylglycine, an inhibitor of endogenous H 2 S synthesis, reversed gastric protection induced by L-cysteine. Glibenclamide reversed L-cysteine, NaHS, or Lawesson's reagent gastroprotective effects against ethanol-induced macroscopic damage in a dose-dependent manner. Chemical ablation of sensory afferent neurons by capsaicin reversed gastroprotective effects of L-cysteine or H 2 S donors (NaHS or Lawesson's reagent) in ethanol-induced macroscopic gastric damage. Likewise, in the presence of the TRPV1 antagonist capsazepine, the gastroprotective effects of L-cysteine, NaHS, or Lawesson's reagent were also abolished. Our results suggest that H 2 S prevents ethanol-induced gastric damage. Although there are many mechanisms through which this effect can occur, our data support the hypothesis that the activation of K ATP channels and afferent neurons/TRPV1 receptors is of primary importance.