Toxin Mediated Diarrhea in the 21st Century: The Pathophysiology of Intestinal Ion Transport in the Course of ETEC, V. cholerae and Rotavirus Infection

An estimated 4 billion episodes of diarrhea occur each year. As a result, 2–3 million children and 0.5–1 million adults succumb to the consequences of this major healthcare concern. The majority of these deaths can be attributed to toxin mediated diarrhea by infectious agents, such as E. coli, V. cholerae or Rotavirus. Our understanding of the pathophysiological processes underlying these infectious diseases has notably improved over the last years. This review will focus on the cellular mechanism of action of the most common enterotoxins and the latest specific therapeutic approaches that have been developed to contain their lethal effects

The role of ClC-2 in intestinal chloride secretion

grantor: University of TorontoCystic Fibrosis (CF) is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. Recent studies indicate, however, that additional modifier genes determine intestinal disease severity in both, CFTR knockout mice (CF mice) as well as in CF patients. As the principal function of CFTR is chloride secretion, amelioration of disease severity in the small intestine may reflect compensation by an alternative chloride channel. This thesis assessed the potential of the ClC-2 chloride channel for mediating small intestinal chloride secretion. Immunofluorescent localization and immunogold electronmicroscopy showed that the ClC-2 chloride channel localizes to the apical epithelial pole in the small intestine, primarilly at the tight junction membrane and diffusely to the apical membrane. This localization is consistent with a role in secretion. Wild type mice and CF mice exhibited comparable ClC-2 message levels as well as similar localization pattern of ClC-2 in the small intestine. Electrophysiological studies, using the Ussing chamber technique, revealed that CF mice as well as their wild type siblings exhibit a ClC-2-like secretory chloride current in the small intestine. Further, this current was found to be elevated in the intestine of CF mice with mild intestinal disease when compared to their severely affected siblings, suggesting that ClC-2 mediated chloride secretion may functionally compensate for CFTR function in the small intestine of CF mice with mild disease. To assess the potential of ClC-2 in mediating chloride secretion in the human small intestine, the Caco-2 cell line was utilized as a model epithelium. As in the murine small intestine, Caco-2 cells were found to express ClC-2 near the apical epithelial pole, specifically at the apical aspect of the tight junction membrane. In addition, voltage clamp studies revealed the presence of an endogenous ClC-2-like secretory current in this cell line. The studies presented in this thesis suggest that ClC-2 likely mediates chloride secretion in the murine and human small intestine. Further, elevated chloride secretion by the ClC-2 chloride channel may account for the amelioration of small intestinal disease in mildly affected CF mice. Thus, enhancing the activity of ClC-2 may provide a novel avenue for circumventing the CF defect in the intestine.Ph.D

Temporal expression of prostaglandin endoperoxide H synthase type 2 and prostaglandin receptors at birth

grantor: University of TorontoParturition in the sheep is preceded by increased levels of prostaglandins (PGs) in intrauterine tissues as well as maternal and fetal plasma, reflecting an important role for PGs in the processes leading to the initiation of ovine labour. In order to elucidate the importance of PGs in the mechanisms regulating parturition in the sheep, we examined the pattern of expression of prostaglandin H synthase type 2 (PGHS-2) and prostaglandin receptors for PGE\sb2 and PGF\sb{\alpha 2}, in ovine intrauterine tissues with the onset of labour. We found that PGHS-2 mRNA and protein expression displayed a temporal and tissue specific pattern of regulation, increasing initially in the fetal placental trophoblasts followed by an increase in its expression in the maternal endometrial epithelium and myometrium. On the contrary, receptors for PGE\sb2 (EP-1, EP-2, EP-3, EP-4) and for PGF\sb{2\alpha} (FP) did not display any change in mRNA expression in placentomes or the myometrium, although they were present in these tissues during the initiation of parturition. These results suggest that an increase in intrauterine prostaglandin synthetic capacity, rather than an increase in the upregulation of prostaglandin receptor gene expression, is important in the causal role of PGs in the processes leading to the initiation of ovine parturition.M.Sc

Murine mCLCA6 Is an Integral Apical Membrane Protein of Non-goblet Cell Enterocytes and Co-localizes With the Cystic Fibrosis Transmembrane Conductance Regulator

The CLCA family of proteins consists of a growing number of structurally and functionally diverse members with distinct expression patterns in different tissues. Several CLCA homologs have been implicated in diseases with secretory dysfunctions in the respiratory and intestinal tracts. Here we present biochemical protein characterization and details on the cellular and subcellular expression pattern of the murine mCLCA6 using specific antibodies directed against the amino- and carboxy-terminal cleavage products of mCLCA6. Computational and biochemical characterizations revealed protein processing and structural elements shared with hCLCA2 including anchorage in the apical cell membrane by a transmembrane domain in the carboxy-terminal subunit. A systematic light- and electron-microscopic immunolocalization found mCLCA6 to be associated with the microvilli of non-goblet cell enterocytes in the murine small and large intestine but in no other tissues. The expression pattern was confirmed by quantitative RT-PCR following laser-capture microdissection of relevant tissues. Confocal laser scanning microscopy colocalized the mCLCA6 protein with the cystic fibrosis transmembrane conductance regulator CFTR at the apical surface of colonic crypt cells. Together with previously published functional data, the results support a direct or indirect role of mCLCA6 in transepithelial anion conductance in the mouse intestine. (J Histochem Cytochem 56:495–509, 2008

Glucocorticoid Regulation of Human and Ovine Parturition: The Relationship Between Fetal Hypothalamic-Pituitary-Adrenal Axis Activation and Intrauterine Prostaglandin Production

International audienceBirth in many animal species and in humans is associated with activation of hypothalamic-pituitary-adrenal function in the fetus and the increased influence of glucocorticoids on trophoblast cells of the placenta and fetal membranes. We suggest that in ovine pregnancy glucocorticoids directly increase fetal placental prostaglandin production, and indirectly increase prostaglandin production by maternal uterine tissues through the stimulation of placental estradiol synthesis. The events of ovine parturition are compared with those of human parturition. In the latter, we suggest similar direct effects of glucocorticoids on prostaglandin synthesis and metabolism in fetal membranes and similar indirect effects mediated by glucocorticoid-stimulated increases in intrauterine corticotropin-releasing hormone expression