FAS engagement drives apoptosis of enterocytes of coeliac patients.
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BACKGROUND: Villus atrophy is the most distinctive sign of untreated coeliac disease (CD) and epithelial apoptosis is considered to be involved in this stage of the coeliac lesion. The extent of villus atrophy is, however, not homogeneous and patients with patchy or mild lesions have been described. AIMS: To address: (a) the degree of "patchiness" in untreated CD patients; and (b) to clarify if apoptosis, and eventually which trigger drives it, causes epithelial damage. PATIENTS: Twenty of 40 untreated, 14 treated coeliac patients, and 15 controls received five or more multiple duodenal biopsies; the remaining 20 untreated CD patients had no more than three biopsies. METHODS: All biopsies were analysed to monitor the presence of a "flat" mucosa. Biopsies of 14 untreated, 10 treated coeliacs, and seven controls were cultured with or without gliadin. DNA fragmentation was studied by terminal deoxynucleotidyl transferase (TdT) mediated dUTP digoxigenin nick end labelling (TUNEL), and FAS and Ki67 expression by immunohistochemistry. Antiendomysium antibodies (EMA) were surveyed in biopsy culture supernatants. RESULTS: A pattern of patchy duodenal lesions was observed in all untreated CD patients biopsied up to five times. High enterocyte FAS expression, and a high number of TUNEL+ and Ki67+ enterocytes were detected in areas with villus atrophy but not in those with a normal morphology (p<0.001). Conversely, EMA in culture supernatants and signs of immunological activation were present in all untreated CD biopsies. In vitro gliadin challenge increased the number of TUNEL+ and Ki67+ enterocytes (p<0.001 v cultures with medium alone) only in "flat" biopsies. Neutralising anti-FAS monoclonal antibodies were found to control gliadin induced enterocyte apoptosis (p>0.01) while agonist anti-FAS monoclonal antibody increased it (p<0.001). CONCLUSIONS: Patchy lesions are observed in untreated CD mucosa and epithelial FAS engagement is a key trigger in driving villus atrophy in CD. (+info)
Mycobacterium avium invades the intestinal mucosa primarily by interacting with enterocytes.
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Previous studies have demonstrated that Mycobacterium avium can invade intestinal epithelial cells both in vitro and in vivo. When given to mice orally, M. avium preferentially interacts with the intestinal mucosa at the terminal ileum. We evaluated the mechanism(s) of M. avium binding and invasion of the intestinal mucosa using three different systems: (i) electron microscopy following administration of M. avium into an intestinal loop in mice, (ii) quantitative comparison of the bacterial load in Peyer's patch areas of the terminal ileum versus areas that do not contain Peyer's patches, and (iii) investigation of the ability of M. avium to cause disseminated infection following oral administration using B-cell-deficient mice, lacking Peyer's patches, in comparison with C57BL/6 black mice. By all approaches, M. avium was found to invade the intestinal mucosa by interacting primarily with enterocytes and not with M cells. (+info)
Metabolic characterization of the major human small intestinal cytochrome p450s.
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Human small intestine epithelial cells (enterocytes) provide the first site for cytochrome P450 (CYP)-catalyzed metabolism of orally ingested xenobiotics. CYP3A4 is the major form of CYP expressed in enterocytes and CYP2C is also expressed at a significant level. In this study, we further characterized the expression of CYP3A4 and CYP2C in human enterocytes and their interindividual variations by examining the metabolic activities from 10 individuals. CYP3A4 in human jejunum microsomes, as determined by 6beta-testosterone hydroxylase activity, varied from 0.36 to 2.46 nmol/min/mg. The apparent average K(m) and V(max) values from two representative individuals were 54 microM and 3.2 nmol/min/mg, respectively. CYP2C9 and CYP2C19 in human jejunum microsomes, as determined by diclofenac 4'-hydroxylase and mephenytoin 4'-hydroxylase activities, varied over an 18-fold range (7.3-129 pmol/min/mg) and 17-fold range (0.8-13.1 pmol/min/mg), respectively. The mean apparent K(m) for diclofenac 4'-hydroxylase was 9.9 microM , whereas the apparent mean K(m) for S-mephenytoin 4'-hydroxylase was 79.3 microM . The mean intrinsic clearance (V(max)/K(m)) was approximately 130-fold greater for diclofenac 4'-hydroxylase than for mephenytoin 4'-hydroxylase. The metabolic activities of CYP2C9 and CYP2C19 were confirmed by inhibition by sulfaphenazole for CYP2C9 and ticlopidine for CYP2C19. In addition, CYP2C9 activities did not correlate with CYP3A4 activities, while CYP2C19 activities had a significant but poor correlation with those of CYP3A4. Thus the major CYP activities in human enterocytes have large interindividual variabilities that are not strongly related. (+info)
Na+/K+ATPase activity inhibition and isoform-specific translocation of protein kinase C following angiotensin II administration in isolated eel enterocytes.
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In the eel, angiotensin II (Ang II) has a role at the level of both gill chloride and kidney tubular cells, regulating sodium balance and therefore osmoregulation. The present study extends these findings to another important osmoregulatory organ - the intestine. Enterocytes were obtained from sea-water (SW)-acclimated eels to investigate the role of Ang II on the intestinal Na+/K+ATPase activity, because in SW-acclimated animals the intestine represents an important site of water and NaCl transport from the mucosal to the serosal side. This paper demonstrates that isolated enterocytes stimulated with increasing Ang II concentrations (0.01-100 nM) showed a dose-dependent inhibition of the Na+/K+ATPase activity. The threshold decrease was at 0.01 nM Ang II; it reached a maximum at 10 nM (81.5% inhibition) and did not decrease further with the use of higher hormone doses. These hormonal effects were blocked by a specific competitive antagonist of the AT1 receptor subtype, DuP-753 (100% inhibition at 10 microM), indicating that these effects are mediated by an AT1-like receptor. Isolated enterocytes stimulated with 10 nM Ang II showed a transient increase in intracellular calcium ([Ca2+]i), followed by a lower sustained phase. Removal of extracellular Ca2+ did not reduce the initial transient response and completely abolished the plateau phase. The inhibition of the Na+/K+ATPase activity was dependent on protein kinase C (PKC) activation since PKC antagonists (calphostin C and staurosporine) abolished the inhibitory effect of Ang II, and the PKC activator phorbol 12-myristate 13-acetate reduced transporter activity. Western blot analysis with antibodies to PKC alpha, beta I, beta II, gamma, delta, epsilon, iota, eta and zeta isoforms showed that eel enterocytes expressed the conventional isoforms (alpha and beta I), the novel isoforms (delta and eta) and the atypical isoforms (zeta and iota). Ang II stimulated the translocation from the cytosol to the plasma membrane of PKC alpha, PKC delta and PKC eta isoforms. In conclusion, our results suggest that Ang II modulates intestinal Na+/K+ATPase in SW-acclimated eels via calcium mobilization and PKC activation. (+info)
(+)-Catechin inhibits intestinal tumor formation and suppresses focal adhesion kinase activation in the min/+ mouse.
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Colorectal cancer is sensitive to dietary influences. Epidemiological data linking high intake of fruits and vegetables to decreased cancer risk have prompted the search for specific plant constituents implicated in tumor prevention. This task is difficult because of the complex chemical composition of plant foods and the multifactorial nature of carcinogenesis. Researchers are aided in this effort by the C57BL/6J-Min/+ (Min/+) mouse, an animal bearing a germline defect in Apc that is similar to the initiating genetic event in the majority of human colorectal cancers. In this study, we treated Min/+ mice with (+)-catechin, a phenolic antioxidant abundant in certain fruits. Administration of (+)-catechin in an AIN-76A diet at doses of 0.1 and 1% decreased the intestinal tumor number by 75 and 71%, respectively. Mechanistic studies linked this effect to (+)-catechin-induced changes in integrin-mediated intestinal cell-survival signaling, including structural alteration of the actin cytoskeleton and decreased focal adhesion kinase (FAK) tyrosine phosphorylation. Immunoblot analysis of small intestine scrapings from Min/+ mice and Apc+/+ wild-type C57BL/6J littermates together with excised Min/+ adenomas showed increased expression of phosphorylated FAK in the macroscopically normal enterocytes of untreated Min/+ mice and adenomas. Confirming the relevance of this signaling pathway, treatment of Min/+ mice with (+)-catechin reduced the expression of phosphorylated FAK to a level similar to the wild-type littermate controls. Thus, the natural abundance and favorable bioavailability of (+)-catechin make it a promising addition to the list of potential colorectal cancer chemopreventive agents. (+info)
In situ hybridization for the detection of transmissible gastroenteritis virus in pigs and comparison with other methods.
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Archived formalin-fixed, paraffin-embedded tissues from 25 pigs naturally infected with transmissible gastroenteritis virus (TGEV) were examined by in situ hybridization for TGEV nucleic acid using a nonradioactive digoxigenin-labeled cDNA probe that targeted the nucleocapsid sequence of TGEV strains. The results of in situ hybridization for the detection of TGEV were compared with virus isolation (VI), a fluorescent antibody test (FAT), and transmission electron microscopy (TEM). VI, FAT, and TEM were tested over a course of time before the in situ hybridization was performed. Positive hybridization signals were detected in duodenal, jejunal, and ileal enterocytes from 21 pigs. Hybridization signals were confined to the cytoplasm. Intestinal specimens from 25 piglets were evaluated by 4 tests. Twenty-one of 25 were positive by in situ hybridization. Of these 21 samples, 5 (24%) were positive for TGEV by all 4 tests, 15 (71%) were positive by FAT, 14 (67%) were positive by VI, and 6 (29%) were positive by TEM. In situ hybridization for the detection of TGEV in formalin-fixed, paraffin-embedded tissues provides a rapid means of confirmation of a histopathological diagnosis of TGEV without virus isolation, or when only formalin-fixed intestinal specimens were available. (+info)
An iron-regulated ferric reductase associated with the absorption of dietary iron.
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The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway. (+info)
Plesiomonas shigelloides enters polarized human intestinal Caco-2 cells in an in vitro model system.
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This study provides the first definitive evidence that the gram-negative bacterium Plesiomonas shigelloides adheres to and enters eukaryotic intestinal host cells in vitro. P. shigelloides is increasingly regarded as an emerging enteric pathogen and has been implicated in intestinal and extraintestinal infections in humans. However, the establishment of its true role in enteric disease has been hindered by inadequacies in experimental design, deficiencies in clinical diagnosis, and the lack of an appropriate animal model. In this investigation, an in vitro system was used to evaluate plesiomonad pathogenesis. Differentiated epithelium-derived Caco-2 cell monolayers inoculated apically with 12 isolates of P. shigelloides from clinical (intestinal) origins were examined at high resolution using transmission electron microscopy. Bacterial cells were observed adhering to intact microvilli and to the plasma membrane on both the apical and the basal surfaces of the monolayer. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. This observation suggests that initial uptake may occur through a phagocytic-like process, as has been documented for many other enteropathogens. P. shigelloides also was noted free in the cytosol of Caco-2 cells, suggesting escape from cytoplasmic vacuoles. Differences in invasion phenotypes were revealed, suggesting the possibility that, like Escherichia coli, P. shigelloides comprises different pathogenic phenotypes. (+info)