Recurrent clostridium difficile.
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Clostridioides difficile↗
Biomedical subjects
J Thomas Lamont
Publications and source records attributed to J Thomas Lamont.
Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase.
BACKGROUND & AIMS: Clostridium difficile toxin A causes marked apoptosis of colonocytes in vivo and in vitro, which contributes to the formation of ulcers and pseudomembranes. We investigated the role of p53-dependent pathways and p38 mitogen-activated protein kinase (p38) in toxin A-induced colonocyte apoptosis. METHODS: The effects of the activation of p53 and p53-dependent pathways including p21(WAF1/CIP1) were assessed in nontransformed human colonic NCM460 epithelial cells exposed to toxin A. Phosphorylation of p53 protein by p38 was measured by in vitro kinase assay, whereas p21 induction by activated p53 was determined by gel shift assays and RNA silencing (small interfering RNA). The relationship between colonocyte apoptosis and p38/p53-dependent pathways was studied in intact mice. RESULTS: Toxin A stimulated p38 and p53 activation and induced cell cycle arrest (G(2)-M) with persistent expression of p21(WAF1/CIP1). Blockage of p38 by SB203580 inhibited p53 phosphorylation and induction of p21(WAF1/CIP1). In intact mice, p38 blockade suppressed toxin A-mediated destruction of intestinal villi, p21(WAF1/CIP1) expression, and enterocyte apoptosis. In addition, toxin A-mediated p21(WAF1/CIP1) and Bak induction, cytochrome c release, and caspase-3 activation were markedly attenuated in p53-silenced colonocytes, despite active p38. Overexpression of p21(WAF1/CIP1) triggered apoptosis and increased toxin A-associated colonocyte apoptosis. CONCLUSIONS: The signaling pathway for colonocyte apoptosis following toxin A exposure involves p38-dependent activation of p53 and subsequent induction of p21(WAF1/CIP1), resulting in cytochrome c release and caspase-3 activation through Bak induction.
Animals↗
Effect of chondroitinase ABC on purulent sputum from cystic fibrosis and other patients.
Cystic fibrosis (CF) patients develop chronic lung infections associated with airway obstruction by viscous and insoluble mucus secretions. Although mucus glycoproteins (mucins) are thought to be responsible for mucus plugs, other glycoconjugate components of airway secretions have not been systematically evaluated. The aim of the present study was to determine whether chondroitin sulfate proteoglycans (CSPG) contribute to the insolubility of CF sputum. Sputa obtained from 18 CF patients were incubated with chondroitinase ABC (ChABC) or buffer (control) for 18 h at 37 degrees C, and after centrifugation at 12,000 g, the volume of the insoluble pellet and turbidity of the supernatant were determined as measures of solubility. ChABC caused a 70-90% reduction in supernatant turbidity and a 60-70% decrease in pellet volume of the 13 purulent CF sputa, but had much less effect on the five nonpurulent CF sputa tested. Similar results were obtained with two non-CF purulent and two non-CF, nonpurulent sputa. Gel electrophoresis, Western blot, and slot blot immunoassays with antichondroitin sulfate and antimucin antibodies revealed that purulent sputa (CF and non-CF) contained more CSPG and less mucin than nonpurulent sputa. In vitro mixing experiments showed that mucin in nonpurulent sputa was reduced upon incubation with purulent sputa, presumably because of degradation or a loss of immunoreactive mucin epitopes from leukocyte and/or bacterial enzymes present in purulent sputa. Our results suggest that CSPG contribute more significantly than mucins to the insolubility of purulent tracheobronchial secretions from CF patients. Because purulent sputa from non-CF patients showed a similar pattern, our observations with CF sputa may have wider applicability.
Adult↗
Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction.
We determined whether the cytoprotective heat shock protein HSP72 protects against the injurious effects of Clostridium difficile toxin A (TxA) on intestinal epithelial cells. Colonic epithelial Caco-2/bbe (C2) cells were stably transfected with HSP72 antisense (C2AS) or vector only (C2VC), resulting in low and high HSP72 expression, respectively. Measurements of epithelial barrier integrity, mitochondrial function, and apoptosis activation were assessed after TxA exposure. HSP72 and RhoA interactions were evaluated with immunoprecipitations. In C2AS cells, TxA was associated with a greater decrease in transepithelial resistance (TER), an increase in [(3)H]mannitol flux, and increased dissociation of perijunctional actin. Although HSP72 binds RhoA, it failed to prevent RhoA glucosylation. TxA caused a more rapid decrease in ATP, release of cytochrome c, and activation of caspase-9 in C2AS cells. To determine whether ATP depletion decreases TER, we treated cells with antimycin A, which caused a decline in TER. We conclude that HSP72 may protect intestinal epithelial cells from TxA-mediated damage through several mechanisms, including actin stabilization, mitochondrial protection, and inhibition of apoptosis activation, but not by prevention of RhoA glucosylation.
Actins↗
Clostridium difficile toxin A triggers human colonocyte IL-8 release via mitochondrial oxygen radical generation.
BACKGROUND & AIMS: Clostridium difficile toxin A causes mitochondrial dysfunction resulting in generation of oxygen radicals and adenosine triphosphate (ATP) depletion. We investigated whether mitochondrial dysfunction is involved in nuclear factor kappaB (NF-kappaB) activation and interleukin (IL)-8 release from toxin A-exposed enterocytes. METHODS: NF-kappaB activation and IL-8 release in response to toxin A were correlated with reactive oxygen intermediate (ROI) generation and ATP production in HT-29 monolayers or HT-29 cells exposed to ethidium bromide (EB) to inhibit mitochondrial function. RESULTS: HT-29 cells exposed to EB showed damaged mitochondria and diminished resting levels of ATP. ROI production in EB-treated cells exposed to toxin A for 30 minutes was significantly reduced. Exposure of wild-type HT-29 cells to toxin A resulted in increased oxygen radical generation and IL-8 production (P < 0.01 vs. control) that was inhibited by antioxidant pretreatment. Degradation of IkappaB was observed within 30 minutes of toxin exposure, before ras homologue (Rho) glucosylation, and was followed by nuclear translocation of NF-kappaB. Toxin A did not increase IL-8 levels in EB-treated cells, whereas IL-8 release in response to IL-1beta was not affected. CONCLUSIONS: Our data support an early role for mitochondria-derived ROIs in stimulation of IL-8 release from colonocytes by toxin A. ROI generation is independent of Rho inactivation and involves nuclear translocation of NF-kappaB before release of IL-8.
Adenosine Triphosphate↗
Mechanism of lovastatin-induced apoptosis in intestinal epithelial cells.
We earlier showed that lovastatin potentiated the chemopreventive effects of sulindac against colon neoplasia in a rodent model and augments apoptosis induced by 5-FU and cisplatin in human colon cancer cells. In the present study, we investigated effects of lovastatin in spontaneously immortalized rat intestinal epithelial cells, IEC-18 and their K-ras transformed clones. Lovastatin induced morphologic changes (cell rounding and detachment) and apoptosis that were not influenced by K-ras mutations, but were prevented by geranylgeranyl-pyrophosphate or by mevalonate. Clostridium difficile toxin B, which directly inactivates rho, induced similar morphologic changes and apoptosis. Cycloheximide prevented these effects of lovastatin, but not C. difficile toxin B. Lovastatin decreased the amounts of membrane bound rhoA and rhoB. Cycloheximide and geranylgeranyl-pyrophosphate prevented lovastatin induced morphologic changes and apoptosis but did not inhibit lovastatin-induced changes in membrane translocation of rho. Our data suggest that lovastatin induces morphologic changes and apoptosis by inhibiting geranylgeranylation of small GTPases of the rho family and thereby inactivating them. Restoration of membrane translocation of rho is not necessary for preventing lovastatin-induced morphologic changes or apoptosis.
Animals↗
Theodore E. Woodward Award. How bacterial enterotoxins work: insights from in vivo studies.
Clostridium difficile is a spore forming, gram-positive anaerobic bacillus first described in 1935 by Hall and O'Toole as a commensal organism in the fecal flora of healthy newborn infants (1). The organism was given its unusual name because it grew slowly and was difficult to isolate in pure culture. Its presence in the stool of healthy neonates suggested that C. difficile was a nonpathogen, even though it produced toxins in broth culture. Following its original description, C. difficile passed quickly into relative obscurity in the 1960's and 1970's when antibiotic-associated pseudomembranous colitis became prevalent following the introduction into clinical practice of broad spectrum antibiotics. The frequent association of clindamycin and lincomycin therapy with pseudomembranous colitis led to the term "clindamycin colitis" (2). A breakthrough occurred in 1978 when C. difficile was identified as the source of a cytotoxin in the stool of patients with pseudomembranous colitis (3). During the two decades since its rediscovery, a great deal has been learned about the pathophysiology, epidemiology and management of C. difficile infection, yet many challenges remain. Currently this organism infects over 30% of individuals admitted to United States hospitals, making C. difficile colitis one of the most common nosocomial infections (4). It is estimated that approximately 10-12 million adults are infected with this organism each year in the United States, about a third of whom become symptomatic. The disease burden in the elderly is particularly severe as they are hospitalized more frequently and for longer duration. The pathophysiology of C. difficile diarrhea requires alteration of the colonic microflora by antibiotics, colonization by C. difficile, and release of two potent enterotoxins designated A and B (5). The toxins of Clostridium difficile are required virulence factors in both animals and humans since non-toxigenic strains do not cause disease. Recent cloning and sequencing of the toxin genes reveals extensive amino acid homology between them that is reflected in common molecular and cellular mechanisms. Both toxins damage cells by modifying the rho family of proteins, key regulators of cellular actin. C. difficile infection causes a florid acute inflammatory response seen in patients with pseudomembranous colitis. It is now realized that neurons and immune cells of the lamina propria are major determinants of toxin-induced diarrhea and mucosal damage. Early critical events following toxin exposure are release of the neuropeptides substance P and calcitonin gene related peptide (CGRP) from sensory afferent neurons and activation of lamina propria macrophages and intestinal mast cells. These peptides in turn release a complex cascade of other inflammatory mediators from lamina propria cells (5). The importance of the host immune response, specifically serum IgG directed against toxin A, is now recognized as a critical determinant of disease expression in man.
Actins↗