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C Pothoulakis

Publications and source records attributed to C Pothoulakis.

At least 37 records · Page 2Linked to original sources
PubMed1996

A receptor decoy inhibits the enterotoxic effects of Clostridium difficile toxin A in rat ileum.

BACKGROUND & AIMS: Clostridium difficile toxin A causes secretion and intestinal inflammation in rodents by binding to a specific trisaccharide Gal alpha 1-3Gal beta 1-4 GlcNAc on enterocyte receptors. The purpose of this study was to explore the ability of Synsorb 90 (Synsorb Biotech Inc., Calgary, Alberta, Canada), and inert support carrying this trisaccharide, to bind toxin A in vitro and to inhibit its enterotoxic effects in vivo. METHODS: Binding of [3H]toxin A to Synsorb 90, Synsorb 83 (beta-mannose attached), and Chromosorb P (inert support with no sugar attached) (Synsorb Biotech Inc.) was measured. The inhibitory effects of these compounds on toxin A-mediated fluid secretion, mannitol permeability, and histological damage were measured in ileal loops in vivo. RESULTS: Toxin A showed specific binding to Synsorb 90, bearing the specific trisaccharide that binds toxin A, but not to Synsorb 83 or to Chromosorb P. Pretreatment of rats with Synsorb 90 by gavage (200 mg/kg body wt), but no Synsorb 83 or Chromosorb P at the same doses, dramatically reduced toxin A-associated fluid secretion and permeability. CONCLUSIONS: An immobilized toxin A receptor sequesters toxin A in the intestinal lumen and inhibits its effects of ileal mucosa. These results suggest a potential use for this agent in treating patients with C. difficile colitis.

Animals↗
PubMed1996

Pathogenesis of Clostridium difficile-associated diarrhoea.

Clostridium difficile is now regarded as a major enteric pathogen in hospitals and nursing-home facilities. The pathophysiology of this pathogen involves alterations of the indigenous colonic flora by antibiotics, ingestion of spores and colonization by C. difficile, followed by release of its toxins. Although most of the research on the intestinal effects of C. difficile had been focused on its enterotoxin or toxin A, recent results indicate that toxin B, the cytotoxin of C. difficile, is also active in human colon. The cloning and sequencing of the toxin A and toxin B gene and the identification of the GTP-binding protein Rho as their intracellular target represent major advances in our understanding of the mode of action of these toxins. An important characteristic of C. difficile infection is the dramatic inflammation seen in pseudomembranous colitis. Recent studies indicate that an interplay between lamina propria neuroimmune cells and intestinal epithelial cells may be central in pathogenesis of this toxin-mediated inflammatory response.

Bacterial Toxins↗
PubMed1996

Anti-Clostridium difficile bovine immunoglobulin concentrate inhibits cytotoxicity and enterotoxicity of C. difficile toxins.

Clostridium difficile diarrhea and colitis result from the actions of bacterial exotoxins on the colonic mucosa. This study examined the ability of hyperimmune bovine colostral antibodies to neutralize the biological effects of these toxins. Anti-C. difficile bovine immunoglobulin concentrate was prepared from the colostral milk of Holstein cows previously immunized with C. difficile toxoids. The anti-C. difficile bovine immunoglobulin concentrate contained high levels of bovine immunoglobulin G specific for C. difficile toxins A and B, as evaluated by enzyme-linked immunosorbent assay. Anti-C. difficile bovine immunoglobulin concentrate neutralized the cytotoxic effects of purified toxin A and toxin B on cultured human fibroblasts, whereas control bovine immunoglobulin concentrate had little toxin-neutralizing activity. Anti-C. difficile bovine immunoglobulin concentrate also blocked the binding of toxin A to its enterocyte receptor and inhibited the enterotoxic effects of C. difficile toxins on the rat ileum, as measured by an increased rat ileal loop weight/length ratio (63% inhibition; P < 0.01), increased mannitol permeability (92% inhibition; P < 0.01), and histologic grading of enteritis (P < 0.01 versus nonimmune bovine immunoglobulin concentrate). Thus, anti-C. difficile bovine immunoglobulin concentrate neutralizes the cytotoxic effects of C. difficile toxins in vitro and inhibits their enterotoxic effects in vivo. This agent may be clinically useful in the prevention and treatment of C. difficile diarrhea and colitis.

Animals↗
PubMed1996

Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans.

Animals↗
PubMed1996

Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells.

We determined the effects of immobilization stress on rat colonic mucus release and mast cell degranulation and examined whether corticotropin releasing factor (CRF) was involved in these responses. After 30-min immobilization, rats were killed, colonic mucosal explants were cultured, and levels of rat mast cell protease II (RMCP II) and prostaglandin E2 (PGE2) were measured. Mucin release from explants was assayed by incorporation of [3H]glucosamine into colonic mucin and by histological evaluation of goblet cell depletion. Stress caused significant increases of colonic RMCP II, PGE2, and mucin release and fecal pellet output and caused an approximately 10-fold increase in colonic mucosal levels of cyclooxygenase-2 (COX-2) mRNA. These stress-associated changes were reproduced by intravenous or intracerebral injection of CRF in conscious, nonstressed rats. Pretreatment of rats with the CRF antagonist alpha-helical-CRF9-41, hexamethonium, atropine, or bretylium, or the mast cell stabilizer lodoxamide inhibited stress-induced release of RMCP II, PGE2, and mucin, whereas indomethacin prevented mucin release but not mast cell degranulation. Hexamethonium and CP-96,345, a substance P antagonist, inhibited fecal pellet output caused by stress. We conclude that CRF released during immobilization stress increases colonic transit via a neuronal pathway and stimulates colonic mucin secretion via activation of neurons and mast cells.

Animals↗
PubMed1995

Effects of purified Clostridium difficile toxin A on rabbit distal colon.

BACKGROUND & AIMS: Antibiotic-associated pseudomembranous colitis in humans is caused by proliferation of Clostridium difficile, which elaborates an enterotoxin toxin A that causes epithelial damage and altered motility in rabbit small intestine. The aim of this study was to assess the effects of toxin A on rabbit distal colonic motility and to relate this to histological damage and inflammatory mediator production. METHODS: Two hundred micrograms per milliliter of toxin A was placed in a distal colonic loop in anesthetized rabbits, and myoelectric activity was recorded for the following 7 hours. The colon was histologically evaluated and assayed for eicosanoid production. The effects of toxin A on longitudinal and circular muscle were also assessed in vitro. RESULTS: Beginning 1 hour after instillation, toxin A caused a significant increase in the number of spike bursts without altering slow wave frequency; this was associated with an increase in mucosal neutrophils and increased production of prostaglandin E2 and leukotrienes B4 and C4/D4/E4. Seven hours after administration of toxin A, mediator levels and myoelectric activity remained increased but significant mucosal damage was now also present. Toxin A did not affect longitudinal or circular muscle in vitro. CONCLUSIONS: C. difficile toxin A caused a significant neutrophil infiltration and an increased myoelectric activity before producing mucosal damage. The myoelectric effect may be indirect, resulting from the production of motility-altering arachidonic acid metabolites.

Animals↗
PubMed1995

Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein.

BACKGROUND & AIMS: Goblet cells secrete a combination of trefoil peptides and mucin glycoproteins to form a continuous gel on the mucosal surface. The functional effects of these products remain uncertain. METHODS: Trefoil peptides and/or mucin glycoproteins were added to Transwell monolayers of the human colonic cancer-derived T84 cell line. Intact monolayers permitted penetration of < 4% of the inert marker [3H]mannitol at 4 hours. Exposure to the toxic lectin phytohemagglutinin (1 mg/mL), oleic acid (8 mmol/L) and taurocholic acid (12 mmol/L), or Clostridium difficile toxin A (0.7 microgram/mL) resulted in loss of barrier function with 36%, 62%, and 45% of [3H]mannitol penetration, respectively. RESULTS: Addition of recombinant human intestinal trefoil factor in physiological concentrations (1-5 micrograms/microL) resulted in attenuation of the damage to monolayer integrity by up to 52%. Protection was enhanced (up to 95%) by the copresence of human colonic mucin glycoproteins. Similar effects were observed when rat intestinal trefoil factor or human spasmolysin, another human trefoil peptide, were added alone or in the presence of human mucin glycoproteins. Conversely, mucin glycoproteins isolated from the rat colon or stomach facilitated protection when added with human spasmolysin or human intestinal trefoil factor. CONCLUSIONS: Trefoil peptides and mucin glycoproteins protect gastrointestinal mucosa from a variety of insults.

Bacterial Toxins↗
PubMed1995

Involvement of Ras-related Rho proteins in the mechanisms of action of Clostridium difficile toxin A and toxin B.

Toxins A and B of Clostridium difficile are responsible for pseudomembranous colitis, a disease that afflicts a substantial number of hospitalized patients treated with antibiotics. A major effect of these proteins is the disruption of the actin cytoskeleton. Recently, I. Just, G. Fritz, K. Aktories, M. Giry, M. R. Popoff, P. Boquet, S. Hegenbarth, and C. von Eichel-Streiber (J. Biol. Chem. 269:10706-10712, 1994) implicated Rho proteins as cellular targets of C. difficile toxin B, since pretreatment of cells or purified Rho with toxin prevented subsequent ADP-ribosylation of Rho by exoenzyme C3. Moreover, they showed that overexpression of Rho proteins in cells suppressed cell rounding normally associated with exposure of cells to C. difficile toxin B. Here we expand these findings by showing directly that Rho proteins are covalently modified by both C. difficile toxins A and B. In addition, we demonstrate that the stability of toxin-modified Rho in NIH 3T3 cells is dramatically reduced. Finally, we show that C. difficile toxins A and B do not have similar effects on the closely related Rac and CDC42 GTP-binding proteins.

3T3 Cells↗
PubMed1995

Clostridium difficile toxin B activates calcium influx required for actin disassembly during cytotoxicity.

The principal cellular response to Clostridium difficile toxin B, a protein toxin associated with antibiotic-associated colitis, is the disassembly of actin microfilaments. Although receptor-activated signal transduction mechanisms have been proposed to mediate these effects, the intracellular events that precede actin breakdown are unknown. In NIH-3T3 fibroblasts, toxin B induced an elevation of intracellular calcium possessing either a slow (minutes) or fast (seconds) rise time, followed by a sustained elevation of calcium concentration. Subcellular analysis of steady-state calcium distribution after toxin B demonstrated that the increase of calcium was homogeneous throughout the cytosol and did not vary based on the kinetics of the initial calcium rise. All calcium responses were blocked by substitution with calcium-free buffer or buffer containing lanthanum chloride, indicating that the rise in calcium was attributable to calcium influx from the extracellular space. Quantitatively similar responses were observed in primary cultured gastric smooth muscle and AR42J pancreatic tumor cells, suggesting that toxin-induced calcium signal transduction was conserved between cell types. The morphological response to toxin B consisted of sequential dissociation of the actin cytoskeleton from membrane attachments, retraction of actin stress fibers from the periphery to the perinuclear region, loss of fibre alignment, and cell rounding. The actin reorganization associated with toxin B was blocked by incubation of cells in calcium-free media or the clamping of intracellular calcium with cell-permeant calcium chelating agents. These results demonstrate that the calcium influx activated by C. difficile toxin B is a necessary condition for the breakdown of filamentous actin associated with cytotoxicity.

3T3 Cells↗
PubMed1995

Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro.

Toxin A but not toxin B, appears to mediate intestinal damage in animal models of Clostridium difficile enteritis. The purpose of this study was to investigate the electrophysiologic and morphologic effects of purified C. difficile toxins A and B on human colonic mucosa in Ussing chambers. Luminal exposure of tissues to 16-65 nM of toxin A and 0.2-29 nM of toxin B for 5 h caused dose-dependent epithelial damage. Potential difference, short-circuit current and resistance decreased by 76, 58, and 46%, respectively, with 32 nM of toxin A and by 76, 55, and 47%, respectively, with 3 nM of toxin B, when compared with baseline (P < 0.05). 3 nM of toxin A did not cause electrophysiologic changes. Permeability to [3H]mannitol increased 16-fold after exposure to 32 nM of toxin A and to 3 nM of toxin B when compared with controls (P < 0.05). Light and scanning electron microscopy after exposure to either toxin revealed patchy damage and exfoliation of superficial epithelial cells, while crypt epithelium remained intact. Fluorescent microscopy of phalloidin-stained sections showed that both toxins caused disruption and condensation of cellular F-actin. Our results demonstrate that the human colon is approximately 10 times more sensitive to the damaging effects of toxin B than toxin A, suggesting that toxin B may be more important than toxin A in the pathogenesis of C. difficile colitis in man.

Actins↗
PubMed1994

CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.

Toxin A from Clostridium difficile mediates acute inflammatory enterocolitis in experimental animals, while cholera toxin causes noninflammatory secretory diarrhea. The purpose of this study was to investigate whether an antagonist to the peptide substance P, a constituent of primary sensory neurons known to participate in inflammatory responses, would inhibit toxin A-mediated enteritis in the rat ileum. Pretreatment of rats with CP-96,345 (2.5 mg per kg of body weight), a substance P antagonist, dramatically inhibited fluid secretion (P < 0.01) and mannitol permeability (P < 0.01) in ileal loops exposed to toxin A. The protective effects, which were dose dependent, caused a significant reduction of inflammation in the lamina propria, reduction of the necrosis of intestinal epithelial cells, and complete inhibition of toxin A-mediated release of rat mast cell protease II, a specific product of rat mucosal mast cells. An inactive enantiomer of the substance P antagonist, CP-96,344, had no effect. In contrast, pretreatment with CP-96,345 had no inhibitory effect on the intestinal effects caused by administration of cholera toxin into the ileal loops. From these data, we conclude that the peptide substance P is involved in the secretory and inflammatory effects of toxin A but not of cholera toxin.

Animals↗
PubMed1994

Neuronal involvement in the intestinal effects of Clostridium difficile toxin A and Vibrio cholerae enterotoxin in rat ileum.

BACKGROUND/AIMS: Activation of intestinal mast cells and neurons is involved in intestinal inflammation and diarrhea. This study compared the effects of neuronal inhibitors and inhibition of intestinal sensory afferent nerves on the intestinal actions of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin. METHODS: The effects of lidocaine, hexamethonium, atropine, and long-term pretreatment of capsaicin on fluid secretion, mannitol permeability, myeloperoxidase (MPO) activity, and release of rat mast cell protease II (RMCPII) were measured in toxin A- and cholera toxin-exposed loops in vivo. RESULTS: Lidocaine, hexamethonium, and capsaicin, but not atropine, inhibited toxin A-mediated secretion and MPO activity, but only capsaicin reduced mannitol permeability. Lidocaine, but not capsaicin, reduced secretion and permeability caused by cholera toxin. Toxin A caused release of RMCPII from rat ileum in vivo and in vitro; this was inhibited by lidocaine or capsaicin, whereas cholera toxin had no effect on release of RMCPII. CONCLUSIONS: Neuronal mechanisms are important in the in vivo effects of these two enterotoxins. Capsaicin-sensitive sensory afferent neurons and mast cells are involved in the intestinal mechanism of toxin A, but not cholera toxin.

Animals↗
PubMed1994

IL-8 secretion and neutrophil activation by HT-29 colonic epithelial cells.

This study examines the ability of HT-29 human colonic epithelial cells to stimulate neutrophil migration and adhesion. Interleukin-8 (IL-8), a potent neutrophil chemoattractant, was detected in conditioned media from both unstimulated (1.1 ng/ml) and IL-1 beta-stimulated (16.1 ng/ml) HT-29 cultures. Conditioned medium from IL-1 beta-exposed HT-29 cells stimulated neutrophil migration (395% of control, P < 0.01), and this effect was completely inhibited by anti-IL-8 antibody. HT-29 medium also induced shedding of neutrophil L-selectin and increased expression of neutrophil CD11/CD18 adhesion receptors. Coculture of HT-29 cells with human endothelial cell monolayers resulted in increased neutrophil transendothelial migration (169% of control, P < 0.01), which was blocked by both anti-IL-8 and anti-CD18 antibody. Northern hybridization analysis demonstrated increased levels of mRNA for IL-8 and intercellular adhesion molecule-1 (ICAM-1) in cytokine-treated HT-29 cells. Cytokine stimulation of HT-29 monolayers was also associated with increased neutrophil adhesion to these cells. Neutrophil-HT-29 cell adhesion was blocked by monoclonal antibodies to neutrophil CD18 or to ICAM-1 on the HT-29 cells (86% and 56% inhibition, respectively, P < 0.01 for both). These data suggest that IL-8 secretion by activated colonic epithelial cells may contribute to neutrophil extravasation and tissue infiltration in intestinal inflammation.

Blotting, Northern↗
PubMed1994

Neutrophil recruitment in Clostridium difficile toxin A enteritis in the rabbit.

Neutrophil infiltration is a prominent feature of Clostridium difficile-associated enteritis and colitis. The aim of this study was to examine the importance of neutrophil recruitment and neutrophil-mediated tissue damage in C. difficile toxin A-induced enteritis. Competitive binding experiments using purified 3H-toxin A demonstrated the presence of a single class of medium affinity receptors on rabbit neutrophils (Kd 7 x 10(-8) M). Pertussis toxin and the nonhydrolyzable GTP analog GTPgamma S both inhibited 3H-toxin A binding (by 56 and 65%, respectively), indicating that the rabbit neutrophil toxin A receptor is G protein linked. Toxin A elicited a dose-dependent (25-200 micrograms/ml) stimulation of neutrophil migration in vitro, and this functional effect was also pertussis toxin sensitive (69% inhibition). Treatment of neutrophils with R15.7, a blocking monoclonal antibody to the leuocyte adhesion molecule CD18, inhibited toxin A-stimulated neutrophil migration by 85% in vitro. Pretreatment of rabbits with R15.7 also prevented neutrophil infiltration of toxin A-exposed ileal loops in vivo as determined by histologic examination and by ileal tissue myeloperoxidase levels. Furthermore, R15.7 effected a substantial inhibition of fluid secretion (by 65%), mannitol permeability (by 66%), and histologic damage in toxin A-exposed ileal loops. Anti-CD18 (R15.7) had no inhibitory effect on cholera toxin enterotoxicity. These data demonstrate that C. difficile toxin A is a proinflammatory toxin whose enterotoxic effects are substantially dependent upon neutrophil recruitment.

Animals↗
PubMed1994

Clostridium difficile toxin A-induced microvascular dysfunction. Role of histamine.

Clostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis. Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A. Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A. Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure. These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates. The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin. The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A. The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist. These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules. Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin. The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.

Animals↗
PubMed1993

Effects of purified Clostridium difficile toxin A in the small intestine of the rat in vivo.

The action of highly purified Clostridium difficile toxin A was studied in the jejunum of rats in vivo. C. difficile toxin A reversed dose-dependently net fluid absorption into net fluid secretion, accompanied by an increase in prostaglandin E2 but not 5-hydroxytryptamine output into the gut lumen. Accordingly, indomethacin but not the 5-hydroxytryptamine receptor antagonists ketanserin plus tropisetron were able to inhibit toxin A-induced fluid secretion. Atropine and hexamethonium were without effect on the action of toxin A, such excluding a nervous mechanism. The cyclic nucleotides cyclic AMP and cyclic GMP appear not to be involved in the mediation of the secretory response. The reduced cyclic GMP levels are most likely the result of a complete destruction of the villus membranes, where the guanylate cyclase is located. Histological studies revealed massive damage to intestinal villi, whereas the majority of the crypts seem to be unaffected. In conclusion, toxin A-induced intestinal fluid secretion appears to be caused mainly by severe mucosal damage. PGE2-release may be the consequence of the inflammation accompanying this damage. The mechanism seems to be completely different to those of cholera toxin or Escherichia coli heat stable enterotoxin.

Animals↗
PubMed1993

Saccharomyces boulardii inhibits Clostridium difficile toxin A binding and enterotoxicity in rat ileum.

BACKGROUND: Saccharomyces boulardii is a nonpathogenic yeast used for the prevention and treatment of Clostridium difficile-associated diarrhea and colitis. However, the mechanism by which S. boulardii exerts its protective effects remains unclear. METHODS: The binding of [3H]toxin A to its brush border receptor preincubated with S. boulardii-cultured suspension or filtered conditioned medium was measured in vitro. The effect of toxin A on secretion, epithelial permeability, and morphology in rat ileal loops in vivo was also examined in rats pretreated with S. boulardii. RESULTS: S. boulardii reduced [3H]toxin A-receptor binding in a dose-dependent fashion. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of ileal brush border exposed to S. boulardii-conditioned medium revealed a diminution of all brush border proteins. Treatment of rats with S. boulardii suspension reduced fluid secretion and mannitol permeability caused by toxin A. CONCLUSIONS: S. boulardii may reduce some of the enterotoxic effects of toxin A by inhibiting toxin A-receptor binding. This effect appears to be manifested by a secreted product of the yeast, possibly a protease.

Animals↗