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C Pothoulakis

Publications and source records attributed to C Pothoulakis.

At least 55 records · Page 3Linked to original sources
PubMed1997

Epidermal growth factor attenuates Clostridium difficile toxin A- and B-induced damage of human colonic mucosa.

Epidermal growth factor (EGF) exhibits a cytoprotective effect on gastrointestinal epithelia via a receptor-mediated mechanism. We investigated the effect of EGF on Clostridium difficile toxin A (TxA)- and toxin B (TxB)-induced damage of human colon. Ussing-chambered colonic mucosa was exposed serosally to EGF before and during luminal exposure to TxA and TxB. Resistance was calculated from potential difference and short-circuit current. Epithelial damage was assessed by light microscopy and alteration of F-actin by fluoresceinated phalloidin. Luminal exposure of colonic strips to TxA and TxB caused a time- and dose-dependent decrease in electrical resistance, necrosis and dehiscence of colonocytes, and disruption and condensation of enterocyte F-actin. These effects were inhibited by prior, but not simultaneous, serosal application of EGF (20 nM). Administration of the tyrosine kinase inhibitor genistein (10(-6) M) inhibited the protective effects of EGF. We conclude that EGF protects against TxA and TxB probably by stabilizing the cytoskeleton, the main target of these toxins.

Actins↗
PubMed1997

IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes.

Neutrophil infiltration is central to the pathogenesis of Clostridium difficile toxin A-induced enterocolitis. This study examines whether monocyte activation by C. difficile toxins is instrumental in initiating neutrophil activation and recruitment. Human monocytes were exposed to low concentrations of highly purified C. difficile toxins, and the conditioned media were harvested for cytokine and functional assays. Monocytes exposed to C. difficile toxin A (10(-10) M) or toxin B (10(-12) M) released 100 and 20 times basal levels, respectively, of the neutrophil chemoattractant interleukin-8 (IL-8). Reverse transcriptase-polymerase chain reaction demonstrated a marked increase in IL-8 mRNA expression by monocytes 3 h after toxin exposure. Conditioned media from toxin A- and toxin B-treated monocytes stimulated neutrophil migration (324 and 245% of control, respectively). This effect was completely blocked by IL-8 antiserum. These media also upregulated neutrophil CD11b/CD18 and endothelial cell intercellular adhesion molecule-1 expression. C. difficile toxins, at low concentrations, potently activate monocytes to release factors, including IL-8, that facilitate neutrophil extravasation and tissue infiltration. Our findings indicate a major role for toxin-mediated monocyte and macrophage activation in C. difficile colitis.

Bacterial Proteins↗
PubMed1996

A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats.

We previously reported that short-term immobilization stress of rats causes increased colonic mucin release, goblet cell depletion, prostaglandin E2 secretion, and colonic mast cell activation, as well as increased colonic motility. The purpose of this study was to investigate whether neurotensin (NT), a peptide expressed in both brain and digestive tract, participates in these responses. Rats were pretreated with SR 48692 (1 mg/kg, i.p.), an NT antagonist, 15 min before immobilization (30 min). The administration of the antagonist significantly inhibited stress-mediated secretion of colonic mucin, prostaglandin E2, and a product of rat mast cells, rat mast cell protease II (P < 0.05), but did not alter the increase in fecal pellet output caused by immobilization stress. Immobilization stress also resulted in a quantifiable decrease in the abundance of NT receptor mRNA in rat colon compared with that in colonic tissues from nonimmobilized rats as measured by densitometric analysis of in situ hybridization studies (P < 0.03). We conclude that the peptide NT is involved in colonic goblet cell release and mucosal mast cell activation after immobilization stress.

Animals↗
PubMed1996

Rabbit sucrase-isomaltase contains a functional intestinal receptor for Clostridium difficile toxin A.

The intestinal effects of Clostridium difficile toxin A are inidated by toxin binding to luminal enterocyte receptors. We reported previously that the rabbit ileal brush border (BB) receptor is a glycoprotein with an alpha-d-galactose containing trisaccharide in the toxin-binding domain (1991. J. Clin. Invest. 88:119-125). In this study we characterized the rabbit ileal BB receptor for this toxin. Purified toxin receptor peptides of 19 and 24 amino acids showed 100% homology with rabbit sucrase-isomaltase (SI). Guinea pig receptor antiserum reacted in Western blots with rabbit SI and with the purified toxin receptor. Antireceptor IgG blocked in vitro binding of toxin A to rabbit ileal villus cell BB. Furthermore, anti-SI IgG inhibited toxin A-induced secretion (by 78.1%, P < 0.01), intestinal permeability (by 80.8%, P < 0.01), and histologic injury (P < 0.01) in rabbit ileal loops in vivo. Chinese hamster ovary cells transfected with SI cDNA showed increased intracellular calcium increase in response to native toxin (holotoxin) or to a recombinant 873-amino acid peptide representing the receptor binding domain of toxin A. These data suggest that toxin A binds specifically to carbohydrate domains on rabbit ileal SI, and that such binding is relevant to signal transduction mechanisms that mediate in vitro and in vivo toxicity.

Amino Acid Sequence↗
PubMed1996

A human antibody binds to alpha-galactose receptors and mimics the effects of Clostridium difficile toxin A in rat colon.

BACKGROUND & AIMS: Nearly all human sera contain an immunoglobulin G antibody (antigalactose) that binds the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc expressed on cells from most mammals but not humans. Because the Clostridium difficile toxin A receptor in rodents contains this trisaccharide, the aim of this study was to examine whether antigalactose could mimic the enterotoxic effects of toxin A and bind to receptors containing this trisaccharide. METHODS: Fluid secretion, [3H]-mannitol permeability, and release of rat mast cell protease II and prostaglandin E2 were measured after luminal exposure of rat colon to either purified human anti-galactose, control immunoglobulin G, toxin A, or buffer. RESULTS: Toxin A (5 micrograms) and antigalactose (250 micrograms) but not control immunoglobulin (250 micrograms) stimulated colonic fluid secretion and caused increased mannitol permeability and rat mast cell protease II release. Antigalactose and toxin A and, to a lesser degree, control immunoglobulin G also stimulated release of prostaglandin E2, but only toxin A produced acute inflammation of rat colonic mucosa. Antigalactose and toxin A bound specifically to a single class of colonic brush border receptors with dissociation constants of 10(-6) mol/L and 5.4 x 10(-8) mol/L, respectively. CONCLUSIONS: Fluid secretion, increased permeability, and mast cell activation occur in rat colon when toxin A or human antigalactose immunoglobulin G bind to receptors bearing the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc.

Animals↗
PubMed1996

Nitric oxide inhibits rat intestinal secretion by Clostridium difficile toxin A but not Vibrio cholerae enterotoxin.

BACKGROUND & AIMS: Intestinal inflammation is associated with increased synthesis of nitric oxide, whereas inhibition of NO synthase (NOS) reduces experimental chronic intestinal inflammation. The aim of this study was to test the effects of NO blockers and donors on acute intestinal inflammation induced by Clostridium difficile toxin A in rat ileum. METHODS: Rats received NOS inhibitors or NO donors before measurement of toxin-mediated ileal secretion and permeability changes. Mucosal mast cell and neutrophil activity were measured by release of rat mast cell protease II and myeloperoxidase activity, respectively. RESULTS: NOS inhibitors augmented but an NO donor inhibited toxin A-mediated ileal secretion and permeability when given before but not after toxin administration. Neither an NOS inhibitor nor an NO donor had any effect on cholera toxin-mediated secretion. Mast cell degranulation and neutrophil infiltration occurred after injection of toxin A or an NOS inhibitor, whereas the NO donor blocked both toxin A effects. CONCLUSIONS: NOS inhibitors augmented and an NO donor blocked the intestinal effects of toxin A but not of cholera toxin. NO protects against toxin A by inhibition of intestinal mast cells and neutrophils, which are activated by toxin A, but not by cholera toxin.

Acetylcysteine↗
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↗