Ignazio Castagliuolo

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.

Neurokinin-1 (NK-1) receptor is required in Clostridium difficile- induced enteritis.

Toxin A, a 308,000-Mr enterotoxin from Clostridium difficile, mediates antibiotic-associated diarrhea and colitis in humans. Injection of toxin A into animal intestine triggers an acute inflammatory response characterized by activation of sensory neurons and immune cells of the intestinal lamina propria, including mast cells and macrophages, and migration of circulating neutrophils in the involved intestinal segment. In this study we show that mice genetically deficient in the neurokinin-1 receptor are protected from the secretory and inflammatory changes as well as from epithelial cell damage induced by toxin A. The protective effect of neurokinin-1R deletion correlates with diminished intestinal levels of the cytokine TNF-alpha and its mRNA and the leukocyte enzyme myeloperoxidase. These results demonstrate a major requirement for substance P receptors in the pathogenesis of acute inflammatory diarrhea.

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.

Direct evidence of mast cell involvement in Clostridium difficile toxin A-induced enteritis in mice

BACKGROUND & AIMS The pathogenesis of Clostridium difficile toxin A-induced intestinal inflammation is not completely understood. The aim of this study was to define the contribution of mast cells to the fluid secretion and neutrophil infiltration associated with toxin A-induced enteritis. METHODS Fluid secretion and neutrophil infiltration in toxin A- or buffer-challenged ileal loops were assessed in normal, mast cell-deficient, and mast cell-deficient KitW/KitW-v mice that had undergone selective repair of their mast cell deficiency. The effect of a specific substance P-receptor antagonist was also studied. RESULTS Intestinal fluid secretion and neutrophil recruitment were significantly diminished in mast cell-deficient KitW/KitW-v and mast cell-deficient MgfSl/MgfSl-d mice compared with the respective normal mice. Mast cell-reconstituted KitW/KitW-v mice showed responses similar to the normal congenic mice. Administration of a specific substance P-receptor antagonist (CP-96,345) reduced toxin A-induced intestinal fluid secretion and inhibited neutrophil infiltration in normal, mast cell-deficient KitW/KitW-v, and mast cell-reconstituted KitW/KitW-v mice. CONCLUSIONS C. difficile toxin A elicits intestinal fluid secretion and neutrophil infiltration by both mast cell-dependent and -independent pathways, and substance P participates in both pathways.

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.

Ketotifen inhibits Clostridium difficile toxin A-induced enteritis in rat ileum.

BACKGROUND Clostridium difficile toxin A is the principal mediator of inflammatory enterocolitis in experimental animals. The purpose of this study was to explore the effect of ketotifen, an anti-inflammatory drug, on toxin A-induced enterotoxicity in rat ileum. METHODS The effects of intragastric administration of ketotifen on secretion, mannitol permeability, histological damage, and mucosal levels of leukotriene B4, leukotriene C4, and platelet activating factor in toxin A-exposed rat ileal loops were measured in vivo. The effects of ketotifen on toxin A-mediated release of rat mast cell protease II (rat mucosa mast cell product) release were also measured in rat ileal explants in vitro. The effect of ketotifen on neutrophil migration in vitro was also evaluated. RESULTS Ketotifen pretreatment inhibited toxin A-associated intestinal secretion by 42.5% and mannitol permeability by 56.3% and reduced epithelial cell inflammation and necrosis. These effects were associated with reduced levels of leukotriene B4 by 65.8%, leukotriene C4 by 88.8%, platelet activating factor by 77.8%, and inhibition of rat mast cell protease II by 58.4%. In addition, pretreatment of neutrophils with ketotifen inhibited neutrophil migration in vitro. CONCLUSIONS The protective effect of ketotifen in this animal model was associated with significant inhibition of release of mast cells and neutrophil derived mediators, supporting their involvement in C. difficile enteritis.

Substance P receptor expression in intestinal epithelium in Clostridium difficile toxin A enteritis in rats

We previously reported that the inflammatory effects of Clostridium difficile toxin A on rat intestine can be significantly inhibited with a specific neurokinin-1 receptor (NK-1R) antagonist. In this study we investigated the localization and expression of NK-1R mRNA and protein in rat intestine by in situ hybridization, Northern blot analysis, and immunohistochemistry, respectively, after exposure to toxin A. Northern blot analysis showed increased mucosal levels of NK-1R mRNA starting 30 min after toxin A administration. In situ hybridization showed that toxin A increased NK-1R mRNA expression in intestinal epithelial cells after 30, 120, and 180 min. In rats pretreated with the NK-1R antagonist CP-96345 the increase in NK-1R mRNA levels after exposure to toxin A was inhibited, indicating that NK-1R upregulation is substance P (SP) dependent. One hour after exposure to toxin A many of the intestinal epithelial cells showed staining for NK-1R compared with controls. Specific 125I-SP binding to purified epithelial cell membranes obtained from ileum exposed to toxin A for 15 min was increased twofold over control and persisted for 4 h. This report provides evidence that NK-1R expression is increased in the intestinal epithelium shortly after exposure to toxin A and may be important in toxin A-induced inflammation.

Protective effects of neurokinin-1 receptor during colitis in mice: role of the epidermal growth factor receptor

The role of substance P and its high affinity neurokinin‐1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin‐1 receptor in colitis using the 2,4,6,‐trinitrobenzensulphonic acid and dextran sulphate‐induced animal models of colitis and genetically‐engineered, neurokinin‐1 receptor‐deficient mice. Clinical signs, macroscopic and histologic damage associated with 2,4,6,‐trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin‐1 deficient than in wild‐type mice, while immunoreactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis. Substance P, dose‐dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor‐induced proliferation in intestinal fibroblasts isolated from wild‐type, but not from neurokinin‐1 receptor deficient mice. Substance P‐induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts. Our results indicate that in mice lacking the neurokinin ‐ 1 receptor, substance P plays a protective role in prolonged experimental colitis.

CGRP upregulation in dorsal root ganglia and ileal mucosa during Clostridium difficile toxin A-induced enteritis

We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A. Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure. Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment. Pretreatment of rats with CGRP-(8-37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage. We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons.We have previously reported that pretreatment of rats with capsaicin (an agent that ablates sensory neurons) or CP-96345 (a substance P receptor antagonist) dramatically inhibits fluid secretion and intestinal inflammation in ileal loops exposed to Clostridium difficile toxin A. The aim of this study was to determine whether calcitonin gene-related peptide (CGRP), a neuropeptide also found in sensory afferent neurons, participates in the enterotoxic effects of toxin A. Administration of toxin A was also found to increase CGRP content in dorsal root ganglia and ileal mucosa 60 min after toxin exposure. Furthermore, immunohistochemical studies demonstrated increased neuronal staining for CGRP 2 h after toxin A treatment. Pretreatment of rats with CGRP-(8-37), a specific CGRP antagonist, before instillation of toxin A into ileal loops significantly inhibited toxin-mediated fluid secretion (by 48%), mannitol permeability (by 83%), and histological damage. We conclude that CGRP, like substance P, contributes to the secretory and inflammatory effects of toxin A via increased production of this peptide from intestinal nerves, including primary sensory afferent neurons.

Participation of reactive oxygen metabolites in Clostridium difficile toxin A-induced enteritis in rats

Reactive oxygen metabolites (ROMs) contribute to the pathophysiology of intestinal inflammation. Our aim was to ascertain the involvement of ROMs in experimental ileitis in rats produced by toxin A of Clostridium difficile. Intraluminal toxin A caused a significant increase in hydroxyl radical and hydrogen peroxide production by ileal microsomes starting 1 h following toxin exposure and peaking at 2-3 h, and this was inhibited by pretreatment with DMSO, a ROM scavenger, or superoxide dismutase (SOD), which inactivates ROMs. In contrast, mucosal xanthine oxidase increased only slightly after toxin A exposure, and allopurinol, an inhibitor of xanthine oxidase, had no effect on toxin A-associated intestinal responses. Induction of neutropenia resulted in reduction of toxin-mediated free radical formation, fluid secretion, and permeability. The enterotoxic effects of C. difficile toxin A were associated with increased ROM release in ileal tissues, and the ROM inhibitors DMSO and SOD inhibited these effects. This suggests that ROMs released during toxin A enteritis are released primarily from neutrophils invading the inflamed bowel segment.Reactive oxygen metabolites (ROMs) contribute to the pathophysiology of intestinal inflammation. Our aim was to ascertain the involvement of ROMs in experimental ileitis in rats produced by toxin A of Clostridium difficile. Intraluminal toxin A caused a significant increase in hydroxyl radical and hydrogen peroxide production by ileal microsomes starting 1 h following toxin exposure and peaking at 2-3 h, and this was inhibited by pretreatment with DMSO, a ROM scavenger, or superoxide dismutase (SOD), which inactivates ROMs. In contrast, mucosal xanthine oxidase increased only slightly after toxin A exposure, and allopurinol, an inhibitor of xanthine oxidase, had no effect on toxin A-associated intestinal responses. Induction of neutropenia resulted in reduction of toxin-mediated free radical formation, fluid secretion, and permeability. The enterotoxic effects of C. difficile toxin A were associated with increased ROM release in ileal tissues, and the ROM inhibitors DMSO and SOD inhibited these effects. This suggests that ROMs released during toxin A enteritis are released primarily from neutrophils invading the inflamed bowel segment.