Genome-wide DNA methylation profiling of peripheral blood mononuclear cells in irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a stress-sensitive disorder. Environmental factors including stress can trigger epigenetic changes, which have not been well-studied in IBS. We performed a pilot study investigating genome-wide DNA methylation of IBS patients and healthy controls (HCs) to identify potential epigenetic markers and associated pathways. Additionally, we investigated relationships of epigenetic changes in selected genes with clinical traits. METHODS: Twenty-seven IBS patients (59% women; 10 IBS-diarrhea, 8 IBS-constipation, 9 IBS-mixed) and 23 age- and sex-matched HCs were examined. DNA methylation from peripheral blood mononuclear cells (PBMCs) was measured using HM450 BeadChip, and representative methylation differences were confirmed by bisulphite sequencing. Gene expression was measured using quantitative PCR. Gastrointestinal (GI) and non-GI symptoms were measured using validated questionnaires. Associations were tested using non-parametric methods. KEY RESULTS: Genome-wide DNA methylation profiling of IBS patients compared with HCs identified 133 differentially methylated positions (DMPs) (mean difference ≥10%; p < 0.05). These genes were associated with gene ontology terms including glutathione metabolism related to oxidative stress and neuropeptide hormone activity. Validation by sequencing confirmed differential methylation of subcommissural organ (SCO)-Spondin (SSPO), glutathione-S-transferases mu 5 (GSTM5), and tubulin polymerization promoting protein genes. Methylation of two promoter CpGs in GSTM5 was associated with epigenetic silencing. Epigenetic changes in SSPO gene were positively correlated with hospital anxiety and depression scores in IBS patients (r > 0.4 and false discovery rate <0.05). CONCLUSIONS & INFERENCES: This study is the first to comprehensively explore the methylome of IBS patients. We identified DMPs in novel candidate genes which could provide new insights into disease mechanisms; however, these preliminary findings warrant confirmation in larger, independent studies.

Inhibition of a novel fibrogenic factor Tl1a reverses established colonic fibrosis.

Intestinal fibrostenosis is among the hallmarks of severe Crohn's disease. Patients with certain TNFSF15 (gene name for TL1A) variants over-express TL1A and have a higher risk of developing strictures in the small intestine. In addition, sustained Tl1a expression in mice leads to small and large intestinal fibrostenosis under colitogenic conditions. The aim of this study was to determine whether established murine colonic fibrosis could be reversed with Tl1a antibody (Ab). Treatment with neutralizing Tl1a Ab reversed colonic fibrosis back to the original pre-inflamed levels, potentially as a result of lowered expression of connective tissue growth factor, Il31Ra, transforming growth factor ß1 and insulin-like growth factor-1. In addition, blocking Tl1a function by either neutralizing Tl1a Ab or deletion of death domain receptor 3 (Dr3) reduced the number of fibroblasts and myofibroblasts, the primary cell types that mediate tissue fibrosis. Primary intestinal myofibroblasts expressed Dr3 and functionally responded to direct Tl1a signaling by increasing collagen and Il31Ra expression. These data demonstrated a direct role for TL1A-DR3 signaling in tissue fibrosis and that modulation of TL1A-DR3 signaling could inhibit gut fibrosis.

[Recent advances in Saccharomyces boulardii research]. / Progrès récents dans la recherche sur Saccharomyces boulardii.

This review summarizes the probiotic mechanisms of action of Saccharomyces boulardii (S. boulardii) against inflammatory and non-inflammatory diarrheal conditions. S. boulardii is distributed in lyophilized form in many countries and used for the prevention of diarrhea in children and adults, including Clostridium difficile (C. difficile) associated infection. The main mechanisms of action of S. boulardii include inhibition of activities of bacterial pathogenic products, trophic effects on the intestinal mucosa, as well as modification of host signaling pathways involved in inflammatory and non-inflammatory intestinal diseases. S. boulardii inhibits production of pro-inflammatory cytokines by inhibiting main regulators of inflammation, including nuclear factor κB (NF-κB), and mitogen-activated protein kinases (MAP kinases), ERK1/2 and p38, but stimulates production of anti-inflammatory molecules such as peroxisome proliferator-activated receptor-gamma (PPAR-γ). Moreover, S. boulardii suppresses bacterial infection by inhibiting adhesion and/or overgrowth of bacteria, produces a serine protease that cleaves C. difficile toxin A, and stimulates antibody production against this toxin. Furthermore, S. boulardii may interfere with pathogenesis of Inflammatory Bowel Disease (IBD) by acting on T cells and acts in diarrheal conditions by improving the fecal biostructure in patients with diarrhea. These diverse mechanisms exerted by S. boulardii provide molecular clues for its effectiveness in diarrheal diseases and intestinal inflammatory conditions with an inflammatory component.

Review article: anti-inflammatory mechanisms of action of Saccharomyces boulardii.

BACKGROUND: Saccharomyces boulardii, a well-studied probiotic, can be effective in inflammatory gastrointestinal diseases with diverse pathophysiology, such as inflammatory bowel disease (IBD), and bacterially mediated or enterotoxin-mediated diarrhoea and inflammation. AIM: To discuss the mechanisms of action involved in the intestinal anti-inflammatory action of S. boulardii. METHODS: Review of the literature related to the anti-inflammatory effects of this probiotic. RESULTS: Several mechanisms of action have been identified directed against the host and pathogenic microorganisms. S. boulardii and S. boulardii secreted-protein(s) inhibit production of proinflammatory cytokines by interfering with the global mediator of inflammation nuclear factor kappaB, and modulating the activity of the mitogen-activated protein kinases ERK1/2 and p38. S. boulardii activates expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) that protects from gut inflammation and IBD. S. boulardii also suppresses 'bacteria overgrowth' and host cell adherence, releases a protease that cleaves C. difficile toxin A and its intestinal receptor and stimulates antibody production against toxin A. Recent results indicate that S. boulardii may interfere with IBD pathogenesis by trapping T cells in mesenteric lymph nodes. CONCLUSIONS: The multiple anti-inflammatory mechanisms exerted by S. boulardii provide molecular explanations supporting its effectiveness in intestinal inflammatory states.

Melanin-concentrating hormone (MCH) modulates C difficile toxin A-mediated enteritis in mice.

OBJECTIVE: Melanin-concentrating hormone (MCH) is a hypothalamic orexigenic neuropeptide that regulates energy balance. However, the distribution of MCH and its receptor MCHR1 in tissues other than brain suggested additional, as yet unappreciated, roles for this neuropeptide. Based on previous paradigms and the presence of MCH in the intestine as well as in immune cells, its potential role in gut innate immune responses was examined. METHODS: In human intestinal xenografts grown in mice, changes in the expression of MCH and its receptors following treatment with Clostridium difficile toxin A, the causative agent of antibiotic-associated diarrhoea in hospitalised patients, were examined. In colonocytes, the effect of C difficile toxin A treatment on MCHR1 expression, and of MCH on interleukin 8 (IL8) expression was examined. MCH-deficient mice and immunoneutralisation approaches were used to examine the role of MCH in the pathogenesis of C difficile toxin A-mediated acute enteritis. RESULTS: Upregulation of MCH and MCHR1 expression was found in the human intestinal xenograft model, and of MCHR1 in colonocytes following exposure to toxin A. Treatment of colonocytes with MCH resulted in IL8 transcriptional upregulation, implying a link between MCH and inflammatory pathways. In further support of this view, MCH-deficient mice developed attenuated toxin A-mediated intestinal inflammation and secretion, as did wild-type mice treated with an antibody against MCH or MCHR1. CONCLUSION: These findings signify MCH as a mediator of C difficile-associated enteritis and possibly of additional gut pathogens. MCH may mediate its proinflammatory effects at least in part by acting on epithelial cells in the intestine.

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

1. 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. 2. 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. 3. 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. 4. 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. 5. Our results indicate that in mice lacking the neurokinin - 1 receptor, substance P plays a protective role in prolonged experimental colitis.

Signal transduction pathways mediating neurotensin-stimulated interleukin-8 expression in human colonocytes.

Neurotensin (NT), a neuropeptide released in the gastrointestinal tract in response to several stimuli, is involved in the pathophysiology of colonic inflammation. However, the molecular mechanism(s) mediating this proinflammatory response remains unclear. We found that NCM460, non-transformed human colonocytes, express a functional high affinity NT receptor that mediates NT-induced Erk activation. By using NCM460 cells stably transfected with NTR1, we show that NTR1 activation leads to interleukin (IL)-8 secretion that is mediated via both NF-kappaB- and Erk-dependent pathways. In addition, NT-stimulated NF-kappaB activation is dependent on intracellular calcium release. NT-stimulated Erk activity requires Ras activation because overexpression of the dominant negative Ras mutant Ras-17N almost completely inhibits the Erk activation. Furthermore, NT directly stimulates Ras-GTP formation as shown by a Ras-GTP pull-down assay. By using reporter gene constructs containing targeted substitutions in the IL-8 promoter, we show that the NF-kappaB, AP-1, and to a lesser degree the C/EBP sites in the IL-8 promoter region are required for IL-8 gene expression induced by NT. In summary, our results demonstrate that NT stimulates calcium-dependent NF-kappaB and Ras-dependent Erk pathways that mediate the release of IL-8 from non-transformed human colonocytes. We speculate that these NT-related proinflammatory pathways are important in the pathophysiology of colonic inflammation.

Deletion of neutral endopeptidase exacerbates intestinal inflammation induced by Clostridium difficile toxin A.

Toxin A (TxA) of Clostridium difficile induces acute inflammation of the intestine initiated by release of substance P (SP) and activation of the neurokinin-1 receptor. However, the mechanisms that terminate this response are unknown. We determined whether the SP-degrading enzyme neutral endopeptidase (NEP, EC 3.4.24.11) terminates TxA-induced enteritis. We used both genetic deletion and pharmacological inhibition of NEP to test this hypothesis. In wild-type mice, instillation of TxA (0.5-5 microg) into ileal loops for 3 h dose dependently increased ileal fluid secretion, stimulated granulocyte transmigration determined by myeloperoxidase activity, and caused histological damage characterized by depletion of enterocytes, edema, and neutrophil accumulation. Deletion of NEP reduced the threshold secretory and inflammatory dose of TxA and exacerbated the inflammatory responses by more than twofold. This exacerbated inflammation was prevented by pretreatment with recombinant NEP. Conversely, pretreatment of wild-type mice with the NEP inhibitor phosphoramidon exacerbated enteritis. Thus NEP terminates enteritis induced by C. difficile TxA, underlying the importance of SP degradation in limiting neurogenic inflammation.

Endogenous corticosteroids modulate Clostridium difficile toxin A-induced enteritis in rats.

We examined the role of glucocorticoids in acute inflammatory diarrhea mediated by Clostridium difficile toxin A. Toxin A (5 microg) or buffer was injected in rat ileal loops, and intestinal responses were measured after 30 min to 4 h. Ileal toxin A administration increased plasma glucocorticoids after 1 h, at which time the toxin-stimulated secretion was not significant. Administration of the glucocorticoid analog dexamethasone inhibited toxin A-induced intestinal secretion and inflammation and downregulated toxin A-mediated increase of macrophage inflammatory protein-2. Adrenalectomy followed by replacement with glucocorticoids at various doses suggested that intestinal responses to toxin A were related to circulating levels of glucocorticoids. Administration of the glucocorticoid receptor antagonist RU-486 enhanced toxin A-mediated intestinal secretion and inflammation. We conclude that C. difficile toxin A causes increased secretion of endogenous glucocorticoids, which diminish the intestinal secretory and inflammatory effects of toxin A.

Saccharomyces boulardii stimulates intestinal immunoglobulin A immune response to Clostridium difficile toxin A in mice.

Saccharomyces boulardii is a nonpathogenic yeast that protects against antibiotic-associated diarrhea and recurrent Clostridium difficile colitis. The administration of C. difficile toxoid A by gavage to S. boulardii-fed BALB/c mice caused a 1.8-fold increase in total small intestinal immunoglobulin A levels (P = 0.003) and a 4.4-fold increase in specific intestinal anti-toxin A levels (P < 0.001). Enhancing host intestinal immune responses may be an important mechanism for S. boulardii-mediated protection against diarrheal illnesses.

Microbes and microbial toxins: paradigms for microbial-mucosal interactions II. The integrated response of the intestine to Clostridium difficile toxins.

Clostridium difficile, the major etiologic factor of antibiotic-associated diarrhea and colitis, mediates its effects by releasing two large protein exotoxins, toxins A and B. A major toxin effect is related to the disassembly of actin microfilaments, leading to impairment of tight junctions in human colonocytes. The mechanism of actin disaggregation involves monoglucosylation of the signaling proteins Rho A, Rac, and Cdc 42, which control stress fiber formation directly by toxins A and B. An important aspect of C. difficile infection is the acute necroinflammatory changes seen in patients with pseudomembranous colitis. The early mechanism of toxin-mediated inflammation involves toxin effects on cellular mitochondria, release of reactive oxygen species, and activation of mitogen-activated protein kinases and the transcription factor nuclear factor-kappaB. Injection of toxin A into animal intestine triggers secretion of fluid and intestinal inflammation characterized by epithelial cell destruction and neutrophil activation. A critical feature of C. difficile enterotoxicity is communication between enterocytes and lamina propria nerves, macrophages, and mast cells mediated via release of neuropeptides and proinflammatory cytokines.

Neurotensin stimulates Cl(-) secretion in human colonic mucosa In vitro: role of adenosine.

BACKGROUND & AIMS: Previous studies indicated that the peptide neurotensin (NT) stimulates Cl(-) secretion in animal small intestinal mucosa in vitro. In this study, we investigated whether NT causes Cl(-) secretion in human colonic mucosa and examined the mechanism of this response. METHODS: Human mucosal preparations mounted in Ussing chambers were exposed to NT. Drugs for pharmacologic characterization of NT-induced responses were applied 30 minutes before NT. RESULTS: Serosal, but not luminal, administration of NT (10(-8) to 10(-6) mol/L) induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive short-circuit current (Isc) increase that was inhibited by the specific nonpeptide NT receptor antagonists SR 48692 and SR 142948A, the neuronal blocker tetrodotoxin, and the prostaglandin synthesis inhibitor indomethacin. The mast cell stabilizer lodoxamide and the histamine 1 and 2 receptor antagonists pyrilamine and ranitidine, respectively, did not significantly alter NT-induced Isc increase. In contrast, the adenosine receptor 1 and 2 antagonists inhibited this secretory response, whereas the adenosine uptake inhibitors S-(4-nitrobenzyl)-6-thioguanosine and S-(4-nitrobenzyl)-6-thioinosine and the adenosine deaminase inhibitor deoxycoformycin potentiated NT-induced Isc increase. Serosal adenosine induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive Isc increase. CONCLUSIONS: NT stimulates chloride secretion in human colon by a pathway(s) involving mucosal nerves, adenosine, and prostaglandins.

Altered ion channel activity in murine colonic smooth muscle myocytes in an experimental colitis model.

We have investigated the activity of calcium and potassium channels in a murine model of experimental colitis. Colonic myocytes from dextran sulphate sodium (DSS)-treated mice were examined by whole cell patch clamp techniques. Myeloperoxidase activity was enhanced 3. 5-fold in DSS-treated mouse colon. In whole cell voltage clamp, depolarization predominantly evoked net transient outward currents in DSS-treated mice and inward Ca(2+) currents in control myocytes. Voltage-dependent L-type Ca(2+) currents were studied using intracellular Cs(+) in the patch pipette. Inward Ca(2+) currents were markedly suppressed in inflamed colon. The peak currents at +10 mV depolarization were -3.93 +/- 0.88 pA/pF in control (n = 12) and -1.14 +/- 0.19 (n = 10) in DSS mice. In contrast there was no change in the amplitude, kinetics, or steady-state inactivation properties of the transient outward currents in control or DSS-treated colonic myocytes. Inflammation significantly enhanced activation of the ATP-sensitive K(+) channel. At a holding potential of -50 mV, the K(ATP) channel opener lemakalim induced an inward current of 2.02 +/- 0.5 pA/pF in control (n = 20) and 4.19 +/- 1.17 pA/pF in DSS-treated colon. These currents were abolished by glibenclamide. The present results suggest that inflammation of the colon results in selective changes in ion channel activity of smooth muscle cells.

Clostridium difficile toxin A causes early damage to mitochondria in cultured cells.

BACKGROUND & AIMS: The mechanism by which Clostridium difficile toxin A causes actin depolymerization and cell rounding involves toxin internalization and subsequent monoglucosylation of the Rho family of proteins. This study explored toxin internalization and effects on mitochondrial function before cell rounding. METHODS: Chinese hamster ovary (CHO) cells were exposed to toxin A, and mitochondrial localization was assayed by confocal microscopy. Mitochondrial function was measured by adenosine triphosphate (ATP) concentration, mitochondrial permeability, and leakage of cytochrome c. RESULTS: Confocal microscopy showed toxin A colocalization with the mitochondrial protein GRP 75 at 5 minutes after toxin exposure. Between 5 and 15 minutes, toxin A caused an 80% diminution in cellular ATP levels; cell rounding and Rho glucosylation commenced between 15 and 30 minutes. Toxin A also resulted in reduction of mitochondrial membrane potential and a 2-3-fold increase in reactive oxygen radicals. Preincubation of CHO cells with the antioxidants butylated hydroxyanisole or butylated hydroxytoluene blocked the toxin A-induced increase in oxygen radicals and diminished cell rounding. Western blot analysis of toxin A-exposed isolated mitochondria showed a direct effect of toxin A on leakage of cytochrome c. CONCLUSIONS: The results show that extensive mitochondrial damage occurs within 15 minutes in CHO cells exposed to toxin A. Diminished ATP concentrations and increased oxygen radicals are likely to contribute to cytotoxicity from this bacterial toxin.

Epidermal growth factor receptor transactivation mediates substance P-induced mitogenic responses in U-373 MG cells.

Ligand-induced activation of G protein-coupled receptors is emerging as an important pathway leading to the activation of certain receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR). Substance P (SP) exerts many effects via activation of its G protein-coupled receptor (neurokinin-1, NK-1). SP participates in acute inflammation and activates key proteins involved in mitogenic pathways, such mitogen-activated protein kinases (MAPKs), stimulating DNA synthesis. We tested the hypothesis that SP-induced MAPK activation and DNA synthesis require activation of the EGFR. In U-373 MG cells, which express functional NK-1, SP induced tyrosine phosphorylation of several proteins including EGFR. SP induced formation of an activated EGFR complex containing the adapter proteins SHC and Grb2, but not c-Src. SP activated the MAPK pathway as shown by increased Erk2 kinase activity. SP induced Erk2 activation, and DNA synthesis was inhibited in cells transfected with a dominant negative EGFR plasmid lacking kinase activity, as well as in cells treated with a specific EGFR inhibitor. In addition, pertussis toxin, an inhibitor of Galpha(iota) protein subunits, prevented SP-induced EGFR transactivation and subsequent DNA synthesis. Our results implicate EGFR as an essential regulator in SP/NK-1-induced activation of the MAPK pathway and cell proliferation in U-373 MG cells, and these events are mediated by a pertussis toxin-sensitive Galpha protein. We suggest that this mechanism by which SP controls cell proliferation is an important pathway in tissue restoration and healing.

p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis.

Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury. In cultured cells, toxin A inactivates Rho proteins by monoglucosylation. In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms. We investigated the role of mitogen-activated protein (MAP) kinases in these events. In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascades within 1 to 2 minutes. Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinase was transient. Rho glucosylation became evident after 15 minutes. IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6. SB203580 also blocked monocyte necrosis and IL-1beta release caused by toxin A but not by other toxins. Finally, in mouse ileum, SB203580 prevented toxin A-induced neutrophil recruitment by 92% and villous destruction by 90%. Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis. p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A.

Clostridium difficile toxin A excites enteric neurones and suppresses sympathetic neurotransmission in the guinea pig.

BACKGROUND AND AIMS: Evidence suggests that the intestinal actions of Clostridium difficile toxin A-stimulation of secretion and motility, and an acute inflammatory response-have a neurally mediated component. METHODS: Direct intracellular electrophysiological recording of electrical and synaptic behaviour in enteric neurones was performed in the submucous plexus of guinea pig small intestine during exposure to the toxin. RESULTS: Application of toxin A affected both the electrical behaviour of the neuronal cell bodies and inhibitory noradrenergic neurotransmission to the cell bodies. Altered electrical behaviour included depolarisation and increased excitability. Tetrodotoxin or a histamine H(2) receptor antagonist did not affect the depolarisation evoked by toxin A. Failure of the histamine antagonist to suppress the actions of toxin A is evidence that its actions were not mediated by degranulation of intramural mast cells. The action of toxin A on neurotransmission was suppression of inhibitory postsynaptic potentials evoked in the neuronal cell bodies by stimulation of sympathetic nerve fibres that synapsed with the cell bodies. The inhibitory postsynaptic potentials were mediated by norepinephrine (noradrenaline) acting at postsynaptic alpha adrenoceptors on the cell bodies. Hyperpolarising responses evoked in the cell bodies by micropressure application of norepinephrine were unaffected by toxin A. This fulfils criteria for a presynaptic inhibitory action of toxin A to suppress release of norepinephrine from sympathetic postganglionic axons. CONCLUSIONS: Results suggest that the neural component of the action of toxin A involves both direct excitation of enteric neurones and suppression of norepinephrine release from postganglionic sympathetic nerve fibres in the enteric nervous system.

The role of neuroenteric hormones in intestinal infectious diseases.

It is now well established that communication among the enteric nerves, hormones, and neuropeptides plays a role in the pathogenesis of infectious gastrointestinal conditions. The results of several studies suggest that enteric nerves and hormones modulate important gastrointestinal functions such as intestinal motility and transport, intestinal permeability, fluid secretion, and inflammation in response to infectious agents. During the past year several gut-brain peptides, including substance P, neurotensin, and galanin, emerged as important mediators in the development and progress of intestinal infectious conditions. The intestinal mechanism of neuropeptide and hormone action involves direct effects via binding to receptors on the intestinal epithelium as well as on immune cells localized underneath the epithelial layer. Based on the available evidence from whole animal models it is possible that these new paradigms may offer novel therapeutic strategies in the treatment of gastrointestinal infections. This review summarizes recent progress on the identification of peptide hormones participating in the pathophysiology of infectious intestinal conditions and discusses the possible mechanism(s) of action involved in these processes.

Effects of Clostridium difficile toxins on epithelial cell barrier.

Clostridium difficile is the primary agent responsible for many patients with antibiotic-associated diarrhea and almost all patients with pseudomembranous colitis following antibiotic therapy. C. difficile infection is the most frequent form of colitis in hospitals and nursing homes and affects millions of patients in the United States and abroad. The first event in the pathogenesis of C. difficile infection involves alterations of the indigenous colonic microflora by antibiotics, followed by colonization with C. difficile. C. difficile causes diarrhea and colitis by releasing two high molecular weight protein exotoxins, toxin A and toxin B, with potent cytotoxic and enterotoxic properties. Evidence presented here indicates that C. difficile toxins compromise the epithelial cell barrier by at least two pathophysiologic pathways, one involving disaggregation of actin microfilaments in colonocytes via glucosylation of the Rho family of proteins leading to epithelial cell destruction and opening of the tight junctions, whereas the other appears to involve early release of proinflammatory cytokines from intestinal epithelial cells probably via activation of MAP kinases. We speculate that cytokines released from intestinal epithelial cells in response to toxin A exposure will diffuse into the lamina propria and activate macrophages, enteric nerves, and sensory neurons to release SP, CGRP, and NT, which, in turn, interact with immune and inflammatory cells and amplify the inflammatory response. Dissection of this inflammatory cascade may help us understand the pathophysiology of inflammatory diarrhea caused by this important pathogen.