The current global outbreak of Clostridium difficile infection exemplifies the major public health threat posed by clostridial glucosylating toxins. In the western world, C. difficile infection is one of the most prolific causes of bacterial-induced diarrhea and potentially fatal colitis. Two pathogenic enterotoxins, TcdA and TcdB, cause the disease. Vancomycin and metronidazole remain readily available treatment options for C. difficile infection, but neither is fully effective as is evident by high clinical relapse and fatality rates. Thus, there is an urgent need to find an alternative therapy that preferentially targets the toxins and not the drug-resistant pathogen. Recently, we addressed these critical issues in a Nature Medicine letter, describing a novel host defense mechanism for subverting toxin virulence that we translated into prototypic allosteric therapy for C. difficile infection. In this addendum article, we provide a continued perspective of this antitoxin mechanism and consider the broader implications of therapeutic allostery in combating gut microbial pathogenesis.

Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUTase4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a TUTase-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts is restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors while Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function, and have implications for the development of new strategies for cancer therapy.

Background & Aims

Cathelicidin (encoded by Camp) is an anti-microbial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease; we investigated its signaling mechanisms.

Methods

Quantitative, real-time, reverse transcription PCR, bacterial 16S PCR, immunofluorescence, and small interfering (si)RNA analyses were performed. Colitis was induced in mice using sodium dextran sulfate (DSS); levels of cathelicidin were measured in human primary monocytes.

Results

Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis, compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to E coli DNA induced expression of Camp mRNA, which required signaling by ERK; expression was reduced by siRNAs against toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9 −/− mice with DSS-induced colitis. Compared with wild-type mice, Camp −/− mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice.

Conclusions

Cathelicidin protects against colitis induction in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9–ERK signaling by bacterial DNA. This pathway might be involved in pathogenesis of ulcerative colitis.

Purpose

Psychological stress plays a role in the exacerbation of functional lower urinary tract disorders such as painful bladder syndrome and overactive bladder. To better understand the mechanism underlying this relationship, we characterized changes in micturition, anxiety-related behavior, and bladder pathology in rats exposed to repeated water avoidance (WA) stress.

Methods

Twenty-four Wistar rats were subjected to WA stress or sham. Immediately following acute (day 1) and chronic (day 10) stress or sham, rats were placed in a metabolic cage for a 2-hour voiding behavior assessment. Voiding parameters were compared to baseline values obtained prior to stress. Four animals from each group were sacrificed on day 10 and bladders harvested for histologic and gene expression studies. The remaining 8 animals per group underwent repeated voiding assessment every 3 days for 1 month followed by 10 days of repeat WA stress or sham. Bladder histology and gene expression were studied.

Results

Rats exposed to WA stress developed a significant increase in micturition frequency and decrease in latency to void, voiding interval and volume of first void compared to sham and baseline. Alterations in micturition persisted for approximately 1 month. Stressed rats showed increased fecal pellet excretion and anxiety-like behavior. Additionally, bladder specimens from stressed animals revealed increased angiogenesis, and increased total and activated mast cells.

Conclusions

In rats, repeated psychological stress results in lasting alterations in micturition frequency, interval, and volume. This rodent model may represent a valid tool for studying syndromes characterized by increased urinary frequency.

Background & Aims

Corticotropin-releasing factor receptor-1 (CRF1) mediates the stress-induced colonic motor activity. Less is known about the role of CRF2 in the colonic response to stress.

Methods

We studied colonic contractile activity (CCA) in rats and CRF2-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute, partial-restraint stress (PRS), and/or intraperitoneal (IP) injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF1 and CRF2 using immunohistochemical and immunoblot analyses. We measured phosphorylation of ERK1/2 by CRF ligands in primary cultures of LMMP-neurons (PC-LMMPn) and cAMP production in HEK-293 cells transfected with CRF1 and/or CRF2.

Results

In rats, a selective agonist of CRF2 (urocortin 2) reduced CRF-induced defecation (>50%), CCA, and Fos expression in the colonic LMMP. A selective antagonist of CRF2 (astressin2-B) increased these responses. Urocortin 2 reduced PRS-induced CCA in wild-type and CRF-overexpressing mice, whereas disruption of CRF2 increased PRS-induced CCA and CRF-induced defecation. CRF2 co-localized with CRF1 and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of ERK in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF1 (NBI35965) or astressin2-B, respectively. The EC50 for the CRF-induced cAMP response was 8.6 nM in HEK-293 cells that express only CRF1; this response was suppressed 10-fold in cells that express CRF1 and CRF2.

Conclusions

In colon tissues of rodents, CRF2 activation inhibits CRF1 signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF2 function impairs colonic coping responses to stress.

Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.

OBJECTIVES

Low-grade colonic mucosal inflammation has been postulated to have an important role in the pathophysiology of irritable bowel syndrome (IBS). The objectives of this study were (i) to identify serum and tissue-based immunological and neuroendocrine markers associated with mucosal inflammation in male (M) and female (F) patients with non-post-infectious IBS (non-PI-IBS) compared with healthy controls and (ii) to assess possible correlations of such markers with IBS symptoms.

METHODS

Sigmoid mucosal biopsies were obtained from 45 Rome II positive IBS patients without a history of PI-IBS (26 F, 35.5% IBS-C, 33.3% IBS-D, 31.1% IBS-A/M) and 41 healthy controls (22 F) in order to measure immunological markers (serum cytokine levels, colonic mucosal mRNA levels of cytokines, mucosal immune cell counts) and neuroendocrine markers associated with mucosal inflammation (corticotropin releasing factor- and neurokinin (NK)-related ligands and receptors, enterochromaffin cells). Symptoms were measured using validated questionnaires.

RESULTS

Of all the serum and mucosal cytokines measured, only interleukin-10 (IL-10) mRNA expression showed a group difference, with female, but not male, patients showing lower levels compared with female controls (18.0 ± 2.9 vs. 29.5 ± 4.0, P = 0.006). Mucosal mRNA expression of NK-1 receptor was significantly lower (1.15 ± 0.19 vs. 2.66 ± 0.56, P = 0.008) in female, but not male, patients compared with healthy controls. No other significant differences were observed.

CONCLUSIONS

Immune cell counts and levels of cytokines and neuropeptides that are associated with inflammation were not significantly elevated in the colonic mucosa of non-PI-IBS patients, and did not correlate with symptoms. Thus, these findings do not support that colonic mucosal inflammation consistently has a primary role in these patients. However, the finding of decreased IL-10 mRNA expression may be a possible biomarker of IBS and warrants further investigation.

Several clinical trials and experimental studies strongly suggest a place for Saccharomyces boulardii as a biotherapeutic agent for the prevention and treatment of several gastrointestinal diseases. S. boulardii mediates responses resembling the protective effects of the normal healthy gut flora. The multiple mechanisms of action of S. boulardii and its properties may explain its efficacy and beneficial effects in acute and chronic gastrointestinal diseases that have been confirmed by clinical trials. Caution should be taken in patients with risk factors for adverse events. This review discusses the evidence for efficacy and safety of S. boulardii as a probiotic for the prevention and therapy of gastrointestinal disorders in humans.

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins1–4. Virulence is dependent on the autoactivation of a toxin cysteine protease5–9, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6)10–17. Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins18,19. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.

BACKGROUND & AIMS

The corticotrophin-releasing hormone (CRH) family of peptides modulates intestinal inflammation and the CRH receptor 2 (CRHR2) suppresses postnatal angiogenesis in mice. We investigated the functions of CRHR1 and CRHR2 signaling during intestinal inflammation and angiogenesis.

METHODS

The activities of CRHR1 and CRHR2 were disrupted by genetic deletion in mice or with selective antagonists. A combination of in vivo, ex vivo, and in vitro measures of angiogenesis were used to determine their activity. CRHR1−/− mice and CRHR2−/− mice with dextran sodium sulfate-induced colitis were analyzed in comparison with wild-type littermates (controls).

RESULTS

Colitis was significantly reduced in mice in which CRHR1 activity was disrupted by genetic deletion or with an antagonist, determined by analyses of survival rate, weight loss, histological scores, and cytokine production. Inflammation was exacerbated in mice in which CRHR2 activity was inhibited by genetic deletion or with an antagonist, compared with controls. The inflamed intestines of CRHR1−/− mice had reduced microvascular density and expression of vascular endothelial growth factor (VEGF)-A, whereas the intestines of CRHR2−/− mice had increased angiogenesis and VEGF-A levels. An antagonist of VEGFR2 activity alleviated colitis in CRHR2−/− mice. Ex vivo aortic vessel outgrowth was reduced when CRHR1 was deficient but increased when CRHR2 was deficient. The CRHR1 preferred agonist CRH stimulated tube formation, proliferation, and migration of cultured intestinal microvascular endothelial cells by phosphorylating Akt whereas the specific CRHR2 agonist Urocortin III had opposite effects.

CONCLUSION

CRHR1 promotes intestinal inflammation, as well as endogenous and inflammatory angiogenesis whereas CRHR2 inhibits these activities.

TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, a subset of Crohn's disease (CD) patients with the risk TL1A haplotype is associated with elevated TL1A expression and a more severe disease course. To investigate the in vivo role of elevated TL1A expression, we generated two transgenic (Tg) murine models with constitutive Tl1a expression in either lymphoid or myeloid cells. Compared to wildtype (WT) mice, constitutive expression of Tl1a in either lymphoid or myeloid cells showed mild patchy inflammation in the small intestine, which was more prominent in the ileum. In addition, mice with constitutive Tl1a expression exhibited enhanced intestinal and colonic fibrosis compared to WT littermates. The percentage of T cells expressing the gut homing chemokine receptors CCR9 and CCR10 was higher in the Tl1a Tg mice compared to WT littermates. Sustained expression of Tl1A in T cells also lead to increased Foxp3+ Treg cells. T cells or antigen presenting cells (APC) with constitutive expression of Tl1a were found to have a more activated phenotype and mucosal mononuclear cells exhibit enhanced Th1 cytokine activity. These results indicated an important role of TL1A in mucosal T cells and APC function and showed that up-regulation of TL1A expression can promote mucosal inflammation and gut fibrosis.

C. difficile toxin A impairs tight junction function of colonocytes by glucosylation of Rho family proteins causing actin filament disaggregation and cell rounding. We investigated the effect of toxin A on focal contact formation by assessing its action on focal adhesion kinase (FAK) and the adapter protein paxillin. Exposure of NCM460 human colonocytes to toxin A for 1 hour resulted in complete dephosphorylation of FAK and paxillin, while protein tyrosine phosphatase activity was reduced. Blockage of toxin A-associated glucosyltransferase activity by co-incubation with UDP 2′3′dialdehyde did not reduce toxin A-induced FAK and paxillin dephosphorylation. GST-pull down and in vitro kinase activity experiments demonstrated toxin A binding directly to the catalytic domain of Src with suppression of its kinase activity. Direct binding of toxin A to Src, independent of any effect on protein tyrosine phosphatase or Rho glucosylation, inhibits Src kinase activity followed by FAK/paxillin inactivation. These mechanisms may contribute to toxin A-inhibition of colonocyte focal adhesion that occurs in human colonic epithelium exposed to toxin A.

Background/Aims

Urocortin II (UcnII) is a neuropeptide that binds with high affinity to the corticotropin‐releasing hormone receptor 2 (CRHR2) in peripheral tissues. UcnII is synthesised in the intestine, but its role in human intestinal inflammation is largely unknown.

Methods

Responses of human colonic epithelial cells expressing CRHR2 to stimulation by UcnII were measured using ELISA, western blot analysis, real‐time reverse transcription‐PCR (RT‐PCR) and interleukin (IL)8 promoter activity. Expression levels of CRHR2 and UcnII in human colitis were determined by immunofluorescence and real‐time RT‐PCR in mucosal biopsies from patients with Crohn's and ulcerative colitis, and in human intestinal xenografts after exposure to Clostridium difficile toxin A.

Results

It is reported here that expression of CRHR2 mRNA and protein in human colonic epithelial cells (HT‐29) are increased by exposure to C difficile toxin A or tumour necrosis factor (TNF)α. Stimulation of non‐transformed NCM460 colonocytes overexpressing CRHR2α receptor with UcnII resulted in a time‐ and concentration‐dependent increase in IL8 production. UcnII stimulation also led to activation of nuclear factor‐κB (NF‐κB) and mitogen‐acivated protein (MAP) kinase in these cells, as evidenced by degradation of IκBα and phosphorylation of the p65 subunit of NF‐κB and extracellularly regulated kinase (ERK) 1/2. Furthermore, expression of UcnII and CRHR2 mRNA was increased in mucosal samples of patients with inflammatory bowel disease, and after exposure of human intestinal xenografts to C difficile toxin A.

Conclusions

These results suggest that UcnII has pro‐inflammatory effects in human intestinal cells via the CRHR2α receptor and may play an important role in the pathophysiology of colitis in humans.

Saccharomyces boulardii (Sb) is a probiotic yeast with anti-inflammatory and antimicrobial activities and has been used for decades in the prevention and treatment of a variety of human gastrointestinal disorders. We reported previously that Sb modulates host inflammatory responses through down regulation of Erk1/2 MAP kinase activities both in vitro and in vivo. The aim of this study was to identify upstream mediators responsible for Erk1/2 inactivation and to examine the effects of Sb on tumor development in ApcMin mice. We found that the EGF receptor was deactivated upon exposure to Sb leading to inactivation of both the EGFR-Erk and EGFR-Akt pathways. In human colonic cancer cells, Sb prevented EGF induced proliferation, reduced cell colony formation and promoted apoptosis. HER-2, HER-3 and IGF-1R were also found to be inactivated by Sb. Oral intake of Sb reduced intestinal tumor growth and dysplasia in C57BL/6J Min/+ (ApcMin) mice. Thus, in addition to its anti-inflammatory effects, S. boulardii inhibits EGFR and other receptor tyrosine kinase signaling and thereby may also serve a novel therapeutic or prophylactic role in intestinal neoplasia.

Background & Aims

Toll-like receptor (TLR)-dependent signaling pathways have been proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Here we investigated whether TLR5-dependent signaling modulates colonic tumor development in a mouse xenograft model of human colon cancer.

Methods

The expression of MyD88 or TLR5 was stably knocked down in human colon cancer cells (DLD-1). Nude mice were subcutaneously implanted with MyD88-KD, TLR5-KD, or control cells (n=16) to examine the pathophysiology of tumor xenografts. Protein micro-array assessed the differential expression of cytokines in these tumors. Leukocyte infiltration and tumor angiogenesis were assessed by immunohistochemistry with antibodies against neutrophil (Gr-1, 7/4) or macrophage specific antigens (CD68, F4-80), and the vascular endothelial cell marker PECAM-1/CD31, respectively. Tumor xenografts from DLD-1 cells were treated with flagellin (5.0 µg/kg, one injection/every 2 days for 3 weeks) and tumor regression and histopathology of these tumors were examined.

Results

Lack of MyD88 or TLR5 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenograft of human colon cancer. In contrast, TLR5 activation by peritumoral flagellin treatment substantially increased tumor necrosis, leading to significant tumor regression. Tumors from MyD88- or TLR5-KD cells revealed the reduced production of neutrophil attracting chemokines (ENA-78, MIP3α, and IL-8). Consequently, neutrophil infiltration was dramatically diminished in MyD88 or TLR5 deficient tumor xenografts, while tumor-associated macrophage infiltration or angiogenesis was not changed.

Conclusions

TLR5 engagement by flagellin mediates innate immunity and elicits potent anti-tumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate colonic tumors.

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor κ B (NF-κB)-driven proin-flammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-κB activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10−7 M) induced an early (1 min) phosphorylation of the PKC isoforms PKCδ, PKCθ, and PKCϵ, followed by I-κB kinase, IκBα, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 µM) blocked SP-induced IκBα and p65 phosphorylation and IL-8 production. The PKCδ inhibitor rottlerin at a low concentration (1 µM), but not pseudosubstrate PKCθ and PKCϵ inhibitors (10 µM), significantly reduced IL-8 secretion. PKCδ silencing by RNA interference reduced PKCδ protein expression and SP-induced PKCδ phosphorylation that was associated with diminished IL-8 promoter and NF-κB luciferase activities in response to SP. Moreover, overexpression of wild-type PKCδ increased SP-induced IL-8 promoter- and NF-κB-driven luciferase activities that were rottlerin-sensitive. We conclude that PKCδ plays an important role in SP-induced proinflammatory signaling in human colonocytes.

Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Cθ (PKCθ), but not PKCε and PKCδ, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.

Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.

Clostridium difficile-associated colitis is an increasing cause of morbidity and mortality in hospitalized patients, with high relapse rates following conventional therapy. We sought to determine the efficacy of rifaximin, a novel nonabsorbed antibiotic, in the hamster model of C. difficile-associated diarrhea (CDAD). Hamsters received clindamycin subcutaneously and 24 h later were infected by gavage with one of two C. difficile strains: a reference strain (VPI 10463) and a current epidemic strain (BI17). Vancomycin (50 mg/kg of body weight) or rifaximin (100, 50, and 25 mg/kg) were then administered orally for 5 days beginning either on the same day as infection (prevention) or 24 h later (treatment). Therapeutic effects were assessed by weight gain, histology, and survival. We found that rifaximin was as effective as vancomycin in the prevention and treatment of colitis associated with the two C. difficile strains that we examined. There was no relapse after treatment with vancomycin or rifaximin in hamsters infected with the BI17 strain. Hamsters infected with the VPI 10463 strain and treated with rifaximin did not develop relapsing infection within a month of follow-up, whereas the majority of vancomycin-treated animals relapsed (0% versus 75%, respectively; P < 0.01). In conclusion, rifaximin was found to be an effective prophylactic and therapeutic agent for CDAD in hamsters and was not associated with disease recurrence. These findings, in conjunction with the pharmacokinetic and safety profiles of rifaximin, suggest that it is an attractive candidate for clinical use for CDAD.

Background & Aims

We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis.

Methods

We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333.

Results

TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%−28% and tissue and fluid myeloperoxidase by 31%−71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis.

Conclusions

PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms.

Background and Aims:

Substance P (SP) is an 11-amino acid peptide that belongs to the tachykinin family of peptides. SP acts in the brain and in the periphery as a neuropeptide, neurotransmitter and hormone affecting diverse physiological pathways, mainly via its high affinity neurokinin-1 receptor (NK-1R). Its presence in the hypothalamus and other areas of the brain that regulate feeding as well as in the stomach and small intestine prompted us to investigate its role on appetite control and energy balance.

Methods:

CJ 012,255 (CJ), a SP antagonist which binds to NK-1R, was injected in lean, diet induced (DIO), and genetically obese mice ob/ob and its effects on body weight, adipocity and insulin sensitivity were investigated.

Results:

CJ administration prevented weight gain and accumulation of fat after two weeks of high fat feeding, while similar CJ treatment in obese mice (following two moths of high fat diet) resulted in weight loss, reduction in adiposity and improvement of insulin sensitivity, in part due to inhibition of food intake. The effects of SP in the control of energy balance are, at least in part, leptin independent, since CJ treatment was also effective in leptin deficient mice. Peripheral SP administration resulted in a mild, dose-dependent increase in food intake, evident 3 hrs post-SP injection.

Conclusions:

SP per se acts as an orexigenic neuropeptide and promotes weight gain in mice via NK-1R coupling. We speculate that NK-1R antagonists, already tested in clinical trials for various diseases, may represent a potential target against obesity.

Although vancomycin and metronidazole effectively treat Clostridium difficile-associated diarrhea and colitis (CDAD), their use is associated with a high incidence of relapsing C. difficile infection. Rifalazil is a new benzoxazinorifamycin that possesses activity against Mycobacterium tuberculosis and gram-positive bacteria. Here we compared rifalazil and vancomycin for effectiveness in preventing or treating clindamycin-induced cecitis in a hamster model of CDAD. Golden Syrian hamsters were injected subcutaneously with clindamycin phosphate (10 mg/kg), followed 24 h later by C. difficile gavage. Hamsters received by gavage for 5 days vehicle, vancomycin (50 mg/kg), or rifalazil (20 mg/kg) either simultaneously with (prophylactic protocol) or 24 h after C. difficile administration (treatment protocol). While all vehicle-administered animals became moribund within 48 h of C. difficile administration, no rifalazil- or vancomycin-treated animals in either protocol showed signs of morbidity after 7 days. Ceca of rifalazil-treated animals showed absence of epithelial cell damage, significantly reduced congestion and edema, and less, but not statistically significantly less, neutrophil infiltration compared to those of vehicle-treated animals. In contrast, vancomycin-treated animals demonstrated severe epithelial cell damage and mildly reduced congestion and edema. Moreover, hamsters relapsed and tested C. difficile toxin positive (by enzyme-linked immunosorbent assay) 10 to 15 days after discontinuation of vancomycin treatment. None of the rifalazil-treated hamsters showed signs of disease or presence of toxins in their feces 30 days after discontinuation of treatment. Our results indicate that once daily rifalazil may be superior to vancomycin for curative treatment of CDAD.

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.

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 NH2-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-1β 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.

The neuropeptide neurotensin mediates several intestinal functions, including chloride secretion, motility, and cellular growth. However, whether this peptide participates in intestinal inflammation is not known. Toxin A, an enterotoxin from Clostridium difficile, mediates pseudomembranous colitis in humans. In animal models, toxin A causes an acute inflammatory response characterized by activation of sensory neurons and intestinal nerves and immune cells of the lamina propria. Here we show that neurotensin and its receptor are elevated in the rat colonic mucosa following toxin A administration. Pretreatment of rats with the neurotensin receptor antagonist SR-48,692 inhibits toxin A–induced changes in colonic secretion, mucosal permeability, and histologic damage. Exposure of colonic explants to toxin A or neurotensin causes mast cell degranulation, which is inhibited by SR-48,692. Because substance P was previously shown to mediate mast cell activation, we examined whether substance P is involved in neurotensin-induced mast cell degranulation. Our results show that neurotensin-induced mast cell degranulation in colonic explants is inhibited by the substance P (neurokinin-1) receptor antagonist CP-96,345, indicating that colonic mast activation in response to neurotensin involves release of substance P. We conclude that neurotensin plays a key role in the pathogenesis of C. difficile–induced colonic inflammation and mast cell activation.