Background & Aims Clostridium difficile (C.difficile) is the leading cause of nosocomial infectious diarrhea. Increasing incidence, antibiotic resistance and more virulent strains have dramatically increased the number of C.difficile-related deaths worldwide. The innate host response mechanisms to C.difficile are not resolved; however, we hypothesize that hypoxia-inducible factor (HIF-1) plays an innate protective role in C.difficile colitis. Thus, we assessed the impact of C.difficile toxins on the regulation of HIF-1 and evaluated the role of HIF-1α in C.difficile-mediated injury/inflammation. Methods In vitro studies assessed HIF-1α mRNA, protein levels and DNA binding events in human mucosal biopsies and Caco-2 cells exposed to C.difficile toxins. In vivo studies employed the murine ileal loop model of C.difficile toxin-induced intestinal injury. Mice with targeted deletion of HIF-1α in the intestinal epithelium were used to assess the impact of HIF-1α signaling in response to C.difficile toxin. Results Mucosal biopsies and Caco-2 cells exposed to C.difficile toxin displayed a significant increase in HIF-1α transcription and protein levels. Toxin-induced DNA binding was also observed in Caco-2 cells. Toxin-induced HIF-1α accumulation was attenuated by nitric oxide synthase inhibitors. In vivo, deletion of intestinal epithelial HIF-1α resulted in more severe toxin-induced intestinal injury and inflammation. In contrast, stabilization of HIF-1α, with dimethyloxallyl glycine, attenuated toxin-induced injury and inflammation. This was associated with an induction of HIF-1-regulated protective factors including VEGFa, CD73 and intestinal trefoil factor and down-regulation of proinflammatory molecules TNF and KC. Conclusions Our study is the first to describe the innate protective role for HIF-1α in response to C.difficile toxins. Harnessing the innate protective actions of HIF-1α in response to C.difficile toxins may represent a novel form of therapy for C.difficile-associated disease.

Short-type DBE combined with several technical innovations enabled us to perform ERCP in most patients with altered gastrointestinal anatomy.

The pathogenesis of irritable bowel syndrome (IBS) is considered to be multifactorial and includes psychosocial factors, visceral hypersensitivity, infection, microbiota and immune activation. It is becoming increasingly clear that low-grade inflammation is present in IBS patients and a number of biomarkers have emerged. This review describes the evidence for low-grade inflammation in IBS and explores its mechanism with particular focus on gastrointestinal motor dysfunction. Understanding of the immunological basis of the altered gastrointestinal motor function in IBS may lead to new therapeutic strategies for IBS.

2ϩsensitization in rat ileal longitudinal smooth muscle. Am J Physiol Gastrointest Liver Physiol 293: G699-G710, 2007. First published July 26, 2007; doi:10.1152/ajpgi.00214.2007.-We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca 2ϩ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 M microcystin induced LC20 diphosphorylation and contraction of ␤-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 M, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca 2ϩ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle. ileum; protein kinase C; myosin phosphatase; mitogen-activated protein kinase; protein kinase C-potentiated phosphatase inhibitor protein of 17 kDa; myosin-targeting subunit of myosin light chain phosphatase SMOOTH MUSCLE CONTRACTION is a dynamic and highly regulated process. The contractile state of smooth muscle is mainly regulated by phosphorylation of the 20-kDa myosin regulatory light chain (LC 20 ] i )} is the primary determinant of smooth muscle contraction. Force can be further increased through signaling pathways that modulate MLCK and/or MLCP activities. The Ca 2ϩ sensitivity of contraction can be affected by any change in the ratio of MLCK:MLCP activity. A decrease in MLCP activity will shift the balance in favor of MLCK, resulting in a greater degree of LC 20 phosphorylation and contraction. The phenomenon of ...

Inflammation and immune activation in the gut are usually accompanied by alteration of gastrointestinal (GI) motility. In infection, changes in motor function have been linked to host defense by enhancing the expulsion of the infectious agents. In this review, we describe the evidence for inflammation and immune activation in GI infection, inflammatory bowel disease, ileus, achalasia, eosinophilic esophagitis, microscopic colitis, celiac disease, pseudo-obstruction and functional GI disorders. We also describe the possible mechanisms by which inflammation and immune activation in the gut affect GI motility. GI motility disorder is a broad spectrum disturbance of GI physiology. Although several systems including central nerves, enteric nerves, interstitial cells of Cajal and smooth muscles contribute to a coordinated regulation of GI motility, smooth muscle probably plays the most important role. Thus, we focus on the relationship between activation of cytokines induced by adaptive immune response and alteration of GI smooth muscle contractility. Accumulated evidence has shown that Th1 and Th2 cytokines cause hypocontractility and hypercontractility of inflamed intestinal smooth muscle. Th1 cytokines downregulate CPI-17 and L-type Ca(2+) channels and upregulate regulators of G protein signaling 4, which contributes to hypocontractility of inflamed intestinal smooth muscle. Conversely, Th2 cytokines cause hypercontractilty via signal transducer and activator of transcription 6 or mitogen-activated protein kinase signaling pathways. Th1 and Th2 cytokines have opposing effects on intestinal smooth muscle contraction via 5-hydroxytryptamine signaling. Understanding the immunological basis of altered GI motor function could lead to new therapeutic strategies for GI functional and inflammatory disorders.

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Inflammatory bowel disease (IBD) is associated with intestinal smooth muscle dysfunction. Many smooth muscle contractile events are associated with alterations in Ca 2ϩ -sensitizing pathways. The aim of the present study was to assess the effect of colitis on Ca 2ϩ sensitization and the signaling pathways responsible for contractile dysfunction in murine experimental colitis. Colitis was induced in BALB/c mice by providing 5% dextran sulfate sodium (DSS) in drinking water for 7 days. Contractile responses of colonic circular smooth muscle strips to 118 mM K ϩ and carbachol (CCh) were assessed. DSS induced a T H 2 colitis [increased interleukin (IL)-4 and IL-6] with no changes in T H 1 cytokines. Animals exposed to DSS had increased CCh-induced contraction (3.5-fold) and CCh-induced Ca 2ϩ -sensitization (2.2-fold) responses in intact and ␣-toxin permeabilized colonic smooth muscle, respectively. The contributions of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) to CCh-induced contractions were significantly increased during colitis. Ca 2ϩ -independent contraction induced by microcystin was potentiated (1.5-fold) in mice with colitis. ERK and p38MAPK (but not Rho-associated kinase) contributed to this potentiation. ERK1/2 and p38MAPK expression were increased in the muscularis propria of colonic tissue from both DSS-treated mice and patients with IBD (ulcerative colitis Ͼ Ͼ Crohn's disease). Murine T H 2 colitis resulted in colonic smooth muscle hypercontractility with increased Ca 2ϩ sensitization. Both ERK and p38MAPK pathways contributed to this contractile dysfunction, and expression of these molecules was altered in patients with IBD.It has been hypothesized that an imbalance of the T H 1/T H 2 immune response plays a central role in the pathogenesis of inflammatory bowel disease (IBD) (Neurath et al., 2002;Xavier and Podolsky, 2007). The mediators of intestinal inflammation are also known to impair gastrointestinal motility in human disease and animal models, which may be a reflection of altered smooth muscle function. Alterations in gastrointestinal motility with resultant changes in transit contribute to the abdominal pain, intestinal cramping, and diarrhea characteristically associated with IBD. Furthermore, defects in smooth muscle function can lead to the development of toxic megacolon. While it is commonly accepted that smooth muscle contractility is altered in inflamed intestine, there is still considerable disagreement whether intestinal contractility is increased or decreased in IBD. T H 1 and T H 2 immune responses play different roles in dysfunction of smooth muscle contractility. Tumor necrosis factor

This adoptive transfer model of colitis was associated with augmented Th1 and Th17 responses, and Treg were capable of suppressing colonic inflammation by downregulating Th17 responses as well as Th1 responses via TGF-β. Consequently, Treg transfer therapy is expected to be efficacious for IBD even if Th17 is involved in the pathogenesis.