Eikichi Ihara

Hypoxia-Inducible Factor Signaling Provides Protection in Clostridium difficile-Induced Intestinal Injury

BACKGROUND & AIMS Clostridium difficile is the leading cause of nosocomial infectious diarrhea. Antibiotic resistance and increased virulence of strains have increased the number of C difficile-related deaths worldwide. The innate host response mechanisms to C difficile are not resolved; we propose that hypoxia-inducible factor (HIF-1) has an innate, protective role in C difficile colitis. We studied the impact of C difficile toxins on the regulation of HIF-1 and evaluated the role of HIF-1alpha in C difficile-mediated injury/inflammation. METHODS We assessed HIF-1alpha mRNA and protein levels and DNA binding in human mucosal biopsy samples and Caco-2 cells following exposure to C difficile toxins. We used the mouse ileal loop model of C difficile toxin-induced intestinal injury. Mice with targeted deletion of HIF-1alpha in the intestinal epithelium were used to assess the effects of HIF-1alpha signaling in response to C difficile toxin. RESULTS Mucosal biopsy specimens and Caco-2 cells exposed to C difficile toxin had a significant increase in HIF-1alpha transcription and protein levels. Toxin-induced DNA binding was also observed in Caco-2 cells. Toxin-induced HIF-1alpha accumulation was attenuated by nitric oxide synthase inhibitors. In vivo deletion of intestinal epithelial HIF-1alpha resulted in more severe, toxin-induced intestinal injury and inflammation. In contrast, stabilization of HIF-1alpha with dimethyloxallyl glycine attenuated toxin-induced injury and inflammation. This was associated with induction of HIF-1-regulated protective factors (such as vascular endothelial growth factor-alpha, CD73, and intestinal trefoil factor) and down-regulation of proinflammatory molecules such as tumor necrosis factor and Cxcl1. CONCLUSIONS HIF-1alpha protects the intestinal mucosa from C difficile toxins. The innate protective actions of HIF-1alpha in response to C difficile toxins be developed as therapeutics for C difficile-associated disease.

Low-grade inflammation plays a pivotal role in gastrointestinal dysfunction in irritable bowel syndrome

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.

Thrombin causes endothelium‐dependent biphasic regulation of vascular tone in the porcine renal interlobar artery

Using a method employing front‐surface fura‐2 fluorometry to measure the cytosolic Ca2+ concentration, [Ca2+]i, the mechanism of endothelium‐dependent regulation of vascular tone by thrombin was studied in porcine renal interlobar arterial strips. At concentrations lower than 3 u ml−1, thrombin evoked only early transient relaxation, while at 3 u ml−1 and higher concentrations, thrombin caused an early relaxation and a subsequent transient contraction. Both thrombin‐induced relaxation and contraction were abolished by removing the endothelium. Similar biphasic responses were observed with a protease‐activated receptor‐1‐activating peptide. Early relaxation was associated with a decrease in [Ca2+]i, while the transient contraction was not associated with a change in [Ca2+]i of smooth muscle cells. A thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10−5 M ONO‐3708) completely inhibited the thrombin‐induced contraction, whereas a thromboxane A2 synthase inhibitor (10−5 M OKY‐046) only partly inhibited it. When the thrombin‐induced contraction was inhibited by ONO‐3708, either pretreatment with Nω‐nitro‐L‐arginine methylester (L‐NAME) or an increase in the amount of external K+ to 40 mM did not abolish thrombin‐induced relaxation during phenylephrine‐induced sustained contraction. However, the combination of pretreatment with L‐NAME and an elevation of external K+ to 40 mM completely abolished the relaxation. There was no significant difference in the concentration‐dependent effects of thrombin on the initial early relaxation between conditions in which the contractile components either were or were not inhibited. Thrombin is thus considered to mainly activate protease‐activated receptor‐1 and cause a biphasic response, early relaxation and a transient contraction, in the porcine renal interlobar artery in an endothelium‐dependent manner. The thrombin‐induced endothelium‐dependent relaxation was mediated by nitric oxide and hyperpolarizing factors, while the contraction was mediated by TXA2 and PGH2.

Mitogen-activated protein kinase pathways contribute to hypercontractility and increased Ca2+ sensitization in murine experimental colitis.

Inflammatory bowel disease (IBD) is associated with intestinal smooth muscle dysfunction. Many smooth muscle contractile events are associated with alterations in Ca2+-sensitizing pathways. The aim of the present study was to assess the effect of colitis on Ca2+ 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+ and carbachol (CCh) were assessed. DSS induced a TH2 colitis [increased interleukin (IL)-4 and IL-6] with no changes in TH1 cytokines. Animals exposed to DSS had increased CCh-induced contraction (3.5-fold) and CCh-induced Ca2+-sensitization (2.2-fold) responses in intact and α-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. Ca2+-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 ≫ Crohns disease). Murine TH2 colitis resulted in colonic smooth muscle hypercontractility with increased Ca2+ sensitization. Both ERK and p38MAPK pathways contributed to this contractile dysfunction, and expression of these molecules was altered in patients with IBD.

Improved techniques for double-balloon-enteroscopy-assisted endoscopic retrograde cholangiopancreatography

AIM To investigate the clinical outcome of double balloon enteroscopy (DBE)-assisted endoscopic retrograde cholangiopancreatography (DB-ERCP) in patients with altered gastrointestinal anatomy. METHODS Between September 2006 and April 2011, 47 procedures of DB-ERCP were performed in 28 patients with a Roux-en-Y total gastrectomy (n = 11), Billroth II gastrectomy (n = 15), or Roux-en-Y anastomosis with hepaticojejunostomy (n = 2). DB-ERCP was performed using a short-type DBE combined with several technical innovations such as using an endoscope attachment, marking by submucosal tattooing, selectively applying contrast medium, and CO₂ insufflations. RESULTS The papilla of Vater or hepaticojejunostomy site was reached in its entirety with a 96% success rate (45/47 procedures). There were no significant differences in the success rate of reaching the blind end with a DBE among Roux-en-Y total gastrectomy (96%), Billroth II reconstruction (94%), or pancreatoduodenectomy (100%), respectively (P = 0.91). The total successful rate of cannulation and contrast enhancement of the target bile duct in patients whom the blind end was reached with a DBE was 40/45 procedures (89%). Again, there were no significant differences in the success rate of cannulation and contrast enhancement of the target bile duct with a DBE among Roux-en-Y total gastrectomy (88 %), Billroth II reconstruction (89%), or pancreatoduodenectomy (100%), respectively (P = 0.67). Treatment was achieved in all 40 procedures (100%) in patients whom the contrast enhancement of the bile duct was successful. Common endoscopic treatments were endoscopic biliary drainage (24 procedures) and extraction of stones (14 procedures). Biliary drainage was done by placement of plastic stents. Stones extraction was done by lithotomy with the mechanical lithotripter followed by extraction with a basket or by the balloon pull-through method. Endoscopic sphincterotomy was performed in 14 procedures with a needle precutting knife using a guidewire. The mean total duration of the procedure was 93.6 ± 6.8 min and the mean time required to reach the papilla was 30.5 ± 3.7 min. The mean time required to reach the papilla tended to be shorter in Billroth II reconstruction (20.9 ± 5.8 min) than that in Roux-en-Y total gastrectomy (37.1 ± 4.9 min) but there was no significant difference (P = 0.09). A major complication occurred in one patient (3.5%); perforation of the long limb in a patient with Billroth II anastomosis. CONCLUSION Short-type DBE combined with several technical innovations enabled us to perform ERCP in most patients with altered gastrointestinal anatomy.

The mechanism of bradykinin-induced endothelium-dependent contraction and relaxation in the porcine interlobar renal artery

The mechanism of endothelium‐dependent regulation of vascular tone of bradykinin was investigated by simultaneously monitoring the changes in the cytosolic Ca2+ concentration and the force of smooth muscle in fura‐2‐loaded strips of the porcine renal artery with endothelium. During phenylephrine‐induced sustained contraction, bradykinin (>3×10−9 M) caused endothelium‐dependent triphasic changes in the force of the strips, composed of an initial relaxation, a subsequent transient contraction and a late sustained relaxation. At low concentrations (10−10–10−9 M), bradykinin caused an endothelium‐dependent biphasic relaxation with no contraction. A thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor antagonist (10−5 M ONO‐3708) completely inhibited, while a TXA2 synthase inhibitor (10−5 M OKY‐046) only partially inhibited, the transient contraction induced by bradykinin. Under conditions where the bradykinin‐induced contraction was inhibited by ONO‐3708 during the phenylephrine‐induced contraction, bradykinin induced only a transient relaxation in the presence of NΩ‐nitro‐L‐arginine methyl ester (L‐NAME). This transient relaxation was inhibited when the precontraction was initiated by phenylephrine plus 40 mM extracellular K+. The removal of L‐NAME from this condition caused a partial reappearance of the initial relaxation and a complete reappearance of the sustained relaxation. In conclusion, bradykinin caused the endothelium‐dependent triphasic regulation of vascular tone in the porcine renal artery. The concentrations of bradykinin required to induce a contraction was higher than that required to induce relaxation. Both TXA2 and PGH2 were involved in the bradykinin‐induced contraction. The initial relaxation was mediated by nitric oxide and hyperpolarizing factors while the sustained relaxation depended on nitric oxide.

Endoscopic submucosal dissection for removal of superficial gastrointestinal neoplasms: A technical review

Endoscopic submucosal dissection (ESD) is now the most common endoscopic treatment in Japan for intramucosal gastrointestinal neoplasms (non-metastatic). ESD is an invasive endoscopic surgical procedure, requiring extensive knowledge, skill, and specialized equipment. ESD starts with evaluation of the lesion, as accurate assessment of the depth and margin of the lesion is essential. The devices and strategies used in ESD vary, depending on the nature of the lesion. Prior to the procedure, the operator must be knowledgeable about the treatment strategy(ies), the device(s) to use, the electrocautery machine settings, the substances to inject, and other aspects. In addition, the operator must be able to manage complications, should they arise, including immediate recognition of the complication(s) and its treatment. Finally, in case the ESD treatment is not successful, the operator should be prepared to apply alternative treatments. Thus, adequate knowledge and training are essential to successfully perform ESD.

Mechanism of down-regulation of L-type Ca2+ channel in the proliferating smooth muscle cells of rat aorta

The mechanism of down‐regulation of L‐type Ca2+ channel (L‐VOC) was investigated in rat aortic smooth muscle cells in primary culture. On culture days 3–5, the cells actively incorporated the 5‐bromo‐2′‐deoxy‐uridine (BrdU), and did not respond to K+ depolarization nor express α1C subunit of L‐VOC. At confluence on day 8, BrdU incorporation decreased, and the cells up‐regulated α1C subunit mRNA, expressed α1C subunit protein at cell periphery, and responded to K+ depolarization. Treating the proliferating cells on day 3 with serum‐free media or 10 μM PD98059, a MAP kinase kinase inhibitor, for 2 days induced the expression of α1C subunit protein and the responsiveness to K+ depolarization. However, the serum starvation, but not PD98059, decreased the BrdU incorporation and increased the α1C subunit mRNA. It is concluded that the expression of L‐VOC is substantially suppressed in the proliferating cells due to two mechanisms; a MAP kinase‐mediated post‐transcriptional down‐regulation and the transcriptional down‐regulation by additional mitogenic signals. J. Cell. Biochem. 87: 242–251, 2002.

Thapsigargin‐induced endothelium‐dependent triphasic regulation of vascular tone in the porcine renal artery

To elucidate the role of thapsigargin‐induced Ca2+ entry in endothelial cells in the regulation of vascular tone, changes in Ca2+ and force of smooth muscle were simultaneously monitored in fura‐2‐loaded strips of porcine renal artery. During phenylephrine‐induced sustained contraction, thapsigargin caused an endothelium‐dependent triphasic response; an initial relaxation, a subsequent transient contraction, and a sustained relaxation. The initial relaxation and the contraction were associated with a decrease and an increase in [Ca2+]i, respectively. There was no apparent [Ca2+]i decrease during the sustained relaxation. Thapsigargin‐induced responses were observed at 10−8 M and higher concentrations, with the maximum response observed at 10−6 M. The transient contraction was inhibited by a cyclo‐oxygenase inhibitor (10−5 M indomethacin), a thromboxane A2 (TXA2)/prostagrandin H2 (PGH2) receptor antagonist (10−5 M ONO‐3708), and a TXA2 synthase inhibitor (10−5 M OKY‐046). During the phenylephrine‐induced contraction in the presence of indomethacin, thapsigargin caused an initial, but not a sustained relaxation, in the presence of Nω‐nitro‐L‐arginine methylester (L‐NAME). During the contraction induced by phenylephrine plus 40 mM K+‐depolarization in the presence of indomethacin, thapsigargin induced both a transient and a sustained relaxation. However, these relaxations were completely abolished in the presence of L‐NAME. Thapsigargin caused a large Ca2+ elevation in cultured endothelial cells of the renal artery. The concentration‐response relation was thus similar to that for force development in the arterial strips. In conclusion, thapsigargin‐induced Ca2+ entry in endothelial cells led to triphasic changes in the tone of the porcine renal artery. The endothelium‐dependent contraction was mediated mainly by TXA2. Nitric oxide and hyperpolarizing factor are both involved in the initial relaxation. However, a sustained relaxation was observed which mainly depended on nitric oxide.

A case of gallstone ileus with a spontaneous evacuation

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