Kousaku Kawashima

Strategic compartmentalization of Toll-like receptor 4 in the mouse gut.

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1β mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.

Ulinastatin shows preventive effect on post-endoscopic retrograde cholangiopancreatography pancreatitis in a multicenter prospective randomized study

Background and Aim:  Endoscopic retrograde cholangiopancreatography (ERCP) is a useful diagnostic and therapeutic procedure; however, ERCP occasionally causes post‐ERCP pancreatitis. The administration of gabexate mesilate has been reported to be effective for the prevention for post‐ERCP pancreatitis when given during and after the procedure. The aim of the present study was to investigate the preventive effect of the novel protease inhibitor ulinastatin on post‐ERCP pancreatitis.

Pathogenesis of Irritable Bowel Syndrome - Review Regarding Associated Infection and Immune Activation

There is increasing evidence regarding the role of immune activation in the etiology of irritable bowel syndrome (IBS), which has been mainly been shown in studies investigating mechanisms of postinfectious IBS (PI-IBS). Exposure to intestinal infection induces persistent low-grade systemic and mucosal inflammation, which is characterized by an altered population of circulating cells, mucosal infiltration of immune cells and increased production of various cytokines in IBS patients. Recent studies have also indicated an increased innate immune response in these patients by evaluating expression and activation of Toll-like receptors (TLRs). These findings suggest that immune activation may play a crucial role in the pathogenesis of IBS. In addition, psychological stress has been reported to be one of the factors that induces immune activation. However, it remains unknown whether immune activation in IBS patients is largely dependent on infectious gastroenteritis and/or psychological stress. Additional studies are necessary to understand the precise mechanism of immune activation and its relationship to the development of IBS.

Peroxisome proliferator‐activated receptor γ‐dependent and ‐independent growth inhibition of gastrointestinal tumour cells

Peroxisome proliferator‐activated receptor γ (PPARγ) acts as a ligand‐activated transcription factor. Although ligand‐induced cellular differentiation and growth inhibition have been mostly studied on human cancers expressing PPARγ, it is unclear if the transcriptional activation of PPARγ is the main mechanism of growth inhibition. In this study, we investigated whether there is a link between growth inhibitory effect and transcriptional activation of PPARγ in several gastrointestinal tumour cell lines. The transcriptional activation potential of PPARγ was assessed by reporter gene assay employing a PPRE‐luciferase vector, and growth inhibitory effect of PPARγ was investigated by 3H‐thymidine incorporation assay, in the presence or absence of thiazolidinedione ligands, rosiglitazone and troglitazone. As expected, in the case of cell lines positive for the transcriptional activation potential of PPARγ (T.Tn, MKN‐45 and LoVo), both the ligands induced growth inhibition. However, in case of some other cell lines negative for the transcriptional activation potential of PPARγ (TT, AGS and HCT‐15), troglitazone still showed a growth inhibitory effect. Administration of the PPARγ antagonist GW9662 did not reverse this growth inhibitory activity of troglitazone. The introduction of dominant negative mutants of PPARγ did not suppress the activity either. These observations suggest that while rosiglitazone inhibits cellular growth predominantly through transcriptional activation of PPARγ, troglitazone can induce it both in PPARγ‐dependent and ‐independent pathways.

Analysis of gastrin receptor gene expression in proliferating cells in the neck zone of gastric fundic glands using laser capture microdissection

Gastrin stimulates proliferation of progenitor cells in the neck zone of gastric fundic mucosa. However, whether it directly enhances this proliferation through its receptors remains unclear. We investigated the expression of gastrin receptors in neck zone proliferating cells in rat gastric fundic glands using a reverse transcription polymerase chain reaction (RT‐PCR) coupled with laser capture microdissection and in situ RT‐PCR. Gastrin receptor expression was identified in c‐fos‐expressing cells located in the neck zone, and results of the RT‐PCR analysis argued against contamination by other cells, such as enterochromaffin‐like, parietal or D cells. Supporting this finding, gastrin receptor gene expression was identified in the neck zone as well as base glands by in situ RT‐PCR. Therefore, it is suggested that proliferating cells in the neck zone are stimulated directly by gastrin via their gastrin receptors.

Localization of calcitonin gene-related peptide receptors in rat gastric mucosa

The location of calcitonin gene-related peptide (CGRP) receptors in the rat stomach has not been elucidated. It was recently reported that the CGRP receptor is formed when a calcitonin-receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 1 are co-expressed on the cell membrane. The aim of this study was to determine the location and the role of CGRP receptors in the rat gastric mucosa. Gene expressions of CRLR and RAMP1 were investigated by Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization. Immunohistochemical stainings for CGRP, somatostatin, gastrin, and chromogranin A were performed. Gastric endocrine cells were collected by counterflow-elutriation and their responses to CGRP were studied. CRLR and RAMP1 mRNA was expressed mainly in small gastric epithelial cells in the pyloric glands. The mRNA expression had a similar distribution to that of D cells. In cultured gastric endocrine cells, CGRP enhanced somatostatin production, while it inhibited the secretion of histamine and gastrin. Our results suggest that CGRP receptors are expressed in D cells in the rat gastric mucosa and control production and secretion of somatostatin.

Prevalence of gastroesophageal reflux disease in children, adults, and elderly in the same community.

The prevalence of gastroesophageal reflux disease (GERD) in adults is increasing in Japan as well as worldwide likely due to increasing obesity and the decreasing rate of Helicobacter pylori infection. However, data regarding the prevalence of GERD in children and adolescents in Japan are lacking. We investigated the prevalence of GERD in children, adults, and elderly living in the same community.

Role of regulatory B cells in chronic intestinal inflammation: association with pathogenesis of Crohn's disease.

Abstract:The role of regulatory B cells (Bregs) producing interleukin (IL)-10 in the pathogenesis of inflammatory bowel diseases remains unknown. We investigated IL-10 production in B cells from patients with inflammatory bowel diseases and immunoregulatory functions of Bregs in experimental colitis mouse models. CpG DNA-induced IL-10 production in peripheral blood B cells isolated from patients with inflammatory bowel diseases and control subjects was examined. CD19 and CD1d were used for evaluating possible cell surface markers of Bregs. Colitis models of severe combined immunodeficiency mice were established by adoptive transfer of whole CD4+ T cells or regulatory T cell (Treg)-depleted T cells (CD4+CD25−) isolated from SAMP1/Yit mice and the function of Bregs in intestinal inflammation was elucidated by evaluating the effects of cotransfer of whole or Breg-depleted B cells. CpG DNA-induced IL-10 production was significantly decreased in B cells from patients with Crohns disease (CD), as compared with those from healthy controls, whereas Bregs were found to be enriched in a population of CD19hi and CD1dhi B cells isolated from both human and mouse samples. The severity of intestinal inflammation was significantly increased in the Breg-depleted mice, with similar results also found in adoptive transfer colitis model mice even after Treg depletion. Our findings show that Bregs, characterized by the cell surface markers CD19hi and CD1dhi, significantly reduced experimental colitis regardless of the presence or absence of Tregs. These results suggest that a deficiency or decrease of Bregs function exacerbates intestinal inflammation, which may be associated with the pathogenesis of CD.

T Cell-Mediated Cytotoxicity via Fas/Fas Ligand Signaling in Helicobacter pylori-Infected Gastric Corpus

Helicobacter pylori infection induces T helper‐1 immune responses in inflamed mucosa. However, the role of T cell‐mediated cytotoxicity in the induction of epithelial apoptosis is still unclear. The aim of this study was to investigate the involvement of the Fas/Fas ligand (Fas/Fas‐L) system in the H. pylori‐infected gastric corpus.

Hepatocyte Growth Factor Expression in Dextran Sodium Sulfate-Induced Colitis in Rats

Hepatocyte growth factor (HGF), a potent inducer of cell migration with morphogenic and mitogenic actions was reported to have key roles in the repair of various tissues. In order to evaluate the role of HGF in the repair process of inflammatory bowel disease, we have investigated the HGF expression in a dextran sodium sulfate (DSS) colitis model. We randomly assigned rats to a colitis group or to a placebo group; the former received a 7-day course of 5% DSS (mw 5 kDa) in drinking water. DSS-induced severe colitis in rats manifested with weight loss, diarrhea, and intestinal bleeding. Animals were killed from day 1 through 7 and on days 9 and 14 after the end of DSS administration. After DSS was withdrawn, disease activity subsided gradually and HGF expression was significantly enhanced along with the augmented expression of IL-1β, TNF-α, and cyclooxygenase-2, accompanied by an increased number of proliferating epithelial cells in colon. These findings suggest that proinflammatory cytokines and cyclooxygenase-2 may have an important role in the mucosal repair in inflammatory bowel disease through increased production of HGF.