Mohammad Azharul Karim Rumi

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.

Essential Role of MD-2 in TLR4-Dependent Signaling during Helicobacter pylori-Associated Gastritis

TLR4, a member of pattern recognition receptors, is the main receptor of LPS. MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. Helicobacter pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated in this study the role of MD-2 in TLR4-dependent signaling in H. pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H. pylori infection and four gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-κB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H. pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-κB and IL-8 promoter upon stimulation with H. pylori LPS. Live H. pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H. pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H. pylori LPS in the stomach.

Predominant nocturnal acid reflux in patients with Los Angeles grade C and D reflux esophagitis

Nocturnal gastric acid breakthrough (NAB) is defined as an intragastric pH < 4.0 lasting more than 1 h during the night in patients taking a proton pump inhibitor (PPI). Gastroesophageal reflux disease (GERD) patients with nocturnal gastroesophageal acid reflux accompanied by NAB are thought to be refractory to PPI treatment. The aim of this study was to endoscopically identify the patients with predominant nocturnal gastroesophageal acid reflux.

MFG-E8 Attenuates Intestinal Inflammation in Murine Experimental Colitis by Modulating Osteopontin-Dependent αvβ3 Integrin Signaling

MFG-E8 (milk fat globule-epidermal growth factor 8) deficiency is strongly associated with acquisition of immune-mediated disorders due to the loss of tissue homeostasis. However, comparatively little is known regarding its functions in gastrointestinal tract disorders, in which immune homeostasis is a major concern. Herein, we report altered MFG-E8 expression in inflamed colons during the acute phase of murine experimental colitis and found that treatment with recombinant MFG-E8, but not its arginine-glycine-aspartate mutant counterpart, ameliorated colitis by reducing inflammation and improving disease parameters. To reveal the MFG-E8-mediated antiinflammatory mechanism, we employed an in vitro system, which showed the down-regulation of NF-κB in an LPS-dependent manner. Additionally, MFG-E8 altered αvβ3 integrin-mediated focal adhesion kinase phosphorylation by impeding the binding of one of its potent ligands osteopontin, which becomes activated during colitis. Taken together, our results indicated that MFG-E8 has a novel therapeutic potential for treatment of colitis.

BRAF and K-ras gene mutations in human pancreatic cancers

We investigated the frequency of BRAF mutations in human pancreatic cancer specimens to determine its role in the development of pancreatic cancer. Nine pancreatic cancer samples without a K-ras codon 12 mutation and 19 with a K-ras mutation were included in the study. Analyses of the BRAF sequence revealed mutations in exon 15 (V599E) in two cases, both of which also exhibited a K-ras codon 12 mutation. No BRAF mutation was found in cases without a K-ras mutation. The BRAF V599E mutation was not found to be a major mutation in pancreatic cancers that had no K-ras codon 12 mutation.

Crystal Violet Chromoendoscopy with Mucosal Pit Pattern Diagnosis is Useful for Surveillance of Short-Segment Barrett's Esophagus

BACKGROUND:Because of a rapid increase in the incidence of Barretts cancer, the appropriate surveillance method for Barretts esophagus is of interest. Methylene blue chromoendoscopy has been reported to be an effective and inexpensive method to improve biopsy surveillance of Barretts epithelium. However, the usefulness of this method in short-segment Barretts esophagus cases is still controversial.AIMS:This study was undertaken to evaluate the abilities of crystal violet and methylene blue chromoendoscopy to detect potentially dysplastic Barretts epithelium in cases with short-segment columnar-appearing epithelium of the esophago-gastric junction.PATIENTS AND METHODS:Four hundred patients with endoscopically suspected short-segment Barretts esophagus were enrolled and randomly assigned to receive chromoendoscopy with 0.05% crystal violet, 0.1% crystal violet, 0.5% methylene blue, or 1.0% methylene blue. During crystal violet and methylene blue chromoendoscopy, biopsy specimens were obtained from stained and unstained columnar-appearing epithelium of the esophago-gastric junction, and the detection rates of Barretts epithelium were evaluated. The value of pit pattern diagnosis was also evaluated as a possible way to detect dysplastic Barretts epithelium.RESULTS:Chromoendoscopy with 0.05% crystal violet detected histologically confirmed Barretts epithelium with the highest sensitivity (89.2%) and specificity (85.7%). Crystal violet clearly stained both dysplastic and nondysplastic Barretts epithelia and made the surface pit pattern easy to observe without using magnifying endoscopy.CONCLUSIONS:The combination of crystal violet chromoendoscopy and pit pattern diagnosis is considered to be useful for the surveillance of short-segment Barretts esophagus.

Epithelial toll-like receptor 5 is constitutively localized in the mouse cecum and exhibits distinctive down-regulation during experimental colitis.

ABSTRACT We recently demonstrated that the pattern recognition receptors (PRRs) toll-like receptor 2 (TLR2), TLR4, and CD14 are expressed in mouse colonic epithelium in a compartmentalized manner. Here we report the localization of TLR5, the receptor for bacterial flagellin, and its distinctive down-regulation during experimental colitis. Guts from normal BALB/c mice and those with dextran sodium sulfate (DSS)-induced colitis were compared. Each gut was divided into seven segments (stomach, small intestine [three parts], and colon [three parts]), and epithelial cells and crypt units were collected by scraping and EDTA treatment, respectively. Northern blotting showed that TLR5 mRNA was preferentially expressed in the epithelium of the proximal colon in normal mice. Laser capture microdissection coupled to reverse transcriptase PCR confirmed this localization. TLR5 protein expression reflected mRNA expression, as evidenced by Western blotting. In mice with acute colitis, inflammation occurred mainly in the distal colon. Interestingly, while TLR2, TLR4, and CD14 were up-regulated in the inflamed colon, TLR5 was down-regulated at both the mRNA and protein levels. Decreased TLR5 expression was more evident during chronic colitis. Additional in vitro studies using a mouse cell line, Colon-26, showed that gamma interferon (IFN-γ) time- and dose-dependently down-regulates TLR5. In conclusion, epithelial cells, mainly in the proximal colon, constitutively express TLR5. TLR5 expression is down-regulated in vivo during acute and chronic DSS-induced colitis, in contrast to the expression of TLR2, TLR4, and CD14. The mechanism governing TLR5 regulation may therefore differ from that controlling other PRRs. Finally, IFN-γ may be involved in down-regulating TLR5 expression.

Cardiovascular risk factors in subjects with Helicobacter pylori infection.

Background. It has been proposed that Helicobacter pylori infection is related to cardiovascular disease, although this has not been fully investigated. The aim of this study was to investigate whether H. pylori in‐fection is associated with cardiovascular risk factors.

Therapeutic Targeting of Toll-Like Receptors in Gastrointestinal Inflammation

Toll-like receptors (TLRs) are sensors of microbial products that initiate host defense responses in multicellular organisms. They are mainly linked to innate immunity and bridging to adaptive immunity, signaling through different TLRs responsible for a wide range of biological responses. The intracellular signaling pathways through Toll/interleukin-1 receptor (IL-1R) domains result in recruitment of the cytoplasmic adaptor molecules, with subsequent activation of a signaling cascade leading to nuclear factor-kappa B (NF-kappaB). TLR-signaling induces host inflammatory response and the inflammation becomes more severe in the absence of several extra and intra cellular negative regulators of TLR-signaling. In the intestine, TLR-dependent activation of NF-kappaB plays a vital role in maintaining epithelial homeostasis as well as regulating infections and inflammation, while dysregulation of TLR-signaling is associated with the pathogenesis of inflammatory bowel diseases (IBD). Recent findings regarding innate immunity-mediated regulation of intestinal pathophysiology prove that development of new drugs targeting TLRs including antagonists of TLR-signaling and agonists of their negative regulators has a potential impact on therapeutic strategies for intestinal inflammatory diseases.

Nocturnal gastric acid breakthrough during the administration of rabeprazole and ranitidine in Helicobacter pylori-negative subjects: effects of different regimens

Background. Nocturnal gastric acid breakthrough (NAB) is defined as nocturnal intragastric pH less than 4 for more than 1 h during proton pump inhibitor (PPI) administration. A bedtime dose of an H2 receptor antagonist (H2RA) inhibites NAB, but the efficacy of the H2RA decreases with continuous administration. We carried out the present study to investigate the effect of 14-day H2RA administration on NAB. Methods. Ten male volunteers without Helicobacter pylori infection received four different 14-day regimens of rabeprazole and ranitidine (study a, morning dose of 20 mg rabeprazole; study b, morning dose of 20 mg rabeprazole with a single bedtime dose of 150 mg ranitidine only on the last day; study c, continuous 20 mg morning dose of rabeprazole and 150 mg at bedtime; study d, morning and evening doses of 10 mg rabeprazole). Ambulatory 24-h gastric pH monitoring was conducted on the last day of each regimen. Results. NAB in studies a, b, c, and d was observed in 9, 1, 4, and 4 subjects, respectively, and the longest periods of nocturnal gastric pH at less than 4.0 were 102.5, 14.0, 37.5, and 52.5 min, respectively (study b vs study c, P < 0.05). Conclusions. The continuous inhibitory effect of ranitidine combined with rabeprazole on nocturnal gastric acid secretion declined during 14-day-long administration in H. Pylori-negative subjects. Split dosing of rabeprazole was more effective than the single morning dose for inhibiting nocturnal gastric acid secretion.