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Background & Aims: Helicobacter pylori infection is a risk factor for gastric cancer. Ammonia/ammonium (A/A) is a cytotoxin generated by H pylori that kills gastric epithelial cells. We investigated whether A/A cytotoxicity occurs by activating N-methyl d-aspartate (NMDA) channels, which results in Ca2+permeation and epithelial cell death. Methods: Gastric epithelial cells were cultured to confluence and then incubated with A/A and NMDA channel or cell signaling antagonists. Cells were incubated with wild-type H pylori or mutant strains that do not produce A/A. Changes in intracellular Ca2+were examined in living cells by confocal microscopy. Biochemical and histochemical techniques were used to examine the relationship between A/A-induced cell death and intracellular levels of Ca2+. Results: A/A increased Ca2+permeation in gastric epithelial cells; the increase was blocked by NMDA receptor and cell signaling antagonists. Wild-type, but not mutant H pylori, also caused extensive Ca2+permeation of gastric epithelial cells, which was blocked when NMDA-receptor expression was repressed. Ca2+that entered cells was initially cytoplasmic and activated proteases. Later, the Ca2+was sequestered to cytoplasmic vacuoles that are dilatations of the endoplasmic reticulum. Inositol-3-phosphatedependent release of Ca2+from the endoplasmic reticulum and protease activity damaged mitochondria, reduced levels of adenosine triphosphate, and transcriptionally up-regulated cell death effectors. Expression of the NMDA receptor was altered in stomachs of mice infected with H pylori. Conclusions: A/A affects gastric epithelial cell viability by allowing excessive Ca2+permeation through NMDA channels. NMDA channels might thereby regulate cell survival and death pathways during development of gastric cancers associated with H pylori infection., National Institutes of Health (U.S.) (Grant R01AI/RR037750)

Background & Aims: K + recycling at the apical membrane of gastric parietal cells is a prerequisite for gastric acid secretion. Two K + channels are currently being considered for this function, namely KCNQ1 and inwardly rectifying K + channels (Kir). This study addresses the subcellular localization, trafficking, and potential functional significance of KCNQ1 and Kir4.1 channels during stimulated acid secretion. Methods: The effect of pharmacologic KCNQ1 blockade on acid secretion was studied in cultured rat and rabbit parietal cells and in isolated mouse gastric mucosa. The subcellular localization of KCNQ1 and Kir4.1 was determined in highly purified membrane fractions by Western blot analysis as well as in fixed and living cells by confocal microscopy. Results: In cultured parietal cells and in isolated gastric mucosa, a robust acid secretory response was seen after complete pharmacologic blockade of KCNQ1. Both biochemical and morphologic data demonstrate that Kir4.1 and KCNQ1 colocalize with the H + /K + -ATPase but do so in different tubulovesicular pools. All Kir4.1 translocates to the apical membrane after stimulation in contrast to only a fraction of KCNQ1, which mostly remains cytoplasmic. Conclusions: Acid secretion can be stimulated after complete pharmacologic blockade of KCNQ1 activity, suggesting that additional apical K + channels regulate gastric acid secretion. The close association of Kir4.1 channels with H + /K + -ATPase in the resting and stimulated membrane suggests a possible role for Kir4.1 channels during the acid secretory cycle.

Background & Aims: Serotonin (5-HT) is a critical signaling molecule in the gut. 5-HT released from enterochromaffin cells initiates peristaltic, secretory, vasodilatory, vagal, and nociceptive reflexes. Despite being pathophysiologically divergent, ulcerative colitis (UC) and irritable bowel syndrome (IBS) are both associated with clinical symptoms that include alterations in the normal patterns of motility, secretion, and sensation. Our aim was to test whether enteric 5-HT signaling is defective in these disorders. Methods: Rectal biopsy specimens were obtained from healthy controls and patients with UC, IBS with diarrhea (IBS-D), and IBS with constipation (IBS-C). Key elements of 5-HT signaling, including measures of 5-HT content, release, and reuptake, were analyzed with these samples. Results: Mucosal 5-HT, tryptophan hydroxylase 1 messenger RNA, serotonin transporter messenger RNA, and serotonin transporter immunoreactivity were all significantly reduced in UC, IBS-C, and IBS-D. The enterochromaffin cell population was decreased in severe UC samples but was unchanged in IBS-C and IBS-D. When 5-HT release was investigated under basal and mechanical stimulation conditions, no changes were detected in any of the groups relative to controls. Conclusions: These data show that UC and IBS are associated with similar molecular changes in serotonergic signaling mechanisms. While UC and IBS have distinct pathophysiologic properties, these data suggest that shared defects in 5-HT signaling may underlie the altered motility, secretion, and sensation. These findings represent the first demonstration of significant molecular alterations specific to the gut in patients with IBS and support the assertion that disordered gastrointestinal function in IBS involves changes intrinsic to the bowel.

Background & aims CD1d is a major histocompatibility complex class I-like molecule that presents glycolipid antigens to a subset of natural killer (NK)1.1(+) T cells. These NK T cells exhibit important immunoregulatory functions in several autoimmune disease models. Methods To investigate whether CD1d and NK T cells have a similar role in intestinal inflammation, the effects of the glycolipid, alpha-galactosylceramide (alpha-GalCer), on dextran sodium sulfate (DSS)-induced colitis were examined. Wild-type (WT), CD1d(-/-), and RAG(-/-) mice were examined for their response to either alpha-GalCer or the control analogue, alpha-mannosylceramide (alpha-ManCer). Results WT mice, but not CD1d(-/-) and RAG(-/-) mice, receiving alpha-GalCer had a significant improvement in DSS-induced colitis based on body weight, bleeding, diarrhea, and survival when compared with those receiving alpha-ManCer. Elimination of NK T cells through antibody-mediated depletion resulted in a reduction of the effect of alpha-GalCer. Furthermore, adoptive transfer of NK T cells preactivated by alpha-GalCer, but not alpha-ManCer, resulted in diminished colitis. Using a fluorescent-labeled analogue of alpha-GalCer, confocal microscopy localized alpha-GalCer to the colonic surface epithelium of WT but not CD1d(-/-) mice, indicating alpha-GalCer binds CD1d in the intestinal epithelium and may be functionally active at this site. Conclusions These results show an important functional role for NK T cells, activated by alpha-GalCer in a CD1d-restricted manner, in regulating intestinal inflammation.

We created gene-targeted pigs with mutations in the adenomatous polyposis coli ( APC ) gene ( APC ) that are orthologous to those responsible for human familial adenomatous polyposis (FAP). One-year-old pigs with the APC 1311 mutation (orthologous to human APC 1309 ) have aberrant crypt foci and low- and high-grade dysplastic adenomas in the large intestine, similar to the precancerous lesions that develop in patients with FAP. Dysplastic adenomas accumulate β-catenin and lose heterozygosity of APC . This large-animal, genetic model of FAP will be useful in the development of diagnostics and therapeutics for colorectal cancer. DNA sequence data: NCBI accession number GU951771.

Introduction: Tumors at the gastroesophageal junction can be treated by gastrectomy or esophagectomy. Oncologic outcomes are reported to be similar, therefore the aim of this study was to evaluate quality of life (QOL) and alimentary satisfaction after gastrectomy versus esophagectomy. Methods: A retrospective chart review was performed of patients that had gastrectomy with Roux-en-Y esophagojejunostomy or esophagectomy with gastric pull-up from 2000-2014. Symptoms, alimentary satisfaction and QOL were assessed by telephone interview and questionnaires (RAND 36-item Short Form Health Survey [SF-36], Gastrointestinal Quality of Life Index [GIQLI], and the Alimentary Satisfaction [AS] Score). In each questionnaire higher scores denote better satisfaction. Follow-up was divided into early (≤ 2years), medium (>2-5 years) and long (>5 years). Patients that could not be contacted or who refused to participate were excluded. Results: There were 20 patients in each group (table). There were no significant differences between groups for age, gender, initial BMI, or BMI at follow-up. All but 3 patients lost weight after gastrectomy and all lost weight after esophagectomy. The median weight loss after gastrectomy was 14 and after esophagectomy was 11.6 pounds. The mean number of meals a day (3) was similar between groups. There were no significant differences in symptoms but more patients used proton pump inhibitors after esophagectomy. Overall, QOL by SF-36 was similar between groups in all but the category role limitation due to physical health which was higher after esophagectomy. However, at early follow-up QOL was better after esophagectomy (Figure). There was no significant difference in the overall scores for GIQLI (97 after gastrectomy versus 101 after esophagectomy, p=0.685) and AS (7.5 in each group, p=0.926), but GIQLI scores were significantly higher at early follow-up after esophagectomy (78.7 for gastrectomy versus 105.6 for esophagectomy, p=0.014). The SF-36 scores were similar between males and females after gastrectomy, but after esophagectomy scores in the categories for general health and physical function were significantly better in females (71.7 and 88.3 for females respectively versus 48.9 and 63.2 for males, p=0.043 and p=0.008). Comparing GIQOL and AS scores by gender, females tended to have higher scores for both questionnaires after a gastrectomy (100 and 8 for females respectively versus 95.4 and 7.2 for males) and an esophagectomy (116 and 9.1 for females versus 94 and 6.8 for males). Conclusions: Overall QOL and alimentary satisfaction were similar after gastrectomy and esophagectomy, but in the first two years the scores for both SF-36 and the GIQLI questionnaires were better after an esophagectomy. Satisfaction was similar by gender, but females tended to score higher than males after both procedures. Demographic and clinical data.

Serotonin (5-HT) is a critical signaling molecule in the gut. 5-HT released from enterochromaffin cells initiates peristaltic, secretory, vasodilatory, vagal, and nociceptive reflexes. Despite being pathophysiologically divergent, ulcerative colitis (UC) and irritable bowel syndrome (IBS) are both associated with clinical symptoms that include alterations in the normal patterns of motility, secretion, and sensation. Our aim was to test whether enteric 5-HT signaling is defective in these disorders.Rectal biopsy specimens were obtained from healthy controls and patients with UC, IBS with diarrhea (IBS-D), and IBS with constipation (IBS-C). Key elements of 5-HT signaling, including measures of 5-HT content, release, and reuptake, were analyzed with these samples.Mucosal 5-HT, tryptophan hydroxylase 1 messenger RNA, serotonin transporter messenger RNA, and serotonin transporter immunoreactivity were all significantly reduced in UC, IBS-C, and IBS-D. The enterochromaffin cell population was decreased in severe UC samples but was unchanged in IBS-C and IBS-D. When 5-HT release was investigated under basal and mechanical stimulation conditions, no changes were detected in any of the groups relative to controls.These data show that UC and IBS are associated with similar molecular changes in serotonergic signaling mechanisms. While UC and IBS have distinct pathophysiologic properties, these data suggest that shared defects in 5-HT signaling may underlie the altered motility, secretion, and sensation. These findings represent the first demonstration of significant molecular alterations specific to the gut in patients with IBS and support the assertion that disordered gastrointestinal function in IBS involves changes intrinsic to the bowel.

Clostridium difficile toxin A causes mitochondrial dysfunction resulting in generation of oxygen radicals and adenosine triphosphate (ATP) depletion. We investigated whether mitochondrial dysfunction is involved in nuclear factor kappaB (NF-kappaB) activation and interleukin (IL)-8 release from toxin A-exposed enterocytes.NF-kappaB activation and IL-8 release in response to toxin A were correlated with reactive oxygen intermediate (ROI) generation and ATP production in HT-29 monolayers or HT-29 cells exposed to ethidium bromide (EB) to inhibit mitochondrial function.HT-29 cells exposed to EB showed damaged mitochondria and diminished resting levels of ATP. ROI production in EB-treated cells exposed to toxin A for 30 minutes was significantly reduced. Exposure of wild-type HT-29 cells to toxin A resulted in increased oxygen radical generation and IL-8 production (P0.01 vs. control) that was inhibited by antioxidant pretreatment. Degradation of IkappaB was observed within 30 minutes of toxin exposure, before ras homologue (Rho) glucosylation, and was followed by nuclear translocation of NF-kappaB. Toxin A did not increase IL-8 levels in EB-treated cells, whereas IL-8 release in response to IL-1beta was not affected.Our data support an early role for mitochondria-derived ROIs in stimulation of IL-8 release from colonocytes by toxin A. ROI generation is independent of Rho inactivation and involves nuclear translocation of NF-kappaB before release of IL-8.

While menin shRNA knockdown did not alter HT-29 cell proliferation alone, use of gefitinib in combination with menin shRNA led to decreased proliferation and increased autophagy and apoptosis induction. Similar results were observed with the menin-MLL1 inhibitor MI2-2, whose specificity was independently confirmed using a menin-MLL1 pulldown assay and analysis of Hoxa7/9 mRNA levels. Alone MI-2-2 had no effect on HT-29 cell proliferation, however in combination with gefitinib it produced a dramatic decrease in cell growth, accompanied by significant autophagy and apoptosis induction. Similar results were observed with erlotinib, indicating that the observed effects were not specific to gefitinib. MI-2-2 did not have any direct inhibitory effects on either EGFR phosphorylation or EGF induced signaling through the PI3K-Akt or MAPK pathways. CONCLUSIONS: Menin-MLL1 may be important for colon cancer resistance to EGFR inhibitors, and disruption of this interaction could potentially serve as a method to increase the efficacy of EGFR inhibitors in the treatment of metastatic colon cancer.

endoscopy at least 6 weeks from the index procedure and were either nHw or AA comprising the study group. Ethnic distribution was as follows: 58% nHw (n=75) and 42% AA (n=54). Gender distribution, BMI and age were similar between groups. BE was present in 8% (n= 6) of the nHw group while no AA had BE on repeat EGD (p

Background & Aims: The mechanism by which Clostridium difficile toxin A causes actin depolymerization and cell rounding involves toxin internalization and subsequent monoglucosylation of the Rho family of proteins. This study explored toxin internalization and effects on mitochondrial function before cell rounding. Methods: Chinese hamster ovary (CHO) cells were exposed to toxin A, and mitochondrial localization was assayed by confocal microscopy. Mitochondrial function was measured by adenosine triphosphate (ATP) concentration, mitochondrial permeability, and leakage of cytochrome c . Results: Confocal microscopy showed toxin A colocalization with the mitochondrial protein GRP 75 at 5 minutes after toxin exposure. Between 5 and 15 minutes, toxin A caused an 80% diminution in cellular ATP levels; cell rounding and Rho glucosylation commenced between 15 and 30 minutes. Toxin A also resulted in reduction of mitochondrial membrane potential and a 2-3-fold increase in reactive oxygen radicals. Preincubation of CHO cells with the antioxidants butylated hydroxyanisole or butylated hydroxytoluene blocked the toxin A–induced increase in oxygen radicals and diminished cell rounding. Western blot analysis of toxin A–exposed isolated mitochondria showed a direct effect of toxin A on leakage of cytochrome c . Conclusions: The results show that extensive mitochondrial damage occurs within 15 minutes in CHO cells exposed to toxin A. Diminished ATP concentrations and increased oxygen radicals are likely to contribute to cytotoxicity from this bacterial toxin. GASTROENTEROLOGY 2000;119:139-150