504 PAR-2 Activation Enhances Acid-Induced ATP Release Through TRPV1 and ASICs Sensitization in Human Esophageal Epithelial Cells

erosive esophagitis (EE) and healthy controls (HC), both in the basal state and in response to acid exposure. Methods: 20 patients with chronic GERD - 10 patients with EE (mean age 63) and 10 with NERD (mean age 47) - and 10 HCs (mean age 35) were enrolled. NERD patients were well characterized with 24-h pH-impedance showing abnormal acid exposure (pH<4, >4% of time) and/or positive symptom association probability and no history of esophagitis on previous endoscopy. Before endoscopy, GERD patients discontinued PPI therapy for 7 days. Six esophageal biopsies from macroscopically normal mucosa were obtained at 5 cm above the gastroesophageal junction and directly transferred to a Ussing chamber system. After an equilibration period (40 min), baseline transepithelial electrical resistance (TEER) was assessed. Half of the biopsies were exposed to an acidic solution (pH1 - for 30 min). Changes in TEER during acid exposure and recovery were analyzed relative to baseline TEER. Permeation to the paracellular permeation marker fluorescein (375 DA - 1 mg/ml) was assessed in non-exposed biopsies for 120 minutes. Results: TEER was significantly lower in EE patients when compared to NERD patients (139±10 V vs. 175±10 V, p<0.05) and controls (186±12 V, p<0.01), however no difference was found between NERD patients and HCs. Ex vivo acid exposure provoked a comparable reduction in TEER in all groups (EE: -48±3 %, NERD: -40±5 %, HC: -50±6 % relative to baseline TEER, ns). Recovery of TEER towards baseline after acid exposure was more pronounced in NERD patients when compared to EE (AUC: 389 [347-400] vs. 310 [280-366], p<0.05) (Fig. 1). Mucosal permeability to fluorescein in non-exposed biopsies was not different between groups, indicating that only permeability to ions is increased in EE patients. Conclusion: Esophageal epithelial barrier function in NERD patients evaluated ex vivo in Ussing chambers is not impaired. Therefore, other mechanisms may account for the high symptom burden in NERD patients.

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Table 2. Results of esophageal motility tests.

Results are presented as median [25th -75th percentile]. * survives Bonferroni correction.

502 Impact of Helicobacter pylori on Gastroesophageal Reflux Disease Depends on Its Effects on the Gastric Corpus Mucosa Lars Agreus, Nicholas J. Talley, Michael Vieth, Jukka Ronkainen, Pertti Aro, Anna M. Forsberg, Bengt Wallner, Per M. Hellström, Anna Andreasson Background H. pylori (Hp) increases gastric acid secretion (1) and may predispose to gastroesophageal reflux symptoms (GERS). On the other hand, Barrett's esophagus is inversely correlated with Hp infection (2). The development of gastric atrophy in a subset with Hp infection might explain different outcomes in GERD in the setting of other risk factors. We aimed to determine the link between gastric atrophy and GERD in the general population and the relationship with other risk factors (obesity, hiatal hernia, age, sex, smoking and smokeless tobacco) Methods In two independent Swedish population-based upper endoscopy studies on different populations (Kalixanda 2000-2002, n=1000, 20-81 years of age, mean age 54, 49% men, and LongGerd 2012, n=388, 20-79 years, mean age 54, 48% men), biopsies was taken from the corpus and the antrum, and hiatal hernia (HH) was measured in Kalixanda as a visible separation between the z-line and the diaphragmatic impression ‡ 2 cm (3) (Yes/No), and in LongGerd according to the Hill classification (4). Corpus atrophy (CA) (Yes/No) was classified according to the Sydney classification (5) (Yes/No). Reflux symptoms (GERS) were measured by the validated ASQ questionnaire (6). Smoking and smokeless tobacco (snus) was recorded (Yes/No) Results The prevalence of any GERS the past 3 months was 39% (95% CI 36-42%) in Kalixanda and 24% (95%CI 20-28%) LongGerd. The prevalence of H.p. was 38% (95%CI 29-35%) and 11% (95%CI 9-15%) (p<0.001), and CA 6.4% (95% CI 5-8%, all ‡52 years of age), and 2.6% (95%CI 0.01-4.8, all ‡57 years of age) (p=0.007), in Kalixanda and LongGERD respectively (age and sex taken into account). As shown in the table, Hp infection, HH, male sex and higher BMI increased the risk of GERS, while CA decreased it in both studies: the OR are almost identical, except for H.H which however was not measured identically. Smoking or smokeless tobacco had no significant influence. Conclusion The prevalence of Hp and thus CA appears to be decreasing. In the absence of CA, H.p. infection is associated with GERS, while GERS is less prevalent in the fraction that has CA. This mechanism may explain differences in outcomes in H.p. eradication studies on GERD patients. 1. Kaneko H et al. J Gastroenterol. 2002;37:77-86. 2. Rubenstein JH et al. Clin Gastroenterol Hepatol. 2014;12:239-45. 3. Kahrilas PJ et al. Best Pract Res Clin Gastroenterol. 2008;22:601-16. 4. Hill LD et al. Gastrointest Endosc. 1996;44:541-7. 5. Dixon MF et al. Am J Surg Pathol. 1996;20:116181. 6. Agréus L et al. Scand J Prim Health Care. 1993;11:252-62. Table: Influence of H. pylori infection, corpus atrophy (C.A.), hiatus hernia (H.H.), BMI, gender and age on GERS in the Kalixanda and LongGERD population-based endoscopic studies

Fig. 1 Recovery of TEER after 30 min of luminal acid (pH1) exposure. Area under the curve value was larger in NERD patients when compared to EE patients, p<0.05.

504 PAR-2 Activation Enhances Acid-Induced ATP Release Through TRPV1 and ASICs Sensitization in Human Esophageal Epithelial Cells Liping Wu, Tadayuki Oshima, Jing Shan, Hiroo Sei, Takashi Kondo, Tomoaki Kono, Fumihiko Toyoshima, Katsuyuki Tozawa, Hisatomo Ikehara, Yoshio Ohda, Toshihiko Tomita, Hirokazu Fukui, Jiro Watari, Hiroto Miwa Background and Aims: Protease-activated receptor-2 (PAR-2) is expressed in human esophageal epithelial cells, and is believed to play a role in inflammation. Esophageal visceral hypersensitivity has been proposed to be a pathogenesis of heartburn sensation in non-erosive reflux disease (NERD). However, the mechanisms of heartburn sensation and involvement of PAR-2 in epithelial cells are not clear. PAR-2 activation may modulate these responses through ATP release. The transient receptor potential vanilloid receptor 1 (TRPV1) and acidsensing ion channels (ASICs) are both acid-sensitive nociceptors. Accordingly, we examined whether activated PAR-2 upregulates the sensitivity of TRPV1 and ASIC's function in human esophageal epithelial cells and whether ATP release by esophageal epithelial cells in response to acid is modulated by TRPV1 and ASICs activation. Methods: Primary human esophageal epithelial cells (HEEC) were used in this study. ATP release by HEECs was measured after 5 min acid (pH 5) stimulation using an ATP Bioluminescence Assay Kit CLS II (Roche). Pretreatment with 5-iodoresiniferatoxin (IRTX), a TRPV1 receptor specific antagonist (10-5 M, 30 min) or small interfering RNA (siRNA) against TPRV1 were used to determine the role of TRPV1 in the acid-induced ATP release. Pretreatment with amiloride, a non-selective ASIC blocker (0.3 mM, 30 min) was used to assess the role of ASIC. To evaluate the modulatory effect of PAR-2 activation, pretreatment of trypsin (4 mg/ml, 60 min), tryptase (500 ng/ml, 60 min) or the PAR-2 agonist (SLIGKV, 100 mM, 30 min) was done before

503 Esophageal Epithelial Barrier Function in Non-Erosive Reflux Disease (NERD) Patients: A Barrier Defect? Nicolaas Fedde Rinsma, Ricard Farré, Freddy Troost, Montserrat Elizalde, Ad Masclee, José M. Conchillo Background: Despite the absence of visible mucosal damage in patients with non-erosive reflux disease (NERD), we hypothesized that the esophageal epithelial barrier is functionally impaired and more vulnerable in response to acid exposure. We therefore investigated esophageal epithelial barrier function in NERD patients in comparison to patients with

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and miR-145, 148a and -152 in archived azoxymethane (AOM) and Apc mutant Min mouse tumors. Methods: Mice were fed standard diet (5% fat) or WD (20% fat) and colonic mucosa isolated at 40 wks. Transcripts were measured by real time PCR and protein levels determined by immunostaining and Western blotting. CCD-18Co colonic myofibroblasts were treated with Ang II alone or with ADAM17 inhibitor BMS56634 and EGFR signals measured to assess EGFR transactivation. Cells were transfected with luciferase reporters regulated by ADAM17 3'UTR to dissect miRNA regulation of ADAM17. Results: In studies in vivo, WD significantly up-regulated colonic mucosal RAS pathway components: angiotensinogen, renin, angiotensin converting enzyme (ACE) and AT1. WD also increased ADAM17, EGFR ligand TGF-a and EGFR effector K-Ras. Marimastat, a broad-spectrum ADAM17 inhibitor blocked WD up-regulated EGFR signals in colonic mucosa. In cell culture, another ADAM17 inhibitor, BMS56634 blocked EGFR transactivation by Ang II in CCD-18Co cells. Transfected miR145, miR-148a or miR-152 significantly reduced luciferase regulated by wildtype, but not mutant ADAM17-3'UTR (mutations predicted to abolish interactions with specific miRNAs). These miRNAs were also decreased by WD and significantly down-regulated in AOM and Min mouse tumors, whereas ADAM17 was increased. Conclusions: Taken together, we have demonstrated a potential role for colonic mucosal RAS and ADAM17 in WD-promoted EGFR signals. We also identified inhibitory miRNA-ADAM17 feedback loops that were suppressed by WD and colonic tumorigenesis. Diet-related RAS activation of ADAM17 and concomitant suppression of inhibitory ADAM17-miRNA feedback loops are predicted to increase EGFR signals and promote colonic tumorigenesis. Studies are underway to define the role of myofibroblast ADAM17 in WD effects.

Values are expressed as mean ± SD of at least 3 experiments. 507 505

Intestinal Vitamin D Receptor Protects Mice From Dysbiosis and Tumorigenesis via Modulating JAK/STAT Pathway Shaoping Wu, Yong-Guo Zhang, Rong Lu, Yinglin Xia, Zhongren Zhou, Jun Sun

Impaired Esophageal Mucosa Integrity in Refractory Reflux Disease Patients on Proton Pump Inhibitors. A Role for Residual Acid Reflux? Silvia Cocca, Ans Pauwels, Sabine Roman, Frank Zerbib, Michele Cicala, Jan F. Tack, Ricard Farré

Vitamin D exerts its regulatory roles in mucosal immunity, host defense, and inflammation via vitamin D receptor (VDR). Vitamin D deficiency has been implicated in the pathology of at least 17 types of cancer, including colon cancer. Low VDR expression and diminished vitamin D/VDR signaling are observed in patients with colon cancer. Intestinal epithelial cells are constantly exposed to microbes and equipped with sophisticated protection mechanisms. Nevertheless, how intestinal epithelial VDR is involved in the maintenance of intestinal and microbial homeostasis remains largely unknown. Our recent Gut paper (Wu, et al, 2014) showed that intestinal epithelial VDR deletion leads to microbial dysbiosis and increases susceptibility to dextran sulfate sodium (DSS)-induced colitis. The JAK/STAT pathway plays a critical role in intestinal and microbial homeostasis. We hypothesized intestinal VDR protects mice from dysbiosis via modulating the JAK/STAT pathway in tumorigenesis. In the current study, we used an azoxymethane/ DSS-induced cancer model in intestinal VDR conditional knockout (VDRDIEC) mice and cell culture models. We reported that VDRDIEC mice have higher tumor numbers with tumor location shifted from distal to proximal colon. Fecal microbiota analysis showed that lacking VDR leads to bacterial profile shift from normal to susceptible carcinogenesis. We found enhanced bacterial staining in mouse tumors. These data are consisted with our findings in human tumor sample. VDR deletion decreased Jak2 at protein and mRNA levels. By CHIP assay, we identified that VDR protein bound to the Jak2 promoter, suggesting that VDR transcriptionally regulated Jak2. Taken together, our observations suggest that absence of intestinal epithelial VDR leads to dysbiosis and susceptibility to colon cancer via reduced JAK/STAT signaling. Our study provides new insight into the molecular mechanism of VDR regulating the JAK/STAT pathway in bacterialhost interaction and tumorigenesis. Our studies also indicate a new target—microbiome and VDR signaling for prevention of colon cancer.

Background: Intraluminal impedance baseline (IB) levels are determined by the conductivity of the esophageal wall and have been shown to be decreased in gastro-esophageal reflux disease (GERD) patients. We suggested that IB values may be used as a marker of esophageal mucosal integrity. Altered esophageal mucosal integrity has been proposed as one of the pathological mechanisms associated with persisting symptom perception in GERD patients. Acid is supposed to be the main luminal agent responsible to disrupt mucosal integrity and provoke symptoms in patients off proton pump inhibitors (PPI). Residual acid reflux has been documented in patients with persistent heartburn despite adequate PPI therapy. The aim of this study was to evaluate by using impedance baseline measurements, whether mucosal integrity is impaired in refractory GERD patients on PPI and the possible role of residual acid. Method: Ambulatory 24-h pH-impedance tracings from 27 patients with typical refractory GER symptoms (48±4 years, 7 males) as well as 25 HV (50±3 years, 14 males), both on PPI therapy (40 mg), were analyzed. Mean IB levels at 3, 5 cm and 15 cm were assessed in stable periods of 30 seconds in a time window of 30 minutes (excluding swallows and reflux events) in the upright position. AET was considered pathologic when >0,4% (Zerbib F et al 2013). Values were expressed as mean ±SEM or median (25th, 75th %). Results: Despite the acid suppression therapy, AET was higher in GERD patients compared to HV (median 0% (0, 2) vs 1.1% (0.1, 2.5), P<0.0001). IB at 3 and 5 cm was significantly lower in GERD patients compared to HV both on PPI, while at 15 cm no statistical difference was detected (table I). Positive AET but not negative AET patients had lower distal IB levels at 3 and 5 cm than HV. IB correlates with the AET only in the positive AET group (r=0.43, p=0.01). Conclusions: A subgroup of refractory GERD patients on PPI has a lower IB values in the distal but not it the proximal esophagus probably due to the presence of residual acid reflux. Our findings suggest that in this subgroup of patients, impaired mucosal integrity is related to the presence of residual acid reflux.

508 Association Between Low Fecal Short-Chain Fatty Acids, High Fecal Bile Acids and the High Colon Cancer Risk of the Yup'ik Alaska Native People Tyler Sevco, James P. DeLany, Stephen J. O'Keefe Background: Evidence indicates that diet strongly influences colon cancer risk with meat promoting risk and fiber reducing it. Butyrate is a short-chain fatty acid (SCFA) that is released during microbial fiber fermentation and has been shown to have anti-neoplastic effects. High fat diets stimulate the liver to produce more bile acids (BAs), which have strong carcinogenic properties. The Yup'ik people from Southwest Alaska have one of the highest reported prevalence of colon cancer (>100:100,000) in the world. This population subsists on a diet dominated by fish, marine mammals, and land mammals and is severely deficient in fruit, vegetables, grains, and therefore fiber. We hypothesize that the extremely high colon cancer risk seen in the Yup'ik people is associated with a dietary fiber deficiency, which results in a butyrate deficiency that impairs colonic mucosal host defense against the carcinogenic effects of increased secondary bile acids, a product of their high meat and fat diets. Methods: Fecal samples were collected from ten healthy Yup'ik adults from the YukonKuskokwim Delta Regional Hospital in Southwest Alaska, twelve healthy low colon cancer risk (~5:100,000) rural South Africans (NAs), and twelve healthy high colon cancer risk (~65:100,000) African Americans (AAs) from Pittsburgh, Pennsylvania roughly matched for age and sex. Fecal samples were analyzed for SCFAs using a GC System with a Grace EC1000 capillary column. Fecal BAs were analyzed with HPLC-MS. The group differences were analyzed by non-parametric Mann-Whitney U tests. A level of p £ 0.05 was accepted as statistically significant. Results: The Yup'ik Alaskan, rural Native African, and African American participants were roughly age matched [Age ± SEM: 53 ± 3.6, 57 ± 1.9 and 58 ± 2.5, respectively]. The three chief short-chain fatty acids from saccharolytic fermentation, acetate, propionate, and butyrate were significantly lower in the Yup'ik stool samples (p<0.05) compared with low-risk rural Native Africans (Table). African American acetate, propionate, and butyrate levels were very similar to the Yup'ik samples and no significant differences were found between these two high-risk groups. Fecal bile acid concentrations of the two main secondary bile acids, deoxycholic acid and lithocholic acid, were significantly higher in the Yup'ik stool samples compared to previously collected AA and NA levels (p<0.05). There were no significant differences in cholic acid between the Yup'ik levels and AAs or NAs; however, the group mean value was higher in the Yup'ik samples compared to the

Mean (±Std. Error) IB values ( V) at the three esophageal levels in refractory GERD patients (all patients, positive and negative AET subgropus) vs healthy subjects on PPI

506 Western Diet Up-Regulates ADAM17, a Key Meditator of EGFR Signaling via Activation of Colonic Renin-Angiotensin System and Inhibition of miR-145,148a and -152 Urszula Dougherty, Reba Mustafi, Vesta Valuckaite, Vani J. Konda, Joel Pekow, Farhana Sadiq, Haider I. Haider, Sarbani Adhikari, John Hart, Loren Joseph, Yan Chun Li, Marc Bissonnette Introduction: In prior studies we demonstrated that the renin-angiotensin system (RAS) is up-regulated in experimental and human colon cancer. In colon cancer cells we also showed that RAS signals activate EGFR via angiotensin II receptor, type 1 (AT1), a G-protein coupled receptor. GPCRs can activate EGFR by stimulating the enzyme activity of ADAM17, a metalloprotease that in turn releases membrane-bound EGFR ligands. Since EGFR is required for tumor promotion by Western diet (WD), we asked if WD also increases RAS signaling. In addition to regulation by GPCRs, bioinformatic analysis suggests that ADAM17 expression might be controlled by miR-145, miR-148a and miR-152. The goals of this study were to 1) examine the effects of WD on RAS and ADAM17-EGFR signals and on these miRNAs; 2) assess ADAM17-dependent EGFR transactivation by Ang II in colonic myofibroblasts, the major cell type expressing miR-145 in the colonic mucosa; and 3) measure ADAM17

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acid challenge. TRPV1 was detected by western blotting. The phosphorylation of TRPV1 was assayed by immunoprecipitation and western blotting. To further investigate if PAR2 activation sensitized acid-induced ATP release through TRPV1 and ASICs, pretreatment with IRTX or amiloride for 45 min before trypsin (4 mg/ml, 60 min) was administered. Results: Acid stimulated the release of ATP from HEEC (217 ± 58 % Control, P<0.0001). This was reduced by 60% after pretreatment with IRTX (P<0.0001) and by 79% after amiloride pretreatment (P<0.0001). TRPV1 siRNA transfection also decreased acid-induced ATP release (54%, P<0.01). Pretreatment of HEECs with trypsin, tryptase or the PAR-2 agonist further increased acid-induced ATP release (Table). Trypsin or tryptase led to the phosphorylation of TRPV1, indicating TRPV1 sensitization. Acid-induced ATP release sensitized by trypsin was partially blocked by IRTX and amiloride (39% and 50%, respectively, P<0.0001). Conclusions: Acid-induced ATP release was augmented by TRPV1 and ASICs through PAR2 activation. These findings suggest that the pathophysiology of heartburn sensation or esophageal hypersensitivity may be associated with the activation of PAR-2, TRPV1 and ASICs. Effects of trypsin, tryptase or PAR-2 agonist on acid-induced ATP release in HEEC