Upregulation of Esophageal Prostaglandin E2 Levels in Patients with Heartburn

AGA Abstracts

placebo analgesia. (2) Group 2, had similar pain thresholds on day1 and 10 (n =10; 17.67 ± 1.00 mmHg vs. 16.00 ± 2.67 mmHg, P = 0.544), showing that analgesia was reversed by naloxone, suggestings an opioid pathway. (3) There was no variation in pain threshold for group 3 (P = 0.847), 4 (P = 0.486) and 5 (P = 0.362). (4) Ablation of MOR-expressing neurons blocked analgesia (P = 0.481), suggesting that an endogenous opioid pathway in the ACC is active during visceral placebo analgesia. Conclusions: (1) We confirmed the role of an endogenous opioid pathway in ACC active during visceral placebo analgesia. (2) We successfully established an animal model of visceral placebo analgesia, which can be used to further explore its molecular mechanisms. Table 1 Experimental design used in the study to identify the visceral placebo analgesia in rat model.

min; 200 µL mixed with 500 µL of F12 medium) from colonic biopsies obtained from diarrhea-predominant IBS patients (n=10) or controls (n=10). The neurons were then washed 3x with F12 media and neuronal excitability was measured by perforated patch-clamp. Immediately after washing with F12 media (time 0) or 30 min later (time 30), changes in rheobase (minimum current to trigger an action potential) and the number of action potentials discharged at 2x rheobase were recorded. Antagonists to PAR2 (10 µM I-343), MEK1/2 (50 µM PD8059) or dynamin (30 µM Dyngo4a; endocytosis inhibitor) were applied to neurons 30 min prior to exposure to IBS supernatant or trypsin. Two-way ANOVA and Tukey's post hoc tests were used to analyze the data. RESULTS: IBS supernatants evoked sustained excitability of DRG neurons (decrease in rheobase compared to control; refer to table) and this effect was blocked by I-343, Dyngo-4a and PD8059. Trypsin-mediated sustained excitability was also inhibited by Dyngo-4a and PD8059, but the acute response was unaffected. CONCLUSIONS: Proteases in IBS tissues evoke long-term excitability of nociceptive DRG neurons by activating PAR2. This action is dependent on dynamin-mediated endosomal signaling and ERK1/2. Similarly, the sustained excitability evoked by the serine protease trypsin is ERK1/2 dependent and we have previously shown it is also mediated by PAR2endosomal signaling. Taken together, these data suggest that serine proteases in IBS tissues signal through endosomal pathways in nociceptive DRG neurons to induce sustained signaling and this pathway may provide a novel therapeutic target to treat pain in IBS patients.

Mor: morphine; NS: saline; Nx: naloxone; NT: no treatments. Mo1600 Mo1598 TIBIAL NERVE STIMULATION IMPROVES VISCERAL HYPERSENSITIVITY IN RATS MEDIATED VIA THE AUTONOMIC MECHANISMS Liuqin Jiang, Han Zhang, Nina Zhang, Shengai Zhang, Pankaj J. Pasricha, Jiande Chen

COLONIC SUPERNATANTS FROM CHRONIC MORPHINE EXPOSED MICE INDUCE MORPHINE TOLERANCE IN NAÏVE DORSAL ROOT GANGLION NEURONS THAT IS MITIGATED BY ORAL VANCOMYCIN DELIVERY Ryan Mischel, William L. Dewey, Hamid I. Akbarali

Introduction: Tibial nerve stimulation (TNS) has been applied for the treatment of urinary incontinence and fecal incontinence; however, it has not been investigated in controlling visceral pain or hypersensitivity. The purpose of this study was to determine effects and mechanisms of TNS on visceral hypersensitivity in rats. Methods: (1)Visceral sensitivity during colorectal distension (CRD) was assessed by the measurement of abdominal withdrawal reflux (AWR) and abdominal electromyogram (EMG) in eight regular rats. TNS was performed with different sets of parameters for 30 minutes before the recordings of AWR and EMG. (2)Real/sham TNS was performed in rats with acute visceral hypersensitivity (n= 8) induced by restraint stress and rats (n=10) with colonic hypersensitivity induced by acetic acid during the neonatal period. AWR score, EMG and heart rate variability (HRV) in response to TNS and sham TNS were assessed and compared. Results: (1)In regular rats, five sets of different parameters were tested at a stimulation strength of 90% and 40% of motor threshold (MT) and none showed any significant TNS effect on the AWR during CRD. (2) In restraint stress-induced visceral hypersensitive rats, TNS with the parameters of "14Hz, 330us 40% MT" significantly reduced the AWR scores at different distention pressures (20mmHg: 1.50±0.55 vs 2.83±0.41, 40mmHg: 2.50±0.55 vs 3.83±0.41, and 60mmHg: 3.17±0.41 vs 4.00±0.00; all P<0.01), and decreased EMG responses to CRD (20mmHg: 4.76±10.16 vs 10.47±11.01, 40mmHg: 10.84±12.44 vs 15.92±12.66, 60mmHg: 14.16±14.10 vs 25.02±10.69, and 80mmHg: 17.46±15.11 vs 30.57±10.30; all P<0.05) in the restraint stressed-induced hypersensitized rats. Concurrently, TNS decreased low frequency (LF, sympathetic) by 29.4%, increased high frequency (HF, vagal) by 43.3% (all P<0.05), and reduced LF/HF ratio by 52.1% assessed from the spectral analysis of HRV. (3)In rats with colonic hypersensitivity induced by acetic acid, similar inhibitory effects were noted with TNS: AWR scores were reduced by 40.0%, 37.5% and 15.6% at 20, 40 and 60mmHg (all P<0.05), EMG responses to CRD were significantly reduced at 20mmHg to 80mmHg (all P<0.05), and the inhibitory effect was blocked by naloxone. TNS decreased LF by 18.7% and increased HF by 13.8% (all P<0.05) in the acetic acid induced rats. Conclusions: TNS with optimized parameters of "14Hz 330µs 40%MT" improves visceral hypersensitivity in rodent models of colonic hypersensitivity mediated via the autonomic and opioid mechanisms.

Chronic morphine exposure in mice compromises gut epithelial tight junction integrity, promoting bacterial translocation to the gut wall1 .Bacterial products and secondary inflammatory responses may then modulate the activity of dorsal root ganglion (DRG) neurons with terminal processes in the gut wall. The clinical utility of opioids is limited by the development of analgesic tolerance, and DRG nociceptors play a key role in opioid antinociception. The AIM of this study was to determine if mediators in the gut wall of mice with chronic morphine exposure can induce tolerance in naïve DRG nociceptors, and if oral vancomycin can mitigate this effect. METHODS: Adult male Swiss Webster mice received 10 mg/kg vancomycin (VAN) or saline (SAL) by oral gavage twice daily for 10 days. A 75 mg morphine (MP) or placebo pellet (PP) was implanted on day 5. Full circumference 5 mm colon segments were resected and incubated (37 °C) in 400 µL of neurobasal A medium containing 1% FBS, 1x B-27 supplement, 2 mM L-glutamine, 10 ng/mL GDNF, and penicillin/ streptomycin/ amphotericin B for 24 hours2. The colonic supernatants (400 µL) were then transferred to freshly isolated naïve DRG neuron cultures and incubated (37 °C) for 24 hours before performing patch-clamp investigations. RESULTS: Naïve DRG nociceptors exposed to colonic supernatants from PP+SAL (N=8, n=9) and PP+VAN (N=6, n=8) mice responded to morphine (3 µM) with a positive shift in the action potential threshold (Vthresh; PP+SAL: 3.9±0.8 mV, p<0.001; PP+VAN: 4.7±0.9 mV, p<0.0001), reducing neuronal excitability. In contrast, DRG nociceptors exposed to supernatants from MP+SAL (N=5, n=9) mice did not demonstrate alteration of Vthresh with morphine perfusion, indicative of tolerance formation. This tolerance was prevented in DRG neurons that were incubated in supernatants from MP+VAN (N=7, n=8) mice, evidenced by a positive shift in Vthresh with acute morphine (3.9±0.9 mV, p<0.001). The effects of morphine on DRG nociceptors were blocked by naloxone, a µ-opioid receptor antagonist. Interestingly, incubation in colonic supernatants from both MP groups resulted in increased basal excitability of the DRG nociceptors, manifested by an increased number of action potentials at double rheobase. CONCLUSION: Our results suggest that chronic morphine exposure increases mediators in the colonic wall that induce tolerance to morphine in DRG nociceptors. This effect was mitigated in mice receiving oral vancomycin treatment. Since vancomycin has near 0% oral bioavailability suggests that morphine-induced gastrointestinal dysbiosis and microbial translocation contributes to antinociceptive tolerance development to morphine in DRG nociceptors. 1. Meng, J, et al. (2013). PLoS ONE. 8(1), e54040. 2. Valdez-Morales, EE, et al. (2013). Am J Gastroenterol. 108, 1634-1643

Mo1601 UPREGULATION OF ESOPHAGEAL PROSTAGLANDIN E2 LEVELS IN PATIENTS WITH HEARTBURN Takashi Kondo, Hiroki Okada, Katsuyuki Tozawa, Toshihiko Tomita, Yoshio Ohda, Tadayuki Oshima, Hirokazu Fukui, Takashi Konemura, Ichiro Date, Jiro Watari, Hiroto Miwa

Mo1599 PROTEASES IN COLONIC TISSUE FROM IBS PATIENTS EVOKE SUSTAINED EXCITABILITY OF NOCICEPTIVE DRG NEURONS VIA ENDOSOMAL SIGNALING Nestor N. Jiménez-Vargas, David E. Reed, Nigel W. Bunnett, Stephen Vanner

BACKGROUND: Prostaglandin E2 (PGE2) is a principal proinflammatory and pronociceptive prostanoid and is known to induce sensitization and pain hypersensitivity through the activation of prostanoid receptors on peripheral nerve terminals. Recently, we reported that esophageal PGE2 and EP1 receptor play a crucial role in acid-induced heartburn in healthy subjects (Clin Gastroenterol Hepatol. 2015, DDW 2015 and Am J Physiol Gastrointest Liver Physiol. 2013). Further studies were warranted to examine PEG2 and EP receptor expressions in the esophagus of gastroesophageal disease (GERD). In the present study, we investigated PGE2 and EP receptors in the esophagus of nonerosive reflux disease (NERD), erosive esophagitis (EE) and healthy controls (HCs). METHODS: Endoscopic biopsies were taken from the distal esophagus of 17 patients with NERD, 11 patients with EE, and 15 healthy controls. PGE2 concentration (pg / mg protein) and PGE2 receptor (EP1, EP2, EP3 and EP4) mRNA expression in biopsy samples were measured by enzyme-linked immunosorbent assay (ELISA) and quantitative reverse transcription polymerase chain reaction (RT-PCR). Heartburn symptoms were assessed by a frequency scale for the symptoms of GERD . RESULTS: Esophageal PGE2 levels were significantly higher in patients with NERD or EE than that in HCs (Table). Esophageal EP1-4 mRNA expression was not significantly different

BACKGROUND: Patients suffering from the irritable bowel syndrome (IBS) report exaggerated abdominal pain. We have shown that protease signaling is increased in the colonic tissues of these patients. Moreover, we have demonstrated that activation of protease activated receptor 2 (PAR2) on nociceptive (pain sensing) dorsal root ganglia (DRG) neurons evokes long-term excitability, but the mechanisms behind this phenomenon remain unclear. Recent studies show that certain serine proteases, such as trypsin, trigger canonical PAR2 signaling, leading to receptor endocytosis and in turn, to sustained PAR2-endosomal signaling. In contrast, other proteases such as the cysteine protease cathepsin S, act at non-canonical sites and do not induce receptor endocytosis. It is unknown whether protease signaling in IBS patients is mediated by endosomal-dependent pathways. AIMS: To examine whether proteases in tissues from IBS patients lead to sustained nociceptive signaling and, if so, to identify the intracellular mechanisms involved. METHODS: DRG neurons (T9-T13) from C57BL/6 mice were pre-incubated with trypsin (10 min; 50 nM) or with supernatants (30

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triggers long-lasting changes in the colon with enteric gliosis, decreased number of macrophages, and impaired barrier function. In concert with altered colonic homeostasis, elevated sympathetic dominance after CCI and hyperresponsive peritoneal M1 indicate that CCI induces a pro-inflammatory steady state that may underlie the susceptibility of TBI patients to GI inflammation and associated disorders. NIDDK T32DK067872

Mo1604 PREDICTORS OF INADEQUATE BOWEL PREPARATION FOR COLONOSCOPY: A SYSTEMATIC REVIEW AND META-ANALYSIS Sultan Mahmood, Samid M. Farooqui, Rutaba Tajammal, Salman Nusrat, Mohammad F. Madhoun

Data are expressed as mean (± S.E.)

Background: The quality of colon preparation determines the quality of a colonoscopy. Poor preparation quality can result in increased procedure time, decreased adenoma detection rates and increased risk of rescheduling. Most studies evaluating inadequate bowel preparation are limited by the relatively small sample size and the different factors studied. Methods: Studies were identified by searching ten medical databases including PubMed, Ovid MEDLINE and Cochrane Library Database for reports published between 2000 and 2016, using a reproducible search strategy. References from retrieved articles were also manually reviewed. Only fully published prospective or retrospective studies, evaluating risk factors for inadequate bowel preparation, were included. Two reviewers independently scored the identified studies for methodology and abstracted pertinent data. Pooling was conducted by both fixed-effects and random-effects models; results are presented from the random effects model when heterogeneity was significant. Odd ratios (OR) estimates with 95% confidence interval (CI) were calculated. Heterogeneity was assessed by I-squared index (I2) statistics. Results: A total of 23 studies met the inclusion criteria with a total of 48,085 subjects. Twelve of these studies were from the United States. Polyethylene Glycol solution was the most commonly used bowel preparation (83%). Overall the bowel preparation was inadequate in 9862 (21%) subjects. Age, male gender, inpatient status, diabetes, hypertension (HTN), cirrhosis, constipation, stroke, narcotic and tricyclic anti-depressant use (TCA) use were all associated with inadequate bowel preparation. Other factors studied, which did not significantly affect the quality of bowel preparation, included body mass index (BMI), indication, use of a calcium channel blocker (CCB), previous surgery and history of inflammatory bowel disease (IBD). (Table 1) Conclusions: This is the first meta-analysis to highlights risk factors related to inadequate bowel preparation involving large number of subjects and multiple variables. Interventions to optimize colonoscopy preparation should be targeted at these patient populations. Predictors of Inadequate Bowel Preparation

Mo1602 IDENTIFICATION OF A RET KINASE INHIBITOR, GSK589, AS A PERIPHERAL ANALGESIC FOR THE TREATMENT VISCERAL PAIN ASSOCIATED WITH IRRITABLE BOWEL SYNDROME John Russell, Ehsan Mohammadi, Casey O. Ligon, Rocco Latorre, Gerard Joberty, Nico Zinn, Steve Castellino, Bao Hoang, David L. Krull, Michael P. DeMartino, Mui Cheung, Allen Oliff, Beverley Greenwood-Van Meerveld, Sanjay Kumar Abdominal pain and abnormal bowel habits represent the major symptoms of irritable bowel syndrome (IBS) that are not adequately managed by current treatments. While the etiology of IBS is incompletely understood, exposure to an intestinal inflammatory insult or psychological stress are associated with the development of symptoms through alterations in enteric nervous system (ENS) activity. Neuronal trophic factors such as GDNF dynamically maintain the adult ENS through the initiation and reinforcement of neuronal synapse formation and signal transmission that allows for neuronal plasticity. Stress- or inflammation-induced neuronal growth factor expression increases the potential for hyperinnervation of target tissues that may result in the colonic hypersensitivity observed in IBS. To understand how neuronal trophic factors affect neuronal survival, function and plasticity in IBS, a highly potent and selective small molecule inhibitor of RET kinase, the neuronal growth factor receptor for GDNF, was identified and characterized. Results: GSK589 exhibited 0.07 nM and 21.1 nM IC50 in enzyme and cell-based assays, respectively. Moreover, pharmacokinetic profiling of the RET kinase inhibitor GSK589, demonstrated low systemic oral bioavailability resulting in a GI-restricted distribution potentially minimizing systemic toxicity liabilities. In an animal model of acute colonic hypersensitivity, RET inhibition with GSK589 normalized the visceromotor response, a surrogate for abdominal pain, to colonic distension (Oral dose of 3 or 10 mg/kg GSK589 for 3.5 days BID; at 60 mmHg, 9 and 43% inhibition, respectively). Consistent with its role in the ENS, RET expression was observed in both human and rat colonic neurons of the submucosal and myenteric plexus. Interestingly, RET expression was also observed in a specific population of cells in the epithelial layer of the colonic mucosa likely identifying enteroendocrine cells whose function may be influenced by a RET inhibitor. Conclusions and Inferences: These findings identify a critical role for RET in the function of the ENS and demonstrate RET inhibition may be a therapeutic strategy for the treatment of IBS. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

Mo1603 MECHANISMS INVOLVED IN THE LONG-TERM EFFECTS OF TRAUMATIC BRAIN INJURY ON COLONIC HOMEOSTASIS Elise L. Ma, David J. Loane, Marie Hanscom, Bogdan A. Stoica, Alan Faden, Terez SheaDonohue Introduction: Traumatic brain injury (TBI) initiates processes of neuroinflammation and neurodegeneration that are sustained for years, leading to long-term disability in 2% of the human population. TBI survivors demonstrate life-long impairment of cognitive, sensory, motor functions, and a greater morbidity and mortality by digestive-related diseases. Hypothesis: Chronic brain inflammation post-TBI induces significant and persistent changes in colon mucosal function, which are associated with CNS-mediated disruption of autonomic balance. Methods: Moderate brain injury was induced by controlled cortical impact (CCI) in male C57BL/6 mice. Acute and delayed injury responses in both the brain and proximal colon were assessed. Brain astrocytes and enteric glial cells, microglia, and macrophages were visualized with immunofluorescent staining by glial fibrillary acidic protein (GFAP), CD68, and F4/80. Sympathovagal tone was assessed by non-invasive electrocardiogram (ECG) recordings on two baseline days prior to injury, and periodically between 1 and 28 days post-CCI. Power spectral analyses of heart rate variability (HRV) were conducted using Kubios-HRV to generate low frequency (LF) and high frequency (HF) values. Peritoneal macrophages were elicited by thioglycollate injection on day 1 or day 28 post-CCI. Isolated macrophages were plated and challenged to LPS (10ng/ml) for 4 hours. M1-related genes (TNFα, IL-1β, NOS2) were assessed by real-time QPCR. Paracellular permeability was measured by transepithelial electrical resistance (TEER) and labeled dextran (3kDa) flux in muscle-free colon mounted in microsnapwells. Results: At 28 days post-CCI, there was a significant increase in the number of GFAP+ astrocytes and CD68+ microglia in the brain relative to controls. Similarly, a 41% increase in subepithelial GFAP+ reactivity was observed in the colon relative to controls; however, CD68+ and F4/80+ macrophage densities were reduced by 69% and 45% relative to controls at this time. HRV analysis reveals that CCI significantly increases LF/HF values indicating a shift to sympathetic dominance beginning early as 24 hours and evident still at 28 days. Expression of TNFα, IL-1β, and NOS2 to LPS were elevated significantly in macrophages isolated from CCI mice vs controls at 24 hours as well as 28 days. These neuroimmune changes were associated with increased colonic dextran flux at 28 days post-CCI (7.0±1.8% vs 18.2±2.9%). Conclusions: CCI

Mo1605 EFFICACY OF VARIOUS ENDOSCOPIC MODALITIES IN DETECTING NEOPLASIA IN ULCERATIVE COLITIS: A SYSTEMATIC REVIEW AND NETWORK META-ANALYSIS Bilal Gondal, Yuga Komaki, Fukiko Komaki, David T. Rubin, Dejan Micic, Atsushi Sakuraba Background and Aims: Longstanding ulcerative colitis (UC) is associated with an increased risk of colonic neoplasia. Various endoscopic modalities, such as chromoendoscopy (CE), narrow band imaging (NBI) and random biopsy, have been introduced for surveillance, however, there exists a paucity of direct comparisons between them. In order to provide a comparative evaluation of the efficacy of these modalities, we aimed to conduct a network meta-analysis of randomized controlled trials (RCTs) performed for surveillance of neoplasia in UC. Methods: We searched Medline/PubMed, Web of Science, EMBASE, Google Scholar and Cochrane Central Registry through May 2016 for RCTs evaluating the efficacy of endoscopic modalities for surveillance of neoplasia in UC. The primary outcome of interest were dysplasia detection rates per biopsy and per patient, and dysplasia numbers per patient. Studies were simultaneously analyzed using a random-effects network meta-analysis under Bayesian framework to identify the modality with highest dysplasia detection rate. The best ranking probability for the dysplasia detection rate was analyzed by surface under the

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between the groups. There was a clear correlation between esophageal PGE2 levels and frequency of heartburn (p < .01) CONCLUSION: Overproduction of esophageal PGE2 may play a significant role in the generation of heartburn symptoms not only in patients with EE, but also in patients with NERD without endoscopic inflammatory changes. Control of esophageal PGE2 levels may offer a new therapeutic target for managing heartburn. Esophageal PGE2 levels in the healthy controls (HCs), patients with NERD and EE