data indicate that TJ proteins could be used as sensitive markers of RE instead of DIS. ERK1/2 maybe participate in regulating TJ proteins in the esophageal epithelium of the RE group.

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Impedance Baseline Values in the Upright and Recumbent Period in GERDPatients and Healthy Volunteers on Proton Pump InhibitorsSilvia Cocca, Ans Pauwels, Frank Zerbib, Sabine Roman, Michele Cicala, Jan F. Tack,Ricard Farré

BACKGROUND: Intraluminal impedance baseline (IB) levels are determined by the conduc-tivity of the esophageal wall and have been shown to be decreased in gastro-esophagealreflux disease (GERD) patients. It has been proposed that IB values may be used as a markerof reflux-induced changes in esophageal mucosal integrity (Farre et al. 2011), although thereis no complete agreement on the most appropriate time period to assess IB (upright orrecumbent). Altered esophageal mucosal integrity has been proposed as one of the pathologi-cal mechanisms associated with persisting symptom perception in GERD patients. Neverthe-less, possible alterations in mucosal integrity in refractory GERD patients have not beenassessed. The aims of the present study were: 1) to test whether IB measurements differbetween the upright and recumbent period in healthy volunteers (HV) and refractory GERDpatients on PPI and 2) to evaluate whether mucosal integrity is impaired in refractory GERDpatients. METHOD: Ambulatory 24-h pH-impedance tracings from 16 refractory GERDpatients (46±4 years, 7 male) as well as 20 HV (49±3 years, 11 male) both on proton pumpinhibitor (PPI) therapy (40 mg) were retrospectively analyzed. Mean IB levels in the distalat 3 and 5 cm and in the proximal esophagus at 15 cm were assessed in two stable periodsof 30 seconds in a time window of 30 minutes (excluding swallows and reflux events), bothin the upright and recumbent positions. Values were expressed as mean±SEM. RESULTS:Both in HV and refractory GERD patients on PPI therapy, mean baseline values at 3 and 5cm were comparable in upright and recumbent position. Moreover, at 15 cm, IB valueswere significantly lower in the recumbent period only in HV. In the upright position, IB at3, 5 and 15 cm was significantly lower in GERD patients compared to HV on PPI. Incontrast, in the recumbent position there were no significant differences in IB values betweenGERD patients and HV. Nevertheless, there was a tendency for IB to be lower in patientsboth at 3 (p=0.06) as well as at 15 cm (p=0.06). CONCLUSIONS: Refractory GERDpatients on PPI have an impaired mucosal integrity in the distal and proximal esophagus.IB measurements in the upright period seem superior to evaluate alterations in mucosalintegrity in GERD patients and to distinguish them from health.

**p<0.01 vs upright, ## p<0.01 vs HV

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Esophageal Epithelial-Derived IL-33 Acts As an Exaggerator of Inflammationin the Pathogenesis of GERDTadayuki Oshima, Jing Shan, Taichiro Muto, Koubun Yasuda, Hirokazu Fukui, JiroWatari, Kenji Nakanishi, Hiroto Miwa

Background and Aims: Interleukin-33 (IL-33) is a new tissue-derived cytokine constitutivelyexpressed in endothelial cells and epithelial cells of tissues exposed to the environment.Similar to IL-1α, it seems to act as a dual function protein with proinflammatory cytokineproperties once it is released from the cells and regulatory properties in its nuclear localization.The function of IL-33 in esophagus and in pathological condition including GERD has neverbeen described. Accordingly, we examined the expression of IL-33 in esophagus and itsrole in the pathogenesis of GERD. Methods: The expression of IL-33 in erosive and non-erosive mucosa of GERD and control was examined by real-time RT-PCR and immunofluores-cence. In vitro, normal human esophageal epithelial cells (HEECs) were cultured under theair-liquid interface (ALI) system to establish a stratified squamous epithelial model (Chenet al. Am J Physiol Gastrointest Liver Physiol. 2012). IL-33 level was examined by variousstimuli using real-time RT-PCR, Western blot, ELISA and immunofluorescence. IL-33 siRNAwas transfected to HEECs. Cytokines production was detected by Bio-Plex system. Pharmaco-logical inhibitors were used to identify the underlying cellular signaling pathways involvedin IFNγ-induced IL-33. Results: IL-33 expression was significantly up-regulated in erosivemucosa of GERD compared to control and non-erosive mucosa of GERD, and mainly locatedin the nuclear of basal cell layer. In the esophageal stratified squamous cell model, IFNγup-regulated IL-33 both in mRNA and protein levels time and dose dependently. The up-regulated IL-33 was mainly located in the nuclear of basal cell layer. The release of IL-33was not detected after IFNγ stimulation in vitro. In IL-33 knockdown cells, IFNγ inducedless IL-8, IL-6 and regulated on activation normal T-cell expressed and presumably secreted(RANTES). Deoxycholic acid (DCA) also induced less IL-8 and IL-6 in IL-33 knockdowncells. IFNγ and DCA synergically induced IL-8 and IL-6 in esophageal stratified squamouscell model. IFNγ-induced IL-33 expression was completed inhibited by JAK inhibitor I,SB203580 (a p38 MAPK inhibitor), and EGCG (a STAT1 inhibitor), but not by H89 (a PKAinhibitor) in mRNA and protein levels. Conclusions: Here, we have for the first time shownhigh levels of IL-33 in erosive mucosa of GERD. IFNγ up-regulated intracellular IL-33 inesophageal stratified squamous cell model through JAK/p38MAPK/STAT1 pathway. Intracel-lular IL-33 exacerbated IFNγ or DCA-induced proinflammatory cytokines production. Epi-thelial-derived IL-33 may play an important role to maintain and amplify the chronicinflammation in GERD.

S-863 AGA Abstracts

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Acid Stress-Induced Inhibition of Cellular Protrusion and Filopodia Formationin Human Esophageal Epithelial CellsChien-Lin Chen, Tso-Tsai Liu, Chih-Hsun Yi, Chia-Chin Liu, Lee Jeng-Woei

Background/aim: Dilated intercellular space has been well characterised in patients withgastroesophageal reflux disease (GERD). Our previous work has indicated that besides dilatedintercellular space, ultrastructural alteration of intercellular morphology is associated withincreased severity of GERD (Gastroenterology 2012). Filopodia are thin, actin-rich plasma-membrane protrusions that function as antennae for cells to probe their environment. Inthis study, we investigated whether cellular protrusions and filopodia formation were presentand contributed to intercellular morphology change during acidic stress. Methods: Esophagealsquamous cell carcinoma (ESCC) cells were incubated with normal (pH7.2) or acidifiedgrowth medium (pH4.2). Cellular protrusions with filopodia formation were demonstratedvia scanning electron microscopy and immunofluorescence assay. Expressions of filopodiaregulators were examined by Western blot analysis. In addition, cell behaviour of ESCCcells was also evaluated by cell proliferation, wound healing and anchorage-independentassays. Results: In contrast to normal growth condition, filopodia of esophageal squamousepithelial cells was remarkably inhibited under acidic pH stress. Moreover, decreased expres-sions of stimulators of microfilament, such as member of Rho GTPase family, Cdc42, andvasodilator-stimulated phosphoprotein (VASP) were consistent with filopodia reduction.Accordingly, cell migrating ability of ESCC cells was also repressed. Conclusions: Thisstudy has shown that experimental acid stress induces diminished cellular protrusion withdecreased filopodia formation in ESCC cells. The development of ESCC cell is inhibitedunder acidic pH stress condition. Based on our in vitro findings, this study suggests thatintercellular structural changes with aberrant cellular protrusion and filopodia formationmay contribute to dilated intercellular space between stratified squamous epithelium ofesophagus in patients with GERD.

Figure 1. ESCC cells are incubated in conventional (pH7.2) medium with filopodia formation(white arrow) (A); filopodia formation is inhibited after acidification with cultured medium(pH4.2) (white arrow) (B); recovery of filopodia formation is noticed again with pH7.2growth medium (white arrow) (C); COS-7 cells was used as filopodia-null control (D). Cellmorphology is observed using scanning electron microscopy.

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Understanding the Cause of Persistent GERD Symptoms Despite Proton PumpInhibitor Therapy: Impedance-pH Monitoring RevisitedDaphne Ang, Ikram Hussain, Kwong Ming Fock

Background: Non-response to proton pump inhibitor (PPI) therapy in patients with refluxsymptoms and a normal gastroscopy remains a challenge. Impedance-pH (MII-pH) monitor-ing clarifies the symptom profile and evaluates patients objectively for acid reflux (AR) andnon-acid reflux (NAR). Aim: To study MII-pH characteristics in patients referred for evalua-tion of typical and/or atypical GERD manifestations who remain symptomatic despite PPIs,and study mechanisms related to persistent symptoms. Methods: Between January 2009 andNovember 2013, consecutive patients with persistent typical reflux symptoms (group 1);atypical symptoms (group 2) and non-cardiac chest pain (NCCP, group 3) who remainedsymptomatic despite PPIs and a normal white light gastroscopy underwent 24 hour MII-pH evaluation after PPI washout for 2 weeks. In addition to a negative gastroscopy, patientswith atypical symptoms (group 2) had undergone prior negative evaluation by their respira-tory physicians or ear-nose and throat (ENT) surgeons for evaluation of either unexplainedchronic cough or throat symptoms. Prevalence of (i) oesophageal acid exposure time(AET)>4.2%; (ii) bolus exposure (BE>1.4%), (iii) high reflux numbers (>73) and/or (iv)positive symptom marker based symptom index (SI ≥50%) and/or symptom associationprobability (SAP ≥95%) for AR or NAR events was compared between groups by chi-squareand student t-testing. Results: 208 patients (80M, 139Chinese/28 Malay/22 Indians/19 others,mean age 45.9 ± 12.5) were studied (Table 1). Elevated AET occurred in 24 (11.5%). 120(57.7%) recorded a positive study on MII-pH evaluation despite a normal overall AET.Group 1 and 3 patients had significantly more symptomatic AR events (p<0.05) comparedto group 2. Symptomatic NAR related events did not differ significantly different betweengroups. Patients with a positive symptom association for AR events were more likely to haveabnormal BE (p=0.01) and abnormal reflux numbers (p<0.05). Summary: Acid and non-acid reflux events account for persistent symptoms in patients with typical and atypicalGERD manifestations. Use of MII-pH in PPI non-responders remains an important diagnostic

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