AGA Abstracts Little is known of how Th2 cytokines cause eotaxin-3 secretion by esophageal fibroblasts, however, and we have found that PPIs do not block their eotaxin-3 expression. Topical steroids might not reach subepithelial fibroblasts, and thus the most common medications for esophageal eosinophilia (PPIs and topical steroids) might not prevent fibrostenotic compli- cations of EoE. In this study, we used non-neoplastic esophageal fibroblast cell lines (FEE4- T and BEF-T) to study their mechanism of Th2 cytokine-stimulated eotaxin-3 expression, and we explored whether that expression could be blocked by a selective inhibitor of STAT6 (AS1517499) or by a non-selective tyrosine kinase inhibitor (leflunomide, which blocks JAKs). Methods: We stimulated FEE4-T or BEF-T cells with IL-13 (1-100ng/ml) or IL-4 (1-100ng/ml) for up to 48 hours, with or without AS1517499 (600nM), leflunomide (400- 600μM), or omeprazole (50μM). We determined 1) STAT6 phosphorylation and nuclear translocation by Western blot, 2) STAT6 binding to eotaxin-3 promoter by ChIP, 3) eotaxin- 3 mRNA transcription by RT-PCR and 4) eotaxin-3 protein secretion by ELISA. We also explored effects of AS1517499 and leflunomide on IL-13-induced signaling in esophageal epithelial cell lines (EoE1-T, EoE2-T). Results: In fibroblasts, both IL-13 and IL-4 increased STAT6 phosphorylation, STAT6 nuclear translocation, eotaxin-3 mRNA transcription and eotaxin-3 protein secretion. Omeprazole did not inhibit STAT6 binding to eotaxin-3 promoter and did not block Th2 cytokine-induced eotaxin-3 mRNA and protein expression. In contrast, both AS1517499 and leflunomide decreased IL-13-induced STAT6 phosphorylation, eotaxin- 3 mRNA transcription and protein secretion (Figure 1). We observed similar effects of these inhibitors in epithelial cells (Figure 2). Conclusions: In esophageal fibroblasts, Th2 cytokines increase eotaxin-3 expression via STAT6 signaling, which is not blocked by omeprazole. The small molecule inhibitors AS1517499 and leflunomide block IL-13/STAT6 signaling both in esophageal fibroblasts and epithelial cells. These findings provide rationale for clinical studies of agents like AS1517499 and leflunomide that might benefit both mucosal inflammation and tissue remodeling in EoE. Figure 1. Data are represented as mean ± SEM. Figure 2. Data are represented as mean ± SEM. 204 Epithelial Lysyl Oxidase Is an Early Mediator of Fibrotic Remodeling in Eosinophilic Esophagitis Amanda Muir, Kara K. Dods, Kelly A. Whelan, Jamie Merves, Alain J. Benitez, Maureen DeMarshall, Jonathan Spergel, Gary W. Falk, Rebecca Wells, Mei-Lun Wang, Hiroshi Nakagawa Background: Eosinophilic esophagitis (EoE) is a food allergen-induced inflammatory disor- der, featuring dysphagia, food impaction and esophageal stricture associated with a chronic eosinophil infiltrate and progressive esophageal fibrosis. Esophageal fibrosis is characterized by excessive deposition of collagen produced by activated esophageal fibroblasts as well as increased intra- and intermolecular collagen cross-linking mediated by the lysyl oxidase (LOX) family of enzymes. However, to date nothing is known about the role of LOX in esophageal fibrosis or what factors may influence its expression and activity in EoE. Methods: We evaluated esophageal fibrosis and gene expression in esophageal biopsies representing active EoE (>15 Eos/hpf; 7 adults and 6 pediatric) and normal non-EoE control (0 Eos/hpf; 7 adults and 6 pediatric) patients as well as esophagi from an ova albumin-induced murine EoE (Nat Med. 2013; n=5 control and 5 EoE) by trichrome staining, quantitative reverse S-52 AGA Abstracts transcription polymerase chain reaction (qRT-PCR) and immunofluorescence (IF) assays. Normal human immortalized nontransformed esophageal epithelial EPC2-hTERT cells were treated for 1 week with esophageal fibroblast conditioned media (FCM) along with or without an anti-TNF-a antibody (infliximab) or anti-TGF β inhibitor (1D11) in triplicate. Results: Amongst the LOX family members (LOX, LOXL1, LOXL2, LOXL3 and LOXL4) tested, the expression of LOX appeared to be the highest in the esophageal epithelium of active EoE biopsies including adults (25-fold, P<0.01) and pre-fibrotic pediatric EoE (40-fold, P<0.05) tissues compared to non-EoE controls, implicating LOX in the early stage of fibrosis. IF localized LOX expression predominantly in the epithelial compartment both in human and murine EoE mucosa with increased fibrosis. In EPC2-hTERT cells, FCM stimulated LOX expression robustly (P<0.001) with complete antagonism by infliximab (P<0.01), but not 1D11, suggesting TNF-a-mediated epithelial-stromal crosstalk in LOX induction. Conclu- sions: Our data indicate that esophageal epithelial LOX may have a role in the development of fibrosis in both patients and mice with active EoE. LOX upregulation in pediatric EoE patients may implicate LOX in tissue remodeling in the early stage of EoE progression. Moreover, our data suggest that there is a novel positive feedback mechanism in epithelial LOX induction via fibroblast-derived TNF-a secretion. Therefore, our study may have a translational implication in EoE diagnosis and therapeutics. 205 Differential Proteomic Analysis of Esophageal Tissue Reveals Galectin-3 As Prominent Biomarker Candidate for Esophageal Eosinophilia Girish Hiremath, John E. E. Wiktorowicz, Kizhake V. Soman, Christof Straub, Christina Nance, Melinda Mata, Norma M. Quintanilla, Konrad Pazdrak, Anthony Olive, Alexander Kurosky, Carla M. Davis INTRODUCTION: The esophagus is typically devoid of eosinophils. Persistent esophageal eosinophilia (EE) and related eosinophilic inflammation is associated with eosinophilic esophagitis (EoE) - an allergen-mediated condition limited to the esophagus which can eventually result in epithelial-mesenchymal transition and narrowing of the esophageal lumen. Despite the co-occurrence of EE with EoE and its increasing prevalence in morbidity, no biochemical indicators are available for diagnosis and management of this pathophysiologi- cal phenomenon AIMS: Protein extracted from biopsies obtained from children with EE and healthy donors were analyzed to select for differentially expressed and nitrosylated proteins in esophageal tissues with and without eosinophilia. METHODS: Proteins were extracted and separated by 2D-gel electrophoresis (2DE) from 2-4 proximal and distal esophageal (PE and DE, respectively) biopsies obtained from 6 children with EE and 7 Controls (≤ 1 eos/hpf with no histologic evidence of inflammation). Spots exhibiting p ≤ 0.05 and ratios of ratios (RoR) ≥ 1.5 (based on spot normalized volumes) were selected for robotic picking, and analyzed by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI TOF/MS). Protein abundance was analyzed by t-tests, principal component analysis, and unsupervised hierarchial clustering. Candidate proteins were confirmed by western blot of tissue samples. RESULTS: Both groups were comparable for age, gender, and presenting symptoms. Food allergy was noted in 67% of children with EE and the median eosinophils per high power field (eos/hpf) was 38 (range: 17-100). None of the controls reported any food allergy. Of the 648 proteins consistently expressed as protein spots in 2DE resolved EE and control samples, 91 differentially expressed (p < 0.05) protein spots were analyzed by MALDI TOF/MS resulting in identification of 62 proteins with protein scores of 56 or higher. Analysis of PE samples from EE patients showed upregulation of 8 and downregulation of 3 unique proteins. DE biopsies from EE patients had 6 upregulated and 8 down-regulated proteins. Importantly, Galectin-3 (Gal-3), a galac- tose-specific lectin involved in eosinophil trafficking and development of acute allergic reaction was overexpressed both in PE and DE biopsies (>1.6 and > 2.1 fold, respectively) (Figure) obtained from patients with EE. CONCLUSION: Our results demonstrate that systemic proteomics approach is capable of identifying novel indicators of EE and underscore its potential to aid in biomarker selection and pathway analysis of EE-related disorders. Strong correlation of EE with Galectin 3, a critical regulator of eosinophil migration and trafficking warrants further studies of Gal-3 to its explore its diagnostic and therapeutic poten- tial. Figure: A 3-dimensional montage of galectin-3 expression in the biopsy obtained from the distal esophagus 238 Distinct Clinicopathological Features and Prognosis of Helicobacter pylori Negative Gastric Cancer Jyh-Ming Liou, Kun-Feng Tsai, Jeng-Yih Wu, Chun-Ying Wu, Jaw-Town Lin, Ming-Shiang Wu Background: Helicobacter pylori negative gastric cancer (HPNGC) is an existing disease with distinct clinicopathological features. However, the prognostic effect of HPNGC is still controversial. As previous studies in Eastern Asia, we defined atrophic gastritis patients as the ones with past H. pylori infection in investigating HPNGC by pepsinogen method. We aimed to assess the clinicopathological characteristics of HPNGC and evaluate the potential impact of H. pylori status