Tu1822 IL-23 Regulates Gene Expressions via STAT3 Activation in the Human Intestinal Neuroendocrine Cell Line

Design: 137 patients underwent diagnostic analysis of EUS aspirated pancreatic cyst fluid. DNA was extracted from cyst fluid with quantity and quality (extent of degradation) measured by optical density and qPCR. Mutational analysis targeted KRAS point mutation and loss of heterogeneity (LOH) mutations of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 18q, 21q, 22q. Molecular findings were integrated with results from EUS, chemistry and cytology. For each marker, presence or absence of mutation and clonality were determined, determined, with high clonality comprising 75% of more of cells affected by LOH and low clonality comprising 50-75% of cells. Interpretation of combined molecular, imaging, and chemistry results were classified and subsequently compared to surgical outcome or followup. Surgical pathology was classified into indolent biological behavior (benign or low grade disease), and aggressive biological behavior (high grade or malignant disease).

Results: Surgical pathology was available on 81 of these patients. Outcomes included benign cystic neoplasms with low and high grade dysplasia, malignant ductal disease, endocrine neoplasms and pseudocysts. There were 32 aggressive outcomes and 49 indolent outcomes. Molecular findings, including elevated DNA level, and high numbers and clonality of mutations, correlated strongly with aggressive biological behavior. While KRAS mutation was highly specific for mucinous cyst formation, individual molecular parameters used in isolation showed limited sensitivity for detection of aggressive disease. Combining all molecular parameters together, sensitivity was 94%, and specificity was 100%, with overall accuracy of 97%.

Conclusions: The molecular biology of pancreatic cystic disease involves multiple pathways that can each lead to cancer. The majority of pancreatic cysts do not progress to cancer and the diagnosis and biological potential of individual lesions can be effectively characterized by integrating broad panel molecular findings with information from first line testing. The presence or absence of aggressive molecular changes, taken in the context of overall findings provides an effective means to individualize patient management when first line results are unclear.

promoter regions exist in genomic databases for the SLCO1A2 gene. Four putative VDR response elements (VDREs) were predicted in silico within the SLCO1A2 promoter variant 1 and five within the SLCO1A2 promoter variant 2. One predicted VDRE in each SLCO1A2 promoter variant served as a strong binding site for the VDR:RXRα heterodimers In Vitro, and both of these elements were potently activated by VDR in the presence of vitamin D3 in heterologous promoter assays. In reporter assays employing native promoter constructs, only the SLCO1A2 promoter variant 1 was strongly induced by VDR and its ligand vitamin D3. Site-directed mutagenesis of the VDRE within the promoter variant 1 abolished its responsiveness to VDR and vitamin D3. In ChIP assays, VDR bound to this element, but not to the putative VDRE in the SLCO1A2 promoter variant 2, within living cells. Conclusions. We have shown that the expression of the SLCO1A2 gene is induced by vitamin D3 at the transcriptional level via VDR. Our results suggest that pharmacological administration of vitamin D3 may allow modulation of intestinal absorption of OATP1A2 transport substrates. Tu1822 IL-23 Regulates Gene Expressions via STAT3 Activation in the Human Intestinal Neuroendocrine Cell Line Hideyuki Suzuki, Hitoshi Ogawa, Koh Miura, Takeshi Naitoh, Chikashi Shibata Introduction: Interleukin-23 (IL-23) is predominantly secreted by activated dendritic cells, monocytes, and macrophages, and plays an essential role on mucosal immune system. The IL-23 receptor (IL-23R) is expressed on the T helper type 17 (TH17) cells, and IL-23 contributes to expansion and stabilization of TH17 cells via IL-23R and activation of signal transducers and activators of transcription 3 (STAT3). Recently we identified that IL-23R was also expressed in the intestinal neuroendocrine cells by immunohistochemical analysis, which suggest the intestinal neuroendocrine cells may play a role in mucosal immune system via IL-23R. In the present study, we examined the effect of IL-23 on the intestinal neuroendocrine cells using the human intestinal neuroendocrine (Enterochromaffin) cell line KRJ-I. Material and Methods: KRJ-I cells were kindly provided by Dr. Mark Kidd (Yale University School of Medicine) and cultured with or without 10 μg/ml of recombinant human IL-23. Phosphorylation of STAT3 was examined by Western blotting. Using Affymetrix GeneChip Human Genome U133 Plus 2.0 array, mRNA expressions of a significant number of genes were detected beyond the threshold level, and their changes were confirmed with quantitative RT-PCR. Results: Phosphorylation of STAT3 in KRJ-I cells was induced by IL-23 stimulation. The mRNA expression levels of the several genes including suppressor of cytokine signaling 3 (SOCS3) and IL23R were increased by IL-23 stimulation. Conclusion: Our results indicate that IL-23 regulates gene expressions of the intestinal neuroendocrine cells In Vitro. The altered gene expression was observed in accordance with STAT3 activation. The intestinal neuroendocrine cells may participate in the mucosal immune system and pathogenesis of intestinal immune disorders via IL-23R and STAT3 activation.

Tu1514 Limited Application of New International Consensus Diagnostic Criteria for Autoimmune Pancreatitis in Clinical Practice James J. Farrell, Jonathan L. Wong, Krisztina Kisfalvi Background: The International Association of Pancreatology recently published the International consensus diagnostic criteria for diagnosing and categorizing autoimmune pancreatitis(AIP)(Pancreas April , 2011). We aimed to assess the applicability of these guidelines using our cohort of patients with presumed AIP. Methods: We reviewed our prospectively collected AIP database to identify clinical-radiologic-pathologic features which are necessary for the diagnostic criteria for both definitive and probable type 1 or 2 AIP, at both levels 1 and level 2 criteria, per the consensus guidelines. We sought to classify our patients as either definitive or probable type 1 or 2 AIP, or as unclassifiable. Results: We currently follow 52 patients with a presumed diagnosis of AIP. Of the 52 patients, we could categorize 27 (52%) as definitive Type 1 or Type 2 AIP, according to the new guidelines. 21 patients fell into the definitive Type 1 category (78%) while only 6 patients proved to be Type 2 AIP cases (22%). The majority of the cases of definitive Type 1 AIP were defined by the combination of imaging, serology, other organ involvement and response to steroid, with only 5 patients having level 1 histology criteria. Histology was the primary basis for a definitive diagnosis of Type 2 AIP. In 3 patients (50%), although the histology was in agreement with level 1 histology for Type 2, the combination of non-pathology factors qualified the cases for the definitive Type 1 category also. No patients could be classified as either probable Type 1 or 2. 25 (48%) of our AIP patients, however, could not be definitively categorized as Type 1 or Type 2 AIP. 19 of these cases (76%) had atypical imaging, and 9 (36%) normal or low IgG4 serology. 15(60%) of these patients had histology, including 8 with distinctive histological features for either Type 1 or Type 2 AIP without IgG4 staining. Diagnostic ERP was not performed on 12 patients. 3 patients had spontaneous resolution in radiologic features, which is not a criterion in the current guidelines. Conclusion: While it is possible that certain of this cohorts' patients do not in fact have AIP, the Consensus guidelines' need for IgG4 immunohistochemistry and diagnostic ERCP findings, as well as no emphasis on spontaneous resolution of symptoms and imaging findings, may limit the universal application of these criteria.

Tu1823 MiR-133a Regulates the Endocytosis of a G Protein-Coupled Receptor in Human Colonocytes Ivy Ka Man Law, Kyriaki Bakirtzi, Dimitrios Iliopoulos, Charalabos Pothoulakis Background and Aims: MicroRNAs (miRNAs) are short non-coding RNAs involved in different pathophysiological functions at the post-transcriptional level. However their role in G proteincoupled receptor internalization and recycling has not been determined. Neurotensin (NT) and its high affinity G protein -coupled receptor 1 (NTR1) mediates intestinal inflammation and proliferation In Vivo and In Vitro. NTR1 undergoes endocytosis in response to ligand exposure in target cells. We have recently identified a group of 8 miRNAs (miR21, miR23a, miR-23b, miR-133a, miR-140, miR-155, miR210 and miR-331-5p) that are up-regulated upon NT exposure in NTR1-expressing human colonic NCM460 epithelial cells (NCM460NTR1) (Gastroenterology 2011;141:1749-61). Here we sought to identify miRNA(s) which they regulate NTR1 endocytosis and recycling and determine their gene target(s). Methods: Cellular NTR1 internalization was examined in NCM460-NTR1-GFP cells transfected with different miRNA inhibitors. The miRNAs targeted gene candidates were identified using online databases (TargetScanHuman, PicTar and miRBase). Plasmid constructs with luciferase reporter and miRNA Target 3'UTRs were transfected to NCM460-NTR1 cells with or without miRNA inhibitors 48 h prior to NT exposure. Luciferase activity was also examined in HEK293 cells overexpressing miRNAs. si-RNAs of target genes were transfected to NCM460NTR1-GFP cells for NTR1 localization. The amounts of surface NTR1 internalized and recycled to plasma membrane after NT exposure were assessed by cell-based ELISA. Cellular localizations of target gene candidates were examined by immunocytochemistry. Results: Out of the 8 NT-regulated miRNAs inhibited by antisense miRNAs, only miR-133a inhibition altered NTR1 cellular localization and attenuated NTR1 receptor endocytosis (p< 0.05). In silico analysis suggested that miR-133a has a binding site in the 3'UTR of the aftiphilin gene (AFTN) (a protein containing clathrin-binding motifs). Inhibition of miR-133a impeded NTinduced reduction of AFTN mRNA (p < 0.05%) and AFTN 3'UTR luciferase activity (p < 0.01%). Overexpression of miR-133a reduced AFTN 3'UTR luciferase in HEK293 cells. Gene silencing of AFTN in NCM460 colonic epithelial cells not only affected NTR1 cellular localization, but also reduced the number of NTR1 internalized. Conclusions: Our results indicate that miR-133a regulates endocytosis and recycling of NTR1 in human colonocytes by targeting aftiphilin. This is the first report in the literature of a microRNA regulating endocytosis of a G-protein coupled receptor in colonocytes or any cell type. This is also the first report that aftiphilin is a direct target of miR-133a. Supported by NIH grant DK60729 (CP), the Blinder Research Foundation for Crohn's Disease (IKML), and the Eli and Edythe Broad Chair (CP).

Tu1821 The Gene Encoding the Human Organic Anion Transporting Polypeptide Oatp1a2 (Gene Symbol Slco1a2) is Transactivated by the Vitamin D Receptor (VDR) Jyrki J. Eloranta, Christian Hiller, Moritz Jüttner, Gerd A. Kullak-Ublick Background. The organic anion transporting polypeptide 1A2 mediates cellular uptake of a wide range of endogenous substrates, as well as of drugs and xenobiotics. Transport substrates include bile acids, conjugated sex steroids, thyroid hormones, prostaglandin E2, the endothelin receptor antagonist BQ-123, the thrombin inhibitor CRC-220, the opioid receptor agonist deltorphin II, magnetic resonance imaging contrast agents, and the cyanobacterial toxin microcystin. OATP1A2 is expressed in several tissues, notably at the apical membrane of small intestinal epithelial cells, at the blood-brain barrier, in cholangiocytes, and at the apical domain of distal nephrons in the kidney. Little is known about the transcriptional regulation of the human SLCO1A2 gene. Given its role in intestinal drug absorption, a detailed analysis of the mechanisms that regulate OATP1A2 expression is potentially of great pharmacological relevance. Methods. Messenger RNA levels were determined by reverse transcription followed by TaqMan real-time PCR. SiRNA transfections were used to suppress endogenous gene expression. In silico promoter analysis was performed by NUBIScan and MatInspector softwares. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays were employed to verify DNA-binding In Vitro and within living cells, respectively. Dual luciferase promoter-reporter assays were used to measure either heterologous or native promoter activities. Results. Treatment of human intestine-derived Caco2 cells with vitamin D 3 markedly increased endogenous OATP1A2 mRNA levels. Suppressing endogenous vitamin D receptor (VDR) expression by siRNAs significantly reduced the OATP1A2 mRNA induction by vitamin D3. Two alternative

AGA Abstracts

S-854