SSAT Abstracts Mo1836 Bile Acid At Low pH Can Cause Dilatation of Inter-Cellular Spaces in In Vitro Stratified Primary Esophageal Cells, Possibly by Modulating Wnt and BMP Signaling Sayak Ghatak, Marie Reveiller, Liana Toia, Andrei Ivanov, Tony Godfrey, Jeffrey H. Peters The pathognomonic feature of reflux esophagitis secondary to gastro-esophageal reflux disease is the presence of dilated intercellular spaces in the stratified squamous lining of the esophagus. Bile acid is a major constituent of gastro-esophageal refluxate. In our present study, we developed a novel in vitro transwell culture model for stratified esophageal squamous epithelium. We grew h-TERT transformed primary esophageal cell line EPC1 on polyester transwell surfaces, apically and basally supplemented with calcium enriched media, and observed that the EPC1 cells gradually stratify into a 11-layered squamous epithelium in 7 days. This epithelium also demonstrated well-formed cell junctions, essential for forma- tion of the stratified epithelium. When the EPC1 cells on transwells were treated with a combination of bile acid and pH5, there was loss of epithelial barrier function. Electron microscopy and confocal imaging of the cell junctions showed disruption of adherens junction, tight junction and desmosomes, thus leading to dilated intercellular spaces. At the cellular level, the combination of bile acid and pH5 induced β-catenin phosphorylation and reduced SMAD-1/5/8 phosphorylation, both of which can lead to loss of cell junction proteins. In conclusion, combination of bile acid at low pH in our trasnwell culture model mimicked the effects of gastro-esophageal reflux in vivo, possibly by modulating WNT and BMP signaling pathways. Mo1837 Beneficial Effects of Diazoxide on Hepatic Ischemia/Reperfusion Injury Mateus A. Nogueira, Ana Maria M. Coelho, Sandra N. Sampietre, Nilza A. Molan, Rosely A. Patzina, Luiz C. D'Albuquerque, Marcel C. Machado Background/Aim: Pretreatment with diazoxide, an opening mitoKATP, increases tissue toler- ance against ischemia/reperfusion (I/R) injury, however, there are no prior studies of the role of diazoxide on hepatic I/R injury. In the present study, we evaluated the effect of diazoxide on local and systemic liver I/R process. Methods: Wistar male rats underwent partial liver ischemia performed by clamping the pedicle from medium and left anterior lateral segments during an hour under mechanical ventilation. They were divided into 2 groups: Control Group (n=26): rats received saline and Diazoxide Group (n=26): rats received IV diazoxide (3.5mg/kg) 15 minutes before liver reperfusion. Four and 24 hours after reperfusion, blood were collected for determinations of AST, ALT, TNF- α, IL-6, IL-10, and TGFβ1. Liver tissues were assembled for mitochondrial oxidation and phosphorylation, malondialdehyde (MDA) content, and histologic analysis. Pulmonary vascular permeability and myeloperoxidade (MPO) were also determined. Results: Four hours after reperfusion Diazoxide Group presented elevation of AST, ALT, TNF- α, IL-6, IL-10 and TGF β1 serum levels significantly lower than Control Group (p ,0.05). A significant reduction on liver MDA content and on mitochondrial dysfunction were observed in Diazoxide Group compared to Control Group (p,0.05). No differences in pulmonary vascular permeability and MPO activity were observed between groups. Twenty four hours after reperfusion Diazoxide Group showed a reduction of AST, ALT, and TGF β1 serum levels when compared to Control group (p,0.05). Conclusion: Diazoxide maintains liver mitochondrial function, increases liver tolerance to I/R injury, and reduces systemic inflammatory response. These effects require further evaluations for using in a clinical setting. Grants from FAPESP2010/19078-1 Mo1838 PTK6 Regulates Migration and Invasion of Pancreatic Cancer Cells With ERK1/2 Dependent Pathway Hiroaki Ono, Hiromichi Ito, Marc D. Basson Background: Protein Tyrosine Kinase 6 (PTK6) is a non-receptor type tyrosine kinase, known to be overexpressed in various cancers including pancreatic cancer. The biological role of PTK6 in cancer remains unclear. We hypothesized that PTK6 is a key regulator of pancreatic cancer invasion. Methods: We used 3 cell lines derived from human pancreatic cancers, BxPC3, Panc1, and MIAPaCa2. PTK6 expression and activation were evaluated using western blotting. PTK6 expression was manipulated using siRNA gene silencing or transfection of expression vector. Cellular migration and invasion were evaluated using a Boyden chamber transmigration assay without or with Matrigel, respectively. Downstream signals associated with the effect of PTK6 on cellular migration and invasion were assayed using western blotting and a specific small molecule inhibitor. Results: Pancreatic cancer cell lines expressed PTK6 at various levels; BXPC3 expressed PTK6 robustly, while Panc1 and MIAPaCa2 expressed much lower levels of PTK6. In all 3 cell lines, suppression of PTK6 expression by siRNA significantly reducted both cellular motility and invasion through matrigel (0.59/ 0.49 fold for BXPC3, 0.61/0.62 for Panc1, 0.42/0.39 for MIAPaCa2, respectively, p ,0.05 for each). In contrast, forced over-expression of PTK6 by transfection of a PTK6 expression vector in Panc1 and MIAPaCa2 cells significantly increased cellular migration and invasion (1.57/1.67 fold for Panc1, and 1.44/1.57 for MIAPaCa2, respectively, p , 0.05). Gene silencing of PTK6 reduced the activation of ERK1/2, but not AKT and STAT3, while overex- pression of PTK6 increased ERK1/2 activation. When the cells were treated with U0126, a specific inhibitor of ERK1/2, the effect of PTK6 overexpression on cellular migration/invasion was completely abolished. Conclusion: PTK6 regulates cellular migration and invasion in pancreatic cancer, via the MAPK/ERK signaling pathway. Our findings suggest that PTK6 may be a novel therapeutic target for pancreatic cancer. S-1112 SSAT Abstracts Mo1839 Osteopontin (Opn) Isoforms, Diabetes, Obesity, and Cancer; What's One Got to Do With the Other? A New Role for Opn Konrad Sarosiek, Elizabeth Jones, Galina Chipitsyna, Mazhar Al-Zoubi, Shivam Saxena, Christopher Y. Kang, Ankit V. Gandhi, David Tichansky, Charles J. Yeo, Hwyda A. Arafat Background: Alternative splicing of osteopontin (OPN) produces three splice variants: OPNa, OPNb, and OPNc. We have previously demonstrated a role for OPNc in pancreatic ductal adenocarcinoma (PDA) inflammation and proposed its potential as a novel therapeutic target to reduce PDA-associated inflammation. The aims of this study were to examine the expression pattern of OPN splice variants in sera from patients with pancreatic lesions and to determine their correlation with the presence of systemic inflammatory conditions, such as obesity and diabetes. In addition, the functional significance of the individual isoforms was evaluated. Methods: Serum samples were obtained from 90 patients undergoing pancreatic surgery at a single institution. Patients were grouped into 8 subgroups based on the disease process and presence of obesity and/or diabetes. Sera from age-matched healthy volunteers were analyzed (n=29). Real-time polymerase chain reaction and ultraviolet light illumination of ethidium-bromide gel staining were used to examine the OPN mRNA and its individual isoforms. In vitro, wound healing, cell proliferation and soft-agar colony formation assays evaluated the functional impact of each isoform in PDA cells transfected with isoform- specific cDNA. A panel of inflammation-related genes was also analyzed. Results: Sera were obtained from PDA patients (mean age 66 ± 1.12(SE) years; 40 male). Histopathology confirmed PDA in 58 patients, IPMN in 32. Diabetes (type 2) alone was detected in 13 PDA and 4 IPMN patients and in combination with obesity in 5 PDA and 1 IPMN patients. In PDA only, the presence of OPNb was seen in 33% of the patients' sera, OPNc in 48%, with both being present in 15%. The presence of diabetes and/or obesity was associated with complete disappearance of OPNb and only expression of OPNc (82% of PDA diabetics, 100% of obese PDA patients, and 100% of obese diabetic patients with PDA). No OPNb or c was detected in the normal sera. OPNc had a significant association with presence of systemic inflammation (OR=6.8 [1.7-65, 95%CI]; p ,0.05). In vitro studies show that overexpression of OPNb and c isoforms significantly (P ,0.05) and (P,0.02), respectively, increased the activity of PDA cells in soft-agar colony formation and wound healing assays compared with controls. Conclusions: Our data show for the first time the significant association between OPN splice variant c (OPNc) and the presence of systemic inflammation in patients with obesity and/or diabetes. In vitro data suggest that increased OPNc expression in PDA cells is associated with increased migration capacity. Unraveling the functional role of OPNc in systemic inflammation is essential to understanding its significance as a marker and a therapeutic target during metastasis development in PDA. Mo1840 Local and Systemic Effects of Aging on Acute Pancreatitis Ana Maria M. Coelho, Marcel C. Machado, Sandra N. Sampietre, Nilza A. Molan, Inneke M. van der Heijden, José Eduardo M. Cunha, Luiz C. D'Albuquerque Background/Aim: Acute pancreatitis (AP) is associated with high morbidity and mortality rates. Aging process has been found to influence the course and outcome of AP. The aim of this study was to evaluate the local and systemic effects of aging on severity of AP in an experimental model. Methods: AP was induced in male Wistar rats by intraductal 2.5% taurocholate injection and divided into 2 experimental groups: GI (n=20): Young (3 month old rats), and GII (n=20): Older (18 month old rats). Two and 24 hours after AP blood were collected for determinations of amylase, AST, ALT, urea, creatinine, glucose, and of plasma ileal fatty acid binding protein (I-FABP). TNF- α and IL-6 levels were determined in serum and ascitic fluid. Liver mitochondrial oxidation and phosphorylation and malondial- dehyde (MDA) contents, and pulmonar myeloperoxidade (MPO) activity were also per- formed. Bacterial translocation was evaluated by bacterial cultures of pancreas expressed in colony-forming units (CFU) per gram. Results: A significant increase in serum amylase, AST, ALT, urea, creatinine, glucose, I-FABP, and IL-6 levels, and a reduction in serum and ascitic fluid TNF-α levels were observed in the elder group compared to the young group (p ,0.05). Liver mitochondrial dysfunction, MDA contents, and pulmonary MPO activity were increased in the older group compared to the young group (p ,0.05). Also, a significant increase in positive bacterial cultures obtained from pancreas tissue in older group was significantly increased compared to young rats (p ,0.05). Conclusion: This study demonstrated that aging influences the course of acute pancreatitis evidenced by increased local and systemic lesions and the increased in bacterial translocation. These findings may have significant therapeutic implication in the clinical setting. Mo2130 Minimally Invasive Full Thickness Colonic Resection: A Novel Localised Excision Procedure Adela Brigic, Paul D. Sibbons, Chris H. Fraser, Susan K. Clark, Robin H. Kennedy Aims: Worldwide introduction of the bowel cancer screening programmes has lead to an increase in the number of patients diagnosed with complex, benign colonic polyps unsuitable for endoscopic resection. A significant proportion is referred for hemicolectomy, which is associated with significant risk of morbidity and mortality. To address this and improve clinical outcomes, we modified a previously reported full thickness laparo-endoscopic exci- sion (FLEX) technique developed in our institution. Methods: Following a series of ex-vivo experiments to standardise procedural steps, surgery was performed in five 70-kg pigs. A simulated colonic polyp was created by endoscopic injection of Spot® and the clearance margin was delineated by circumferential placement of mucosal argon plasma coagulator (APC) marks. Full thickness eversion of the colonic wall that contains the simulated lesion was achieved by endoscopic placement of prototype BraceBars (BBs). The everted segment was excised using a linear laparoscopic stapler placed below the BBs. The first pig was terminated immediately and others 8 days after surgery. Results: Procedure duration was defined from placement of mucosal APC marks to specimen excision with a median time of 26 min (range 20-31 min). All excised specimens contained three pairs of BBs delineating