Minocycline reverse hyperalgesia 887 Characterization of Cholangitis Associated With Experimental Autoimmune Pancreatitis in Mice Masao Yamashina, Akiyoshi Nishio, Shinji Nakayama, Atsushi Nakajima, Takeo Kusuda, Norimasa Fukata, Yutaku Sakaguchi, Katsunori Yoshida, Toshiro Fukui, Kazushige Uchida, Kazuichi Okazaki Background and Aim: Autoimmune pancreatitis (AIP) is an increasingly recognized entity of pancreatitis characterized by a steroid-responsive, fibroinflammatory condition. AIP often accompanies extrapancreatic lesions such as sclerosing cholangitis, sclerosing sialoadenitis and retroperitoneal fibrosis. Recent studies support the concept of AIP as an immunoglobulin G4-associated systemic disease. To study the immune response in this multi-organ disease, we analyzed the relationship of the intra- and extrahepatic cholangitis to pancreatitis using murine experimental models of AIP. Methods: MRL/Mp mice and C57BL/6 IL-10 deficient (IL-10KO) mice were injected intraperitoneously with polyinosinic polycytidylic acid (poly I:C) at a dose of 5 mg/kg of body weight twice a week for up to 12 or 8 weeks, respectively. Mice were serially sacrificed and the severity of pancreatitis and cholangitis was evaluated based on scoring systems. Inflammatory cell infiltration into each organ was also immunohis- tochemically examined. Serum pancreatic and hepatobiliary enzyme levels were measured at a commercial laboratory. Serum cytokine levels were measured by ELISA. Gene expression of pro-inflammatory cytokines of bile duct and pancreatic tissues was examined by real time PCR. CD4 and CD8 T cell subsets were prepared using magnetic cell sorting system from IL-10KO mice with pancreatitis. Each T cell subset was adoptively transferred into C57BL/ 6 scid mice. Results: Administration of poly I:C induced intra- and extrahepatic cholangitis in association with pancreatitis in both of MRL/Mp and IL-10KO mice. Immunohistochemically, abundant B cells and plasmacytes, in addition to CD4 and CD8 T cells, were infiltrated into the pancreas and periportal areas of the hepatic lobules. Infiltration of Foxp3-positve CD4 T cells were also observed in the inflamed areas of pancreas, livers and bile ducts. Gene expression of IL-10 of the pancreas and the extrahepatic bile ducts was increased, coupled with the increase of IFN-g and TNF-a. Adoptive transfer of CD4 T cells, but not CD8 T cells induced both pancreatitis and cholangitis in recipient scid mice. Conclusions: In murine AIP, the coexistence of the bile duct lesion with the pancreatitis suggests that the presence of the common autoimmune mechanism via autoreactive CD4 T cells. IL-10 is supposed to play a protective role in the pathogenesis of AIP and associated cholangitis. 888 CCK and Gastrin Stimulate Collagen Synthesis but Inhibit Proliferation in Rat Pancreatic Stellate Cells Oliver Seiz, Marc J. Berna, Friso Nast, Jan K. Hennigs, Andrea Pace Intr.: Pancreatic fibrosis is a characteristic feature of chronic pancreatitis. studies suggest that activated pancreatic stellate cells (PSC) play a pivotal role in the development of fibrosis. Different stimuli have been identified to activate PSC. In pancreatic acinar cells the GI- hormone CCK , known to mediate apoptotic and mitogenic signals activates different signal- ling pathways through stimulation of the CCK-A-receptor (CCK-A-R). So far only little is known about CCKs role in the activation of PSC, in particular its role in pancreatic fibrogen- esis. Aim: Aim of this study was to elucidate the collagen synthesis upon CCK-and gastrin stimulation in PSC and further to investigate their role in proliferation. Meth.: Rat PSC were isolated by gradient centrifugation and then cultured.. Collagen production was examined by real-time-PCR, ELISA, immunocytochemistry and by WB analysis. Prolifration was exam- ined by 3H-thymidine uptake and MTT assays. TGF-beta-stimulation was used as control. Res.: Upon stimulation with CCK-8 and gastrin there was a marked increase in collagen synthesis. CCK-8 stimulated collagen production 37% above non stimulated control, gastrin 36% and TGF-beta 49%. Similar to TGF-beta, which is known to inhibit proliferation in PSC, CCK inhibited proliferation by 18% and Gastrin by 16%. Concl.: In rat PSC both CCK and gastrin significantly induced type I collagen synthesis as assessed by real-time-PCR, WB, ELISA and immunocytochemistry. The effect is less but comparable to TGF-beta- stimulated collagen synthesis, so far described as the most potent stimulant for collagen production in PSC. These data suggest that CCK and gastrin have a direct activating effect on PSC, and seem to be important regulators of pancreatic fibrogenesis. Moreover, as shown by thymidine uptake and MTT similar to TGF-beta both CCK and gastrin inhibit proliferation of PSC. S-125 AGA Abstracts 889 Cathepsin Activity in Pancreatitis: Imaging, Identification and Contribution to Disease Victoria Lyo, Fiore Cattaruzza, Eileen F. Grady, Matthew Bogyo, Nigel W. Bunnett, Kimberly S. Kirkwood Proteases are major mediators of inflammation. Cathepsins are cysteine proteases that contrib- ute to pancreatitis by processing trypsinogen within acinar cells (cathepsin B, L), and secreted cathepsins cause inflammation and pain by incompletely understood mechanisms (cathepsin S). However, the spectrum of cathepsins that are activated in pancreatitis is not defined, and their contribution to disease remains to be determined. Methods and Results. Since proteases are regulated by post-translational control of activity, we used an activity-based probe (ABP) that covalently binds to active proteases to detect activated cathepsins. GB123 has an acyloxymethyl ketone reactive “warhead” that targets the active site of cysteine cathepsins and a Cy5.5 tag for detection. GB123 interacted with purified cathepsin B, L and S, as determined by SDS-PAGE and gel fluorescence analysis. The cathepsin inhibitor K11777 abolished this interaction, confirming that GB123 binds to active cathepsins. To identify cathepsins that are activated in the inflamed pancreas, C57/Bl6 mice were treated with cerulein or vehicle (12 hourly injections, 50 μg/kg SQ) to induce acute pancreatitis. Mice then received GB123 (25nmol/mouse IV) and were studied 24 h later. Reflectance imaging of the excised pancreas revealed a 5-fold increase in the Cy5.5 signal in mice with pancreatitis (p<0.05 vs. control), indicative of cathepsin activation. Biochemical analysis of the excised pancreas by SDS-PAGE and gel fluorescence indicated activation of proteases with the predicted mass of cathepsins B (10.1-fold), L (5.1-fold) and S (5.7-fold) (all p<0.05), and protease identity was confirmed by immunoprecipitation. To evaluate the contribution of cathepsins to pancreatic inflammation, mice were treated with K11777 or vehicle (control) prior to cerulein. K11777 inhibited cathepsin activity in the inflamed pancreas (35% reduc- tion in excised pancreas fluorescence, and 23-39% reduction by SDS-PAGE, both p<0.05 vs. control), confirming effective inhibition. K11777 reduced serum amylase by 38% and pancreatic/total body weight by 37% (both p<0.01). Conclusions. Acute pancreatitis results in activation of cathepsins B, L and S that can be detected by imaging of intact tissues and proteomic analysis of tissue extracts, and inhibition of cathepsins improves disease. ABPs offer a novel approach for identification of proteases that are predictive biomarkers of disease progression and response to therapy. ABPs with near-infrared fluorophores may be useful as non-invasive clinical tools for the diagnosis and quantification of pancreatic inflammation. Supported by DK46285, 43207 and HHMI. 890 Mutations in the CFTR Gene are Associated With Pancreatic Ductal Stone Formation in Hereditary Pancreatitis. Mark Ellrichmann, Monika S. Janot, Rainer Lebert, Wolfgang E. Schmidt, Waldemar Uhl, Andrea Tannapfel, Jan-Michel Otte Background & Aims: Pancreatic lithiasis is a cardinal finding in chronic pancreatitis (CP). However, its potential pathogenetic relevance in the onset and perpetuation of hereditary pancreatitis is incompletely characterized. Recently, mutations in the cystic fibrosis transmem- brane conductance regulator (CFTR) gene have been identified as an independent risk factor for the development of CP. Mutations of this gene are associated with inspissated secretions which might predispose to ductal plug and stone formation. We therefore sought to systemat- ically clarify a putative association of this and other gene mutations in patients suffering from hereditary pancreatitis with intraductal stone formation. Methods: Genomic DNA was prepared from blood and pancreatic tissue samples of a total of n = 478 Western European patients suffering from CP. DNA was analyzed for mutations in the serine protease inhibitor Kazal type I (SPINK1) gene (N34S), the cationic trypsinogen gene (PRSS1) (R122H and N29I) and the CFTR gene (F508del, R117H, 5-T Allel) by melting curve analysis. PRSS1 (Exon 2) and CTRC (Exon 7) mutations were detected by DNA sequencing. For additional CFTR analysis, samples were tested with a validated commercial kit which simultaneously detects 36 mutations and the Tn polymorphism. Results: In the samples analyzed, CFTR mutations were detected in 7.4% (18/243), a Tn polymorphism was detected in 4.5 % (10/ 222), SPINK1 mutations in 3.1 % (15/478), PRSS1 mutations in 2.0% (9/446) and CTRC mutations in 1.3% (1/77). Among these patients 15 (3.1%) showed stone formation and consecutive pancreatic ductal obstruction with the need of surgical treatment. Five of these patients displayed the CFTR mutation F508del (33.3%, p<0.05). Furthermore a SPINK 1 (N34S), PRSS1 (R122H), CFTR (G551D) and a CTRC (R254W) mutation was detected in one patient respectively (6.7%). Two patients were transheterozygous (SPINK1:N34S / CFTR:F508del and PRSS1:R122H / CFTR:508 del). Conclusion: Pancreatic ductal stone formation is not frequently observed in patients with hereditary pancreatitis. However, in patients carrying the CFTR 508del mutation the risk of developing ductals stones is signific- antly increased. CFTR gene mutations may damage bicarbonate producing intrapancreatic epithelium thereby reducing the pH within the intraacinar space. This acidic environment may predispose to intraductal stone formation thereby promoting chronic inflammation within the pancreas. The processes involved in the onset of hereditary pancreatitis associated with mutations of the CFTR gene might therefore represent an alternative pathogenetic con- cept. 891 Experimental Acute Pancreatitis Causes Pathologic Changes in Lysosomal Membrane Protein and Lipid Composition Olga A. Mareninova, Iskandar Yakubov, Wenzhuo Jia, Ilya Gukovsky, Anna S. Gukovskaya Background and Aims: Our recent study (J Clin Invest, 2009) revealed that pancreatitis causes profound impairment of autophagy (a.k.a. macroautophagy), the main cellular cata- bolic, lysosome driven process. We showed that autophagy impairment in pancreatitis is caused by lysosomal dysfunction a prominent manifestation of which was defective pro- cessing/maturation of cathepsins, major lysosomal hydrolases. Impaired autophagy mediates AGA Abstracts