ology 13 (2013) S2–S98

O-55 Abstract id: 219.

Insights into the functional role of tenascin-C in pancreaticcarcinogenesis

Susanne Haneder, Sonja Berchtold, Katja Steiger, Irene Esposito.

Institute of Pathology, Technische Universit€at M€unchen, Germany

Introduction: Pancreatic cancer (PDAC) is characterized by a desmo-plastic stroma, which is rich in proteins of the extracellular matrix (ECM).Tenascin-C (TNC), a large ECM glycoprotein, is upregulated in pancreaticcancer and precursor lesions.

Aims: In this study the role of TNC was characterized in PDAC pro-gression in a mouse model.

Patients & methods: Triple mutant mice (KC-TNCko and KC-TNChet)were generated by crossing mice of the LSL-KrasG12D/þ;Ptf1aþ/Cre(ex1) (KC)line, a well characterized mouse model for pancreatic cancer, with TNCknockout mice. The corresponding pancreata of the different genotypeswere harvested after different time points (1-15 months). A total of 106mutant mice were extensively examined by conventional morphology(H&E), as well as by special stains (PasaV Alcian, MassonaV Goldner) andimmunohistochemistry (Ki67, CaspaseaV 3, CK19, Claudin-18), focusing onthe degree of architectural distortion of the pancreas and on the type andfrequency of various precursor lesions and of PDAC.

Results: Three months old TNCko/het mice showed a more pro-nounced architectural distortion of the pancreas than KC mice withfibrosis, inflammation and acinar-ductal metaplasia (ADM). Precursor le-sions were detected in all genotypes at the age of one month, but older KC-TNCko/het mice showed a higher incidence of PanIN3 and PDAC was onlyobserved in KC-TNCko and KC-TNChet mice.

Conclusion: Extensive areas with ADM in KC-TNCko/het mice might bedue to a reduced epithelial regeneration of challenged pancreata and afaster progression to invasive carcinoma. This result implicates that TNCmight play a critical role in tissue regeneration and stromal homeostasisand possibly in carcinogenesis.

Abstracts / PancreatS20

Poster Session I

PI-1 Abstract id: 32.

Experimental acute pancreatitis induces mitochondrial dysfunctionin rat pancreas, kidney and lungs but not in liver

Irma Kuliaviene 1, Sonata Trumbeckaite 2, Marius Kincius 3, RasaBaniene 4, Eugene Jansen 5, Limas Kupcinskas 3, VilmanteBorutaite 4, Antanas Gulbinas 6.

1 Lithuanian University of Health Sciences, Department ofGastroenterology, Lithuania2 Lithuanian University of Health Sciences, Institute of Neurosciences,Lithuania3 Lithuanian University of Health Sciences, Institute for DigestiveResearch, Lithuania4 Lithuanian University of Health Sciences, Institute of Neurosciences,Lithuania5National Institute for Public Health and the Environment, Laboratoryfor Health Protection Research, Netherlands6 Lithuanian University of Health Sciences, Institute for DigestiveResearch, Lithuania

Introduction: Excessive systemic inflammatory response syndromeduring severe acute pancreatitis (AP) leads to multiple organ dysfunctionsyndrome, which is the main cause of death and may be associated withprimary mitochondrial disturbances.

Aims: The aim of our study was to evaluate the role of mitochondriaduring experimental AP in pancreas and vital organs like kidney, lungs andliver within the first 48 hours.

Materials & methods: AP was induced in 35 male Wistar rats byintraductal application of sodium taurocholate (5%, 1.75 ml/kg). Animalswere divided into seven groups (control and 1, 3, 6, 12, 24, 48 hours)reflecting the time from induction of the AP till collection of tissues.Mitochondria were isolated by differential centrifugation and mitochon-drial respiration rates were measured oxygraphically.

Results: (1) Mitochondria in pancreas are affected within the first 6hours after onset of AP, (2) kidney mitochondria are affected 24 hours afteronset of AP, (3) lungs mitochondria are affected within 48 hours after onsetof AP whereas (4) liver mitochondria remain well preserved within thefirst 48 hours. Severe AP–induced decrease in the oxidative phosphory-lation of pancreas, kidney and lungs mitochondria was more pronouncedwith Complex I–linked (glutamate/malate) than with Complex II-linked(succinate) substrates and was associated with inhibition of Complex I.

Conclusion: Our data show that the disturbances of mitochondrialenergy metabolism in pancreas, kidney and lungs may play an importantrole in the development and progression of AP as a systemic disease.

PI-2 Abstract id: 309.

Inhibition of pancreatitis-associated cell death using a humanpancreatic acinar cell model

Muhammad Ahsan Javed 1, Rishi Mukherjee 2, Li Wen 2, WeiHuang 2, Michael Chvanov 1, Muhammad Awais 2, David Criddle 1, AlexeiTepikin 3, Michael Raraty 2, Paula Ghaneh 2, John Neoptolemos 2, RobertSutton 2.

11 NIHR Liverpool Pancreas Biomedical Research Unit, & 2 Departmentof Cellular and Molecular Physiology, University of Liverpool, UnitedKingdom2NIHR Liverpool Pancreas Biomedical Research Unit, Royal LiverpoolUniversity Hospital and University of Liverpool, United Kingdom3Department of Cellular and Molecular Physiology, University ofLiverpool, United Kingdom

Introduction: Inhibition of acute pancreatitis (AP) in rodents as amodel for human dug development has largely not been successful. Wesought to develop an approach adding selective testing of potential drugsfor AP using freshly isolated human PACs.

Aims: To evaluate effects of the cyclophilin inhibitors CyclosporinA (CsA) orDebio-025 on human PACs exposed to taurolithocholate sulphate (TLCS).

Materials & methods: Human PACs were isolated by collagenase diges-tion, mechanical dispersion and low-speed centrifugation from normalpancreatic tissue gifted by consenting surgical patients. Fluorescent confocalmicroscopy was used to assess mitochondrial membrane potential (MMP)measured with 50 nM TMRM, excitation 543 nm, emission >550 nm)andnecrotic cell death pathway activation (blind cell uptake count with 1 mMpropidium iodide, excitation 488 nm, emission 630-693 nm).PACs wereincubated with 500 mM TLCS for 30 min in the presence or absence of10 mMCsAor 100 nM Debio-025 to test the effect of cyclophilin inhibition.

Results: CsA or Debio-025 preserved MMP (P<0.05) and preventedpropidium iodide uptake (P<0.05) compared to control cells in response to500 mM TLCS. MMP and cell death profiles of human PACs were similar tomurine PACs in response to this toxin.

Conclusion: CsA and Debio-025 preserved MMP and reduced necrosisin human PACs following TLCS, likely through inhibition of cyclophilin D.Testing of compounds with human PACs may promote evaluation of po-tential drugs for AP.

PI-3 Abstract id: 270.

Decrease of bicarbonate dependent pancreatic ductular secretion af-ter ceruleinaV‘induced acute pancreatitis in ratsCategory: Basic science - acute pancreatitis.

Pilar Hernandez-Lorenzo, Monica Garcia, Jos�e Ignacio San Rom�an, Jos�eJulian Calvo.

University of Salamanca, Spain

Abstracts / Pancreatology

Introduction: The effect of acute pancreatitis (AP) on ductal cells hasnot been so extensively studied as in acinar cells.

Aims: To study the influence of experimental AP on the fluid secretionof pancreatic duct cells.

Patients & methods: AP was induced by cerulein hyperstimulation.Fragments of pancreatic ducts were isolated, cultured for 12aV‘16 hours,and fluid secretion was studied by digital videomicroscopy in sealedducts

Results: Cerulein (100 pM or 1 nM) did not stimulate fluid secretion inpancreatic ducts from control rats. In a perfusion solution with HCO3

- andCl-, fluid secretion of ducts from pancreatitic rats showed a significantreduction (71 � 10 pL$min-1$mm-2), compared to ducts from control an-imals (141 � 16 pL$min-1$mm-2), after forskolin stimulation. Chloridesecretion, after stimulation with forskolin, showed no differences in con-trol (70 � 11 pL$min-1$mm-2) versus pancreatitic (48 � 15 pL$min-1$mm-

2) rats. Similar results were obtained when we analysed HCO3--dependent

secretion, driven by NHE, after forskolin stimulation (91 � 15 vs. 76 � 13pL$min-1$mm-2, in control and pancreatitic animals, respectively). How-ever, we found a significant decrease in the HCO3

--dependent fluid secre-tion, driven by NBC, in ducts stimulated by forskolin, from pancreatitic rats(42� 11 pL$min-1$mm-2) compared to those from control animals (98� 18pL$min-1$mm-2).

Conclusion: Cerulein induced pancreatitis reduces bicarbonatedependent fluid secretion in pancreatic duct cells, specifically affecting itsNBC-driven component.

PI-4 Abstract id: 202.

Interferon alpha (IFN-a) promotes pancreatitis-associated lung injuryin murine experimental acute pancreatitis

Li Wen 1, Wei Huang 1, Tao Jin 2, David Criddle 3, Robert Sutton 1.

1NIHR Liverpool Pancreas Biomedical Research Unit, Royal LiverpoolUniversity Hospital, United Kingdom2Department of Integrated Traditional and Western Medicine, WestChina Hospital, Sichuan University, China3Department of Cellular and Molecular Physiology, University ofLiverpool, United Kingdom

Introduction: Acute pancreatitis (AP) is an inflammatory conditionthat occurs in w50 per 100,000 annually. Interferon alpha (IFN-a) orpegylated INF-a has been associated with drug-induced AP, but mecha-nisms have not been determined.

Aims: To determine the effect of IFN-a on the severity of experimentalAP.

Materials &methods:Murine IFN-a (0.1,1.0, 3.0 or 10.0 MIU/kg sc) wasgiven one day and 30 min before AP induction in male CD1 mice (six pergroup) by (i) seven hourly ip caerulein injections (50mg/kg, CER-AP), (ii)retrograde intraductal infusion (50 ml 5mM taurolithocholic acid-3-sulfate,TLCS-AP] or (iii) two hourly ip injections of palmitoleic acid (150 mg/kg)and ethanol (1.35g/kg FAEE-AP]. Blood, pancreas, lung and liver wereharvested 12h or 24h after AP induction. IFN-a-induced immune re-sponses were examined by liver mRNA expression of interferon-inducedproteinwith tetratricopeptide repeats (IFIT 1 and 2). The severity of AP wasevaluated using standard parameters including blinded assessment ofhistopathology.

Results: Administration of IFN-a at different doses resulted in higherliver inflammatory gene expression and standard AP parameters than inexperimental AP models without IFN-a (p<0.05). Application of IFN-a at 1or 3 MIU/kg caused marked increases of lung MPO activity in all AP models(80% in CER-AP, 280% in TLCS-AP and 40% in FAEE-AP vs. AP without IFN-a,p<0.05).

Conclusion: This study suggests that IFN-a exacerbates AP throughimmune responses that contribute to pancreatic and distant organ injury,confirming that distant organ injury is immune-mediated. The mechanismby which IFN-a initiates AP has not been addressed.

PI-5 Abstract id: 158.

Inhibition of histone deacetylation mediates epigenetic changes andreduces severity in acute pancreatitis

Hannes Hartman, Erik Wetterholm, Henrik Thorlacius, Sara Regn�er.

Department of Surgery, Skane University Hospital Malm€o, LundUniversity, Sweden

Introduction: Severe acute pancreatitis (AP) is characterized by pro-tease activation, inflammation and acute lung injury (ALI). The degree ofseverity depends on the magnitude of the inflammatory activity. Posttranscriptional modulation of histones can alter the transcriptional patternin cancer and inflammatory processes.

Aims: Our aim was to analyze the impact of histone deacetylase(HDAC) inhibition in an experimental model of AP.

Materials &methods:Male C57bl/6 mice were pretreated i.p. with theHDAC inhibitor Trichostatin A (TSA, 2mg/kg). AP was induced by retro-grade infusion of taurocholic acid (5%) into the pancreatic duct. Animalswere sacrificed 24 h after onset of AP. Severity was determined by degreeof pancreatic tissue injury, levels of S-amylase, pancreatic macrophageinflammatory protein (MIP)-2 and myeloperoxidase (MPO). Lungs wereanalyzed for MPO and histological signs of inflammation. RtPCR was usedduring AP to evaluate gene expression, focusing on a panel of pro in-flammatory genes in the pancreas.

Results: Infusion of taurocholate increased s-amylase, MIP-2, MPO,signs of pancreatic tissue injury and ALI. Expression of COX2, MIP-2 andIL1b were increased more than 10 times compared to controls. The sys-temic and local inflammation was significantly reduced by pretreatmentwith TSA and the transcriptional pattern was altered in favour of anti-inflammation.

Conclusion: TSA reduce severity in AP through suppression of pro-inflammatory genes. No significant alterations of non-inflammatory geneswere seen in this model. Taken together HDAC inhibition could serve as anovel therapeutic approach in the management of AP.

PI-6 Abstract id: 323.

L-arginine-induced acute pancreatitis in mice: Revisited

Balazs Kui, Zsolt Balla, Peter Hegyi, Zoltan Rakonczay, Jr..

First Department of Medicine University of Szeged, Hungary

Introduction: Acute pancreatitis (AP) is a sudden inflammation of thepancreas. The pathogenesis of AP is not well understood and it has nospecific therapy. To investigate the pathomechanism of AP, wemainly rely onanimal models such as L-arginine-induced AP. The use of L-arginine toinduce AP inmice is becoming increasingly popular. However, we found highmortalitywith the originally published dose (2x4 g/kg) of L-arginine inmice.

Aims: Thus, we aimed to establish a basic amino acid-induced APmodel with a lower mortality rate.

Patients & methods: AP was induced with various intraperitoneal(ip.) doses of L-arginine in CFLP or C57Bl/6 mice. Control mice wereinjected with physiological saline. Laboratory (serum amylase andpancreatic myeloperoxidase activities) and histological (necrosis andinflammatory infiltration) parameters were measured to determine APseverity.

Results: Ip. injection of micewith 2x4 g/kg L-arginine resulted in a 40 %mortality rate in CFLP and 39 % in C57Bl/6 mice, which was independent ofthe disease. Using 3x3 g/kg L-arginine dose, we found significantly lowermortality (15% in CFLP and 19% in C57Bl/6 mice), and similar degree of APmorbidity compared to 2x4 g/kg L-arginine. The pancreatic myeloperox-idase and serum amylase activities and histological parameters weresignificantly elevated in all L-arginine treated groups compared to controlmice.

13 (2013) S2–S98 S21