ogy 14 (2014) S1eS129

T-030.

Genetic factors in acute pancreatitis

Dorota Koziel a, Stanislaw Gluszek b, Artur Kowalik c, Malgorzata Chlopek c

a The Faculty of Health Sciences of the Jan Kochanowski University inKielce, Polandb Clinical Department of Surgery Hospital in Kielce The Faculty ofHealth Sciences of the JKU in Kielce, Polandc The Laboratory of Molecular Testing of the Holy Cross Cancer Centrein Kielce, Poland

Background: Mutations in the PRSS1, SPINK1, C (CTRC) and CFTR werefound in different types of pancreatitis.

Aims: The aim of study was to determine the frequency of geneticmutations in patients with AP and as well as to investigate their relation-ship to the etiology and clinical course.

Patients&methods: From 142 patients treated for AP peripheral bloodsamples were drawn and DNA was isolated. For the genotyping of muta-tions in the SPINK (p.N34S and p.P55S) and CTRC (p.I259V) HRM methodwas used. Mutations in the CFTR (delF508_CTT) were genotype using ASA-PCR. Mutations in the PRSS1 (A16V, N29I, p.R116C, p.R122C) tested usingcapillary sequencing. The results were compared with the control group of175 healthy subjects.

Results: In patients with AP SPINK 1 detected in 14,3% patients vs 2,9%,CFTR 2,8% vs 4,6% CTRC 2,1% vs 0% in controls. There was not found of thePRSS 1mutation either in the study sample or controls. No relationship wasconfirmed mutations with the severityAP: mild AP mutation of CFTR in3(3,75%) and CTRC in 2(2,5%) patients; in the SAP mutation of CFTR in1(3,22%) and CTRC in 1(3,22%) case. SPINK1 mutation significantly morefrequent in severe 7(11,5%) than in mild AP 3(3,75%) (p<0,05).Alcohol wasthe cause of AP in 6 (11,8%) patients with the SPINK 1 and gallstones in 4(7,3%) case (p¼ 0,08).

Conclusion: Significantly more frequently in SAP SPINK1 mutationswere detected than in mild AP. SPINK1 mutation was more common inalcoholic than biliary and idiopathic AP, without statistical significance.

T-031.

Changes of intestinal microbiota during severe acute pancreatitis

Oleksandr Rotar a, Diana Rotar b, Vasiliy Rotar c

a Bukovinian State Medical University, Department of General Surgery,Ukraineb Bukovinian State Medical University, Department of Microbiology andVirology, Ukrainec Bukovinian State Medical University, Department of Anesthesiologyand Reanimatology, Ukraine

Background: Gut is recognized as main source of bacterial trans-location during severe acute pancreatitis (SAP). Besides other factorschanges of intestinal microbiota directly influence on rate of micro-organisms spreading from intestine and may serve as prognostic factor ofseverity pancreatic infection.

Aims: To investigate the changes of luminal and mucosal microbiota ofgut during SAP.

Patients & methods: In 70 Wistar rats SAP was induced by intra-peritoneal injection of 250mg/100 g of 20% L-arginine solution twice during1 hour. Concentration of luminal and mucosal bacteria in colon and distalileumwere investigated during 24-120 hours by bacteriological methods.

Results: In colon amount of autochtonous physiologically usefulmicroflora decreased during all period of SAP: after 72 hours E. feacaliseliminated, after 120 hours Bifidobacteria spp. disappeared and Lactobac-teria spp. were found only in 2 from 7 animals. In such condition concen-tration of autochtonous facultative and allochthonous microorganismsStaphylococcus spp., Clostridia spp., Enterobacteria spp. and Candida spp.

Abstracts / PancreatolS58

reached 3,5-4,5 log CFU/g. In distal ileum concentration of Lactobacteriaspp., Bifidobacteria spp., E. feacalis felt from 6,51-6,81 log CFU/g till 3,57-4,8log CFU/g after 24 hours, and they absolutely disappeared after 48 hoursuntil 7 day. Due to profound deficit of physiologically useful microfloraamount of Peptococcus spp., Staphylococcus spp., Clostridia spp. andespecially Enterobacteria spp. (Klebsiela, Edvardsiela, Proteus, toxic strainsof E. coli.) reached higher level than in colon.

Conclusion: During SAP changes of distal ileal microbiota, especiallymucosal, were more significant than in colon. Thus bacterial translocationfrom distal ileum may occur in a higher level.

T-032.

Improvement of the L-arginine-induced experimental pancreatitismodel

Eszter S�ara Korm�anyos a, Bal�azs Kui a, Zsolt Balla a, B�ela Iv�anyi b, TiborWittmann a, P�eter Hegyi a, Tam�as Tak�acs a, Zolt�an Rakonczay, Jr. a

a First Department of Medicine, University of Szeged, HungarybDepartment of Pathology, University of Szeged, Hungary

Background: The pathogenesis of acute pancreatitis (AP) remainsunknown and it has no adequate therapy. To investigate the patho-mechanism, we rely on animal models such as L-arginine-induced AP,which is becoming increasingly popular. However, we found only mildinflammation in Balb/c and high mortality in FVB/n and C57BL/6 mousestrains with the originally published method (2x4 g/kg, 8% L-arginine).

Aims: We aimed to establish a basic amino acid-induced APmodel withacceptable morbidity and mortality rate.

Materials & methods: AP was induced with different doses (2x4, 3x3,4x2,5 g/kg) and concentrations (0-10%) of intraperitoneal L-arginineadministration in Balb/c, FVB/n and C57BL/6 mice. Serum amylase-, pan-creatic myeloperoxidase activity and oedema, necrosis, leukocyte infiltra-tionwere measured to determine AP severity. Our findings are representedas effectivity rate (ER¼ number of mice with AP/all treated mice).

Results: In L-arginine treated groups, all parameters were significantlyelevated compared to the control group. In all three strains, the injectionwith 3x3 or 4x2,5 g/kg L-arginine caused similar AP severity with lowermortality vs the 2x4 g/kg dose. In Balb/c mice, 10% L-arginine injectionresulted in moderate morbidity and low mortality (ER¼90%). In FVB/nstrain 5% L-arginine caused low mortality with severe AP (ER¼90%), while10% L-arginine caused greater mortality (ER¼25%). C57BL/6 mice devel-oped mild disease with low mortality due to 5% L-arginine (ER¼90%),however, disease severity and mortality were higher in case of 10% L-arginine administration (ER¼35%).

Conclusion: Mouse strains show different sensitivities to L-arginineand a fine borderline appears between effective and lethal doses andconcentrations.

Grant support: OTKA and T�AMOP.

T-033.

Ursodeoxycholate pretreatment reduces the cytotoxic effect of cheno-deoxycholate on pancreatic ductal epithelial cells

M�at�e Katona a, P�eter Hegyi b, Zolt�an Rakonczay, Jr. a, J�ozsef Mal�eth a, ZsoltR�azga b, Vikt�oria Venglovecz c

a First Department of Medicine, University of Szeged, HungarybDepartment of Pathology, University of Szeged, HungarycDepartment of Pharmacology and Pharmacotherapy, University ofSzeged, Hungary

Background: We have recently shown that chenodeoxycholate (CDC)in high concentration strongly inhibited ion transporters through thedestruction of mitochondrial function in intact guinea pig pancreatic ducts.

Abstracts / Pancreatology

Aims: Since ursodeoxycholic acid (UDC) is known to protect themitochondria against hydrophobic bile acids and have antiapoptotic effect,we investigated whether UDC is able to prevent the CDC-induced celldamage.

Materials & methods: Inta-interlobular ducts were isolated fromguinea pig pancreas. Ducts were then pretreated with UDC (0.1 mM and0.5mM) for 5 h and 24 h and changes in intracellular Ca2+concentration[Ca2+]i, ATP level [ATP]i, pH [pH]i, mitochondrial permeability transitionpore (MPTP) opening were measured by microfluorometry. Mitochondrialtransmembrane potential (MTP) was studied by confocal microscopy.Morphological changes of mitochondria were investigated by transmissionelectron microscopy. Expressions of bile acid transporters were studied byreverse transcriptase PCR (RT-PCR).

Results: 24 h pretreatment with 0.5 mM UDC significantly reduced therate of ATP depletion, mitochondrial injury, MPTP opening and thedecrease of MTP induced by 1mM CDC. In addition, 0.5 mMUDC preventedthe inhibitory effect of CDC on the acid-base transporters, however, had noeffect on the CDC-induced calcium signaling. mRNA expression of Slc10A1and A2 was detected in the ducts by RT-PCR.

Conclusion: Our results indicate that protection of mitochondria withUDC administrationmay represent a novel option against bile acid-inducedductal injury.

This study was supported by Hungarian National Development Agencygrants (T�AMOP-4.2.2.A-11/1/KONV-2012-0035, T�AMOP-4.2.2-A-11/1/KONV-2012-0052, T�AMOP-4.2.2.A-11/1/KONV-2012-0073, T�AMOP-4.2.2./B-10/1-2010-0012, T�AMOP 4.2.4.A/2-11-1-2012-0001 ‘National ExcellenceProgram) and the Hungarian Scientific Research Fund (OTKA NF105758,NF100677, K109756)

T-034.

Determination of cytokine status with multiplex technology in acutenecrotizing pancreatitis (ANP) in Rats

S.O. Abiola a, P.G. Rasputin a, E.V. Haar a, S.D. Andreeva b, N.V. Isaeva c

a General Surgery Department ,Kirov State Medical Academy, Russiab Pathology Department, Kirov state Agricultural University, Russiac Kirov Research Institute for Hematology & Blood Transfusion , Russia

Background: The cause of death in most patients with ANP is often theresult of multi organ system failure which develops due to systemicinflammatory response syndrome (SIRS) .The systemic manifestations ofacute pancreatitis is due to the local and systemic actions of specificproinflammatory cytokines such as TNF-a, IL-1, IL-8, etc

Aims:Objective: The purpose of the investigation is to determine the cyto-

kines level in blood serum of rats with experimental ANP with the aid ofmultiplex technology .Using multiplex technology significantly reduces theamount of blood serum needed for research in experiments on small lab-oratory animals when it is essential to evaluate a large number ofparameters

Patients&methods: The animals were euthanized at 1,3 ,7 and 14daysafter surgery.We determine the next Cytokines: interferon - gamma (INF-g),tumor necrosis factor -alpha (TNF-a), interleukin -1 alpha (IL-1a),inter-leukin -4 (IL- 4), granulocyte -macrophage colony stimulating factor (GM-CSF) , and monocyte chemotactic protein-1 (MCP- 1)

Results: Analysis of the results showed a pronounced proinflammatoryprocess in modeled ANP.This is evidenced by a statistically significantincrease compared with the normal level of TNF-a, MCP-1 and GM- CSF inall observation periods with lower IL-1a and reduction of IL- 4 with asignificant increase only at the 14th day, this dynamics correlates with themorphological data (presence of necrotic focuses in pancreas till the end ofexperiment)

Conclusion: The progression of experimental ANP accompanies thehigh rise of proinflammatory cytokines (as shown by the use of Multiplextechnology) which is the base for the development of SIRS

T-035.

Regulatory T-cells may have a protective role in acute pancreatitis.

Edward Alabraba a, Fiona Cambell b, Diane Lataweic a, JohnNeoptolemos a, Robert Sutton a

a NIHR Liverpool Pancreas Biomedical Research Unit, Royal LiverpoolUniversity Hospital, Liverpool, UK, United KingdombDepartment of Molecular and Clinical Cancer Medicine, University ofLiverpool,Liverpool, UK, United Kingdom

Background: Acute pancreatitis (AP) triggers inflammatory immuneresponses. Regulatory T-cells (Tregs) are CD4+CD25+Foxp3+ and exhibitpotent immunosuppressive actions.

Aims: We tested if adoptively transferred Tregs could reduce pancreaticinjury in experimental AP

Materials & methods: AP was induced by 7 intra-peritoneal caeruelininjections (50micrograms/Kg/hr), either with or without Tregs injected intra-venously into the tail vein of mice 12 hours before the first caerulein injection.Using lymphocytes from spleens of syngeneicmice inducedwithAP, Tregswerepurifiedwithmagneticbeads initiallyenrichingCD4+T-cells fromwhichCD25+

T-cells were later selected. Tregs from several spleenswere pooled for adoptivetransfer immediately after isolation to ensure a homogenous population ofFoxp3+ cells. Pancreatic enzymes, cytokines, immune cells and histopathology(blinded) were assessed at 6, 12 and 24 hours (n¼6 all groups).

Results: At 6 hours there was significantly (p<0.05) reduced histo-pathological severity of pancreatic injury, plasma amylase and myeloper-oxidase when Tregs were adoptively transferred before AP induction. Thesemice showed Th2 polarisation of CD4+ T cells at 6 hours, significantlyreduced serum levels of Th1 cytokines (MCP-1, IL6, IL12, and TNF-alpha),significantly (p<0.05) elevated levels of IL10, and reduced expression ofCCR2 on infiltrating leukocytes. These changes were not maintained at 12and 24 hours when these parameters showed no difference compared tocontrol mice with AP but no adoptively transferred Tregs.

Conclusion: Adoptively transferred Tregs attenuate injury in exper-imental AP for up to 6 hours after the last injection of caerulein, but thisprotection is lost thereafter when treated mice are no different from con-trols with AP.

14 (2014) S1eS129 S59

T-036.

Low dose ciprofloxacin nanoparticles prevents intestinal colonizationwith pathological enterobacteria during severe acute pancreatitis

Oleksandr Rotar a, Vasiliy Rotar b, Miroslav Pilat c, Lilia Salamandyk d

a Bukovinian State Medical University, Department of General Surgery,Ukraineb Bukovinian State Medical University, Department of Anesthesiologyand Reanimatology, Ukrainec edical University,Chernivtsi Emergency Hospital, Adepartment ofAnesthesiology and Reanimatology, Ukrained Chernivtsi Emergency Hospital, Department of Bacteriology, Ukraine

Background: Selective digestive decontamination (SDD) is proven toprevent septic complication in patients with severe acute pancreatitsis(SAP) but may be followed with antibacterial resistance development.Diminishing a number of drugs could prevent it.

Aims: To investigate the ability of chitosan nanoparticles (CNp) loadedwith ciprofloxacin (CIPR) to decrease intestinal colonization (IC) by Gramnegative patholigical enterebacteria (GNPE).

Materials&methods: In 200 Wistar rats SAP was iduced by L-argininemethod. 3 mg/kg of CIPR administrated enterally in 1 group, 3 mg/kg ofCIPR included in CNp in 2 group and normal saline e in control (C) group.Suspension of 9 log CFU/g of GNPE was introduced to all animals by gavage.Concentrations of microorganisms in internal organs and blood wereinvestigated during 12-96 hours.