Search

3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca2+ concentration ([Ca2+]c) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca2+]c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca2+, was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca2+]c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP.

Objectives The aim of this study was to comparatively analyze the effects of different concentrations of cigarette smoke condensate (CSC, a standardized tobacco extract) and ethanol on intracellular enzyme activation, cell necrosis, alteration of cytosolic calcium concentration ([Ca]c), and amylase secretion in pancreatic acinar cells. Methods The effects of CSC (1 μg/mL to 0.4 mg/mL) and ethanol (10-100 mM) on intracellular enzyme activity, cell necrosis, and [Ca]c were measured by fluorescence assays in isolated pancreatic acinar cells. Amylase secretion was evaluated by spectrophotometry. Supramaximal concentrations of cholecystokinin (10-100 nM) were used as positive control. Results Neither CSC nor ethanol induced trypsin or elastase activation. Both CSC (0.1-0.4 mg/mL) and ethanol (10-75 mM) significantly increased [Ca]c. Amylase secretion was increased only in CSC-treated cells (0.3 and 0.4 mg/mL). After 60 minutes, CSC (0.3 and 0.4 mg/mL) significantly increased acinar cell necrosis at a similar percentage to that induced by cholecystokinin. Ethanol did not induce any significant cell necrosis. Conclusions Cigarette smoke condensate induces acinar cell injury and increases [Ca]c and amylase secretion, independently of intracellular enzyme activation, suggesting that tobacco could induce several main early events of pancreatitis in pancreatic acinar cells. However, ethanol only induces increases [Ca]c, having no effect on cell injury, amylase secretion, or intracellular enzyme activation.

Diagnosis of pancreatic exocrine insufficiency (PEI) is hindered by methodological difficulties of pancreatic function tests. The probability of PEI in chronic pancreatitis (CP) increases as pancreatic fibrosis develops. Pancreatic fibrosis in CP may be quantified by EUS elastography.To evaluate whether EUS-elastography can predict PEI in patients with CP.Prospective, observational study.Department of Gastroenterology, University Hospital of Santiago de Compostela, Spain.Patients diagnosed with CP based on EUS and magnetic resonance imaging and MRCP findings.Diagnosis of PEI was based on the (13)C-mixed triglyceride breath test. EUS-elastography was performed with PENTAX echoendoscopes and Hitachi-Preirus US platform. Two areas were selected for elastographic evaluation: area A corresponds to the pancreatic parenchyma and area B to a soft peripancreatic reference area. The quotient B/A (strain ratio [SR]) was considered the elastographic result.Pancreatic SR in CP patients with and without PEI.A total of 115 patients with CP (mean age, 50.2 years, range, 21-81; 92 male) of different etiologies were included; 35 patients (30.4%) had PEI. Pancreatic SR was higher in patients with PEI (4.89; 95% confidence interval, 4.36-5.41) than in those with a normal breath test result (2.99; 95% confidence interval, 2.82-3.16) (P.001). A direct relationship was found between the SR and the probability of PEI, which increases from 4.2% in patients with an SR less than 2.5 to 92.8% in those with an SR greater than5.5.Single-center study.The degree of pancreatic fibrosis as measured by EUS-guided elastography allows quantification of the probability of PEI in patients with CP.

Several recent studies have demonstrated the association between smoking and chronic pancreatitis (CP). However, less is known about the role of smoking in the development of CP-related complications. Our aim was to investigate the impact of smoking and alcohol consumption on age of onset and complications at CP diagnosis.A cross-sectional case-case study was performed within a prospectively collected cohort of patients with CP. Alcohol consumption and smoking habits were assessed using a standardized questionnaire. Morphologic severity was defined based on endoscopic ultrasound criteria for CP and classified as mild (3-4 criteria), moderate (5-6 criteria), and severe (≥7 criteria or calcifications). Pancreatic exocrine insufficiency (PEI) was diagnosed using the C-mixed triglyceride breath test. Odds ratios (OR) with 95% confidence intervals (CI) for CP-related complications were calculated using a case-case design.A total of 241 patients were included. Smoking was associated with PEI (OR [95% CI], 2.4 [1.17-5.16]), calcifications (OR [95% CI], 2.33 [1.10-4.95]), and severe morphologic changes (OR [95% CI], 3.41 [1.31-8.85]) but not with pseudocysts or diabetes. Neither smoking nor alcohol consumption was associated with age of onset.Tobacco, but not alcohol, is associated with PEI, calcifications, and severe morphologic (≥7 criteria or calcifications) CP at diagnosis. Smoking cessation should be encouraged in patients with CP.

Dept. of Surgery, University of TX Medical Branch; Galveston, TX, USA Introduction: Patients with advanced PNET have limited therapeutic options. RAD001, an inhibitor of the mTOR pathway, has been shown to increase progression-free survival, but not overall survival, indicating a need to identify additional therapeutic targets. The BON cell line, derived from a metastatic PNET, has been used as a model to study PNET cell biology. The mTOR pathway contributes to the regulation of proteins involved in apoptosis and cell growth. However, inhibition of mTORC1 by RAD001 may induce upstream AKT upregulation, attenuating its therapeutic effects. We hypothesized that dual inhibition of Akt along with mTOR will overcome the limited activity of RAD001 alone. Methods: Cell proliferation assays were performed with DMSO, Akt (LY29400), and/or mTOR (RAD001) inhibitors. Cell growth was measured over time in replicates; cells were quantified using a coulter counter. For in vivo studies, nu/nu mice were treated daily for 6 wks with RAD001 (oral gavage), LY29400 (IP) one week after intrasplenic ijection of BON cells transfected with luciferase gene. Results: Cellular proliferation was most attenuated with the combination therapy LY29400 and RAD001. Similarly, the volume of liver metastasis was lowest in the group treated with both LY29400 and RAD001. Conclusion: Autocrine activation of Insulin Growth Factor 1 receptor on the BON cell stimulates proliferation through both the PI3K/Akt/mTOR pathways. The combination of LY29400 and mTOR inhibition decreased liver tumor burden compared to single drug inhibition. Our data suggest that combination therapy is more effective in vitro and in vivo than either inhibitor alone. Further pre-clinical rodent survival studies are underway to verify these findings. Ongoing studies are also underway to elucidate the mechanism of dual inhibition on tumor growth and metastasis.

Summary Background Little is known about risk factors for complications in chronic pancreatitis (CP). High fat diet (HFD) has been demonstrated to aggravate pancreatic injury in animal models. The aim of this study was to investigate the role of HFD in age at diagnosis of CP and probability of CP related complications. Methods A cross-sectional case–case study was performed within a prospectively collected cohort of patients with CP. Diagnosis and morphological severity of CP was established by endoscopic ultrasound. Pancreatic exocrine insufficiency (PEI) was diagnosed by 13 C mixed triglyceride breath test. Fat intake was assessed by a specific nutritional questionnaire. Odds ratios (OR) for CP related complications were estimated by multivariate logistic regression analysis. Results 168 patients were included (128 (76.2%) men, mean age 44 years (SD 13.5)). Etiology of CP was alcohol abuse in 89 patients (53.0%), other causes in 30 (17.9%) and idiopathic in the remaining 49 subjects (29.2%). 24 patients (14.3%) had a HFD. 68 patients (40.5%) had continuous abdominal pain, 39 (23.2%) PEI and 43 (25.7%) morphologically severe CP. HFD was associated with an increased probability for continuous abdominal pain (OR = 2.84 (95%CI, 1.06–7.61)), and a younger age at diagnosis (37.0 ± 13.9 versus 45.8 ± 13.0 years, p = 0.03) but not with CP related complications after adjusting for sex, years of follow-up, alcohol and tobacco consumption, etiology and body mass index. Conclusions Compared with a normal fat diet, HFD is associated with a younger age at diagnosis of CP and continuous abdominal pain, but not with severity and complications of the disease.

s / Pancreatology 12 (2012) 502–597 511 genes participated in epigenetic modification to regulate drug resistance. Thesedataareof clinical significanceandmaybehelpful for thedevelopment of new strategy for the treatment of pancreatic cancer.

Background Methods for evaluation of pancreatic exocrine insufficiency (PEI) are expensive, labor intensive, and not available at many institutions. The aim of this study was to investigate if PEI in chronic pancreatitis (CP) can by predicted by nutritional markers in blood. Methods A retrospective analysis of a prospectively collected database of CP patients was performed. Diagnosis of CP was based on endoscopic ultrasonography or magnetic resonance imaging. PEI was investigated by the 13 C-mixed triglyceride breath test. Hemoglobin, mean corpuscular volume, lymphocytes, prothrombin time, and serum levels of total protein, albumin, prealbumin, retinol binding protein, cholesterol, triglycerides, amylase, folic acid, vitamin B12, HbA1C, transferrin, ferritin, magnesium and zinc were analyzed. Results 114 patients were included in the study (97 males, mean age 48.1 years, 54 with alcohol etiology), 38 (33%) suffered from PEI. Magnesium below 2.05 mg/dL, hemoglobin, albumin, prealbumin and retinol binding protein below lower limit of normal and HbA1C above upper limit of normal were associated with PEI in univariate analysis. Magnesium below 2.05 mg/dL detected PEI with a sensitivity, specificity and positive and negative predictive values of 0.88 (95% confidence interval, 0.66–0.97), 0.66 (0.48–0.80), 0.58 (0.39–0.75) and 0.91 (0.73–0.98), respectively. The corresponding values were 1.00 (0.80–1.00), 0.55 (0.38–0.71), 0.52 (0.34–0.69) and 1.00 (0.82–1.00)) if one or more pathological tests among parameters significantly associated with PEI in was used as a positive test for PEI. Conclusion Serum nutritional markers can be used to predict the probability of PEI in CP and provide guidance in decisions on enzyme replacement therapy.

Background and aim: Alcohol has been associated with activation and survival of activated pancreatic stellate cells (PSC). The effect of ethanol is partly mediated by the generation of oxidative stress within the cells, but other factors are required to induce pancreatic fibrogenesis. Tobacco is recognized as an etiologic factor of chronic pancreatitis (CP) and it is related with fibrogenesis in other tissues such as heart, liver and kidney. However its effect on pancreatic fibrogenesis is still unknown. We hypothesized that tobacco in combination with alcohol stimulates pancreatic fibrogenesis by PSC activation through the generation of oxidative stress within the cells. Our aim was to evaluate the effect of tobacco alone and in combination with alcohol on the activation of PSC and production of extracellular matrix (ECM) proteins and the generation of oxidative stress within the cells. Methods: PSC were isolated from rat pancreas Sprague-Dawley and exposed to tobacco (cigarette smoke condensate) alone (0.01mg/ml) or in combination of increasing concentrations of ethanol (5 to 50mM). PSC activation (α-SMA expression) was measured in both early and primary cell culture by Western blot. Fibronectin-1 (FNT-1) was evaluated by western blot and immunochemistry. Collagen-I was measured by western blot and Masson trichrome. Oxidative stress was examined using 2'-7'-dichlorofluorescin diacetate (DCF-DA) and was detected by flow cytometry. Results are expressed as mean±SEM. Statistical analysis was carried out one-way ANOVA followed by Fisher's LSD post hoc test. Results: Tobacco alone (α-SMA 1.83±0.3; p=0.003 versus negative control) or in combination with 50 mM ethanol (α-SMA 1.53±0.2; p=0.04 versus negative control) induced PSC activation in early culture. Tobacco in combination with 50 mM ethanol increased the expression of collagen-I (3.9±1.2, p

s / Pancreatolog S54 Aims: To investigate the effects of LPS and hyperglycemia on PSC activation and effects of simvastatin on LPS induced PSC activation. Materials & methods: Primary cultures of rat PSCs were exposed to various concentrations of glucose and LPS with/without simvastatin. Quantification of proliferation was performed by Brd-U assay. PSC activation was assessed by expression of a-SMA and extracellular signal-regulated kinase (ERK) using Western blot. Results: LPS induced activation and proliferation of PSC. However, glucose did not affect PSC proliferation. The proliferative effect of LPS on PSC was not affected by glucose concentration in culture media. Simvastatin inhibited LPS induced activation and proliferation of PSC. The proliferative effect of LPS was mediated by activation of Erk 1/2 pathwaywhich was effectively inhibited by simvastatin. Conclusion: This study shows that LPS is one of the activating factors toward PSC. Endotoxemia which is frequently encountered in the clinical settings of acute and chronic pancreatitis may act as a triggering factor for initiating and persisting pancreatic fibrosis. Simvastain could be a therapeutic agent for preventing pancreatic fibrosis.

s / Pancreatology 14 (2014) S1eS129 S77 Nicotine Replacement Therapy and Group Therapy were available. The demographics and characteristics of the patients were also collected. The smoking status were checked at week-4, -12 and -24. Results: 16 patients showed interests in engaging with the Quitters. The mean agewas 50 (range 21-63). Therewere 7male and 9 female. All but 1 of the patients have alcoholic CP(15), the other had recurrent acute pancreatitis(1). Most of the patients were cigarette smokers(5-30 cigarettes per day,14/16),with1pipe smokerandother rolledhisown tobacco.Atweek-4, 4 patients(4/16) had stopped smoking and remained smoke-free at week-12. By week-24, 3 patients(3/16) remain smokefree and 1 was lost to follow-up. Conclusion: Although this was a small study, we demonstrated that some CP patients would engage with Southampton Quitters giving the opportunity however longer term follow-up would be needed.

s / Pancreatology 13 (2013) S2–S98 S75 b)When comparedwith abstainers, heavy drinker intake is a risk factor to develope CP (p1⁄40,038; O.R1⁄42,583) c) Pancreas divisum is a risk factor to develope ARP (p1⁄40,000; O.R1⁄410,533) but not CP. d) Moderate alcohol intake is not statistically significant in both ARP and CP. Conclusion: Heavy alcohol consumption and smoking inhabits are independent risks for CP. Pancreas divisum is a risk factor to develope ARP. PII-75 Abstract id: 197. Covered self-expanding metal stents (CSEMS) may offer improved clinical success compared to multiple plastic stents (PS) in strictures secondary to chronic pancreatitis: A systematic review and metaanalysis Antti Siiki , Mika Helminen , Juhani Sand , Johanna Laukkarinen . Dept. of Gastroenterology and Alimentary Tract Surgery, Tampere University Hospital, Tampere, Finland 2 Science Center, Pirkanmaa Hospital District and School of Health Sciences, University of Tampere, Tampere, Finland Introduction: CSEMS are being increasingly used in the endoscopic treatment of benign biliary strictures (BBS). Despite promising results, there is not yet solid evidence to support their routine use. Aims: To evaluate feasibility of CSEMS compared to PS in BBS in terms of clinical success and complications. Materials & methods: A systematic search of Medline, Scopus and Embase database for studies published 2000-2012 combined to handsearch of reference lists resulted 4977 articles. Out of 99 potentially relevant studies selected for full-text review, 12 CSEMS (376 patients) and 13 PS studies (570 patients) met the final inclusion criteria. A systematic review was made using proportion meta-analysis. Results: A tendency to successful use of CSEMS in strictures related to chronic pancreatitis (CP) was shown: clinical success of 77% and 33% (95% CI 61-94% vs. 4-63%, p1⁄40.06) was achieved with CSEMS and PS at 12 months follow-up, respectively. There were no differences in the success rates of other etiologies except CP or in the early complications. In CSEMS, incidence of late adverse events was lower in CP related strictures (3% vs. 67%, 95%CI 0-13% vs. 17-99%, p1⁄40.02). The median number of ERCPs was lower with CSEMSs (1.5 vs. 3.9, p1⁄40.002). Conclusion: Improved clinical success with fewer endoscopic sessions and corresponding complication rate may be achieved with CSEMS compared to PS in BBS secondary to chronic pancreatitis. PII-76 Abstract id: 182. Indirect diagnosis of pancreatic exocrine insufficiency (PEI) in chronic pancreatitis (CP) by endoscopic ultrasound (EUS)-guided elastographic quantification of pancreatic fibrosis: A prospective observational study J. Enrique Dominguez-Mu~ noz , Margarita Casti~ neira-Alvari~ no , Maria Luaces-Regueira , Jos e Lari~ no-Noia , Laura Nieto-Garcia , Julio IglesiasGarcia . University Hospital of Santiago. Foundation for Research in Digestive Diseases, Spain 2 Foundation for Research in Digestive Diseases, Spain Introduction: Diagnosis of PEI is hindered by methodological difficulties of pancreatic function tests. The probability of PEI increases as fibrosis develops. Pancreatic fibrosis in CP can be quantified by EUS-guided elastography. Aims:We aimed at evaluating if pancreatic elastography can be used to predict PEI in patients with CP. Materials & methods: Prospective observational study including patients consecutively diagnosed with CP based on EUS ( 5 criteria) and/or s-MRCP findings. PEI was diagnosed by 13C-mixed triglyceride breath test and defined as a 13C-cumulative recovery rate 5.5). Conclusion: The degree of pancreatic fibrosis as measured by EUSguided elastography allows quantifying the probability of PEI, and thus the need for oral pancreatic enzyme therapy, in patients with CP PII-77 Abstract id: 296. Morphological features of pancreatic cystic lesions in endoscopic ultrasonography (EUS) examination – A single-center experience Malgorzata Degowska, Marek Stobinski, Janusz Milewski, Grazyna

s / Pancreatology such as organ failure, necrosis, and mortality. The amount of fluid over the first 24-48 hours needed to be provided. When combining the various studies, the weighted mean of power (sample size) was used to determine the relative value of significance. Sensitivity analysis evaluated sample sizes, timing, type of fluids and severity of patients included. Ten published studies fulfilled the criteria to be included in the analysis, which included 1495 patients. There were 6 retrospective, 4 prospective studies. While 3 studies concluded that aggressive hydration was beneficial, 7 studies showed no benefit and/or harm. Using a weighted mean analysis, when evaluating the effectiveness of aggressive intravenous hydration beyond 24 hours, there was no significant difference in the development of organ failure, pancreatic necrosis or mortality (OR 1.2, CI 0.4 -1.9). However, in a subgroup analysis excluding patients presenting with severe disease, there was a significant benefit to aggressive intravenous hydration in preventing organ failure and/or pancreatic necrosis (OR 1.8, CI 1.5 -2.8, p 1⁄4 0.02). Additionally, there was a significant decrease in organ failure and/or pancreatic necrosis if the aggressive intravenous hydration was given early, within the first 24 hours (OR 2.1, CI 1.6 – 2.9, p1⁄4 0.03) and 6-12 hours (p 1⁄4 0.02). Based on this meta-analysis, aggressive intravenous hydration appears to be most beneficial when applied to patients with acute pancreatitis early in the course of the disease, within the first 6-24 hours, before severe disease develops. In general, there does not appear to be a benefit to aggressive hydration in patients with acute pancreatitis beyond the first 24 hours. Clinicians should recognize the importance of applying the principles of aggressive hydration as early as possible, especially to patients who have mild disease. The benefit of early aggressive hydration appears to be preventing severe disease, organ failure and/or pancreatic necrosis. O-14 Abstract id: 189. Role of tobacco as compared with alcohol in the activation of pro-inflammatory factors and cytokine release from pancreatic acinar cells Maria Luaces-Regueira , Margarita Casti~ neira-Alvari~ no , Julio IglesiasGarcia , Jos e Lari~ no-Noia , J. Enrique Dominguez-Mu~ noz . 1 Foundation for Research In Digestive Diseases, Spain University Hopital of Santiago de Compostela. Foundation for Research in Digestive Diseases, Spain Introduction: Triggering of the inflammatory process through the activation of pro-inflammatory transcription factors, as NFkappaB, and secretion of pro-inflammatory cytokines is a central pathogenic mechanism of chronic pancreatitis (CP). Previous studies suggest a role for alcohol in this inflammatory process, but the effect of tobacco, which is a recognized risk factor for CP, is unknown. Aims: To analyse the effect of tobacco compared with alcohol in the activation of pro-inflammatory factors and cytokine release from pancreatic acinar cells in culture. Materials & methods: Pancreatic acinar cells were isolated from Swiss mouse pancreas by enzymatic (collagenase) and mechanic degradation, filtration and centrifugation. Cells were stimulated over 3 hours with cholecystokinin (CCK, positive control), alcohol (at 10,25,50,75,100 mM) and tobacco (at 0.001,0.01,0.1,0.2,0.4 mg/ml). NFkappaB activation (translocation of the subunit p65 into the nucleus) was measured by Western blot. Interleukin-1b secretionwas analyzed by ELISA in cellular supernatant. Data are shown as mean and standard error, and analyzed by ANOVA test. Results: Tobacco, but not alcohol, induces activation of NFKappaB (2.69þ1.05 fold increase of p65 translocation at 0.1 mg/ml over the negative control). Neither tobacco nor alcohol induces interleukin-1b release, ranging between 14.1 1.1 pg/ml (negative control) to 20.1 3.6 pg/ml (tobacco 0.1 mg/ml), and to 23.8 9.2 pg/ml (ethanol 100 mM) (n.s.). Conclusion: Tobacco, but not alcohol, initiates the inflammatory process through the activation of NFKappaB in acinar cells. By this mechanism, tobacco can act as a pathogenic factor in chronic pancreatitis. O-15 Abstract id: 248. Serotonin mediates pancreatic acinar cell cytoskeletal remodeling in mice Enrica Saponara, Sabrina Sonda, Kamil e Grabliauskaite, Theresia

J. Larino-Noia , J. Iglesias-Garcia , M. Macias , A. Martin , M. Legaz , P. Rendon , J. Vila , A. Reyes , I. Abdulkader , J.E. Dominguez-Munoz . Gastroenterology Department, Hospital Puerta del Mar de Cadiz, Spain 2 Foundation for Research in Digestive Diseases, Spain University Hospital of Santiago de Compostela, Gastroenterology Department, Hospital Puerta del Mar de Cadiz, Spain Gastroenterology Department, Hospital Morales Meseguer de Murcia, Spain Gastroenterology Department, Hospital La Mancha Centro de Alcazar, Spain Gastroenterology Department, Hospital de Navarra, Spain Gastroenterology Department, University Hospital of Cordoba, Spain 8 Pathology Department, University Hospital of Santiago de Compostela, Spain