not yet been addressed. Therefore, the current study uses an experimental model to examine whether Hpa is involved in the pathogenesis of cerulein-induced AP in mice. Material and Methods: Heparanase over-expressing transgenic mice (hpa-TG) and their wild-type (WT) BALB/c mice, and Hpa knockout mice (hpa-KO) and their WT C57BL mice were intraperitoneally injected with either Cerulein (50 mg/kg, 5 times, at 1 hour apart) or vehicle. Pancreatic Hpa activity, edema, and inflammation along blood amylase and lipase levels, were determined 24 hours following pancreatitis induction. Results: Cerulein-induced pancreatitis in wild type mice was associated with significant rises in the serum levels of amylase and lipase. These increases were characterized by enhancement of Hpa activity and pancreatic inflammation. The elevation in amylase and lipase as well as pancreatic edema/inflammation responses to administration of cerulein were profoundly exaggerated in hpa-TG mice. In contrast, when cerulein was injected to hpa-KO mice, the severity of pancreatitis was attenuated as compared with their wild type controls. Importantly, pretreatment with Hpa inhibitor (PG545) reduced significantly the inflammatory response of acute pancreatitis by ameliorating pancreatic edema, amylase, and lipase serum levels. Conclusions: The hpa-TG mice are more susceptible to acute pancreatitis than their WT controls, indicating a role for Hpa in the pathogenesis of this disease state. The pancreatic-protective effects of Hpa inhibition provide a rational basis for therapeutic application of Hpa inhibitors.
0.817, respectively. (CONCLUSION) SW was a high accurate and non-invasive diagnostic apparatus for diagnosing pancreatic fibrosis.
Involvement of the Calcium-Binding Protein in the Onset of Acute Pancreatitis Hirosato Mashima, Noboru Watanabe, Suguru Arata, Hirohide Ohnishi [Background] We reported that the pancreas of interferon-regulatory factor (IRF) 2 knockout (KO) mice represents an early phase of acute pancreatitis, including the defect of regulatory exocytosis, disturbance of autophagy and intracellular activation of trypsin (Mashima H et al. Gastroenterology 2011). Significantly up- and down-regulated genes in this IRF2 KO pancreas were reported (Hayashi H et al. PNAS 2011). The catalogue of gene transcripts included two kinds of calcium-binding proteins (S100 calcium binding protein G (S100g) and Annexin A10 (Anxa10)), which were highly up-regulated in IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and the disturbance of it is related to pancreatitis, we evaluated the roles of these proteins in the onset of acute pancreatitis. [Method] 1) In order to evaluate the relation of IRF2 and these two genes, we analyzed the mRNA expression by qPCR in IRF2-overexpressing AR42J cells (AR42J-IRF2) and dominant-negative IRF2-overexpressing AR42J cells (AR42J-dnIRF2). 2) We induced cerulein-pancreatitis in wild-type mice and examined the changes in expression of these genes by qPCR, Western blotting and immunohistochemistry. 3) We examined cholecystokinin (CCK)-induced amylase secretion and cytosolic calcium concentration ([Ca2+]i) using fluorescent Ca2+ indicator (fura-2) in S100g-overexpressing AR42J cells (AR42J-S100g) and Anxa10-overexpressing AR42J cells (AR42J-Anxa10). [Results] 1) The mRNA level of S100g was decreased in AR42J-IRF2 cells and increased in AR42J-dnIRF2 cells. The mRNA expression of Anxa10 was not detected in AR42J cells. 2) In ceruleaninduced pancreatitis, the expression of these genes in mRNA and protein level was increased. The acini were stained patchily for S100g in the early phase of pancreatitis and the positivelystained area gradually spread over the pancreas. Anxa10 was evenly stained in acini and the intensity was gradually increased during the course of pancreatitis. 3) In the stimulation of 100pM CCK-8, amylase secretion was inhibited in AR42J-S100g cells and only slightly inhibited in AR42J-Anxa10 cells. The increase in [Ca2+]i was inhibited in AR42J-S100g cells and slightly inhibited in AR42J-Anxa10 cells. [Conclusion] In cerulean-induced acute pancreatitis, the expression of S100g and Anxa10 was increased. The amylase secretion and the increase in [Ca2+]i in response to CCK-8 stimulation were inhibited in AR42J-S100g cells and those effects were weak in AR42J-Anxa10 cells. These results suggested that S100g may play a more important role in the onset of acute pancreatitis. Tu1893 Lyn Is a Key Regulator of Stellate Cell Chemotaxis and Infiltration in Human Chronic Pancreatitis Hung Pham, Vincenzo Cirulli, Stephen Pandol, Andrzej Ptasznik Excessive cell migration and infiltration of activated monocytes/macrophages and myofibroblast-like stellate cells within pancreatic parenchyma is a prerequisite for the development of chronic pancreatitis (CP). The current treatment options of CP are inadequate and reflect the fact that signaling therapeutic targets in infiltrating cells are unknown. Therefore, macrophage and stellate cell infiltration cannot be prevented or reversed, leading to persistent pancreatic inflammation, fibrosis, and often to virtually incurable diseases such as diabetes (T3cDM) and cancer. We screened for the activities of known Src kinases in stellate cells isolated from pancreata of donors with or without CP. We detected a ~10 fold increase in Lyn tyrosine kinase activity in cells isolated from CP compared to cells from normal subjects. Importantly, α-SMA, a marker of activation of stellate cells, was also increased in stellate cells from subjects with CP compared to those from normal subjects. These data indicate that increased Lyn kinase activity is associated with activation of stellate cells in CP patients. We previously showed that Lyn is a downstream signaling target of CXCR4 chemokine receptor in various types of cells. Therefore, we now compared the expression of CXCR4 in stellate cells isolated from CP patients and normal subjects, in cultured primary human cells and human pancreatic sections. The mean percentages of stellate cells expressing surface CXCR4 (as measured by flow cytometry analysis) were significantly increased in CP, as compared to normal cells. Immunocolocalization studies, indicated the significant colocalization of CXCR4 with α-SMA, reaching a ~16 fold increase in CP tissues, as compared to normal pancreata. These results indicate that the number of CXCR4-positive activated stellate cells, infiltrating the pancreatic parenchyma, is significantly increased in CP compared to normal pancreas. We next compared the Lyn-mediated SDF-1/CXCR4-induced chemotactic response of CP and normal stellate cells. Stellate cells from CP showed increased CXCR4induced chemotaxis by ~4-fold as compared to those from normal subjects. The selective ablation of Lyn by siRNA effectively inhibited chemotaxis of CP stellate cells. We also observed strong inhibition of chemotaxis following treatment with Bafetinib. Notably, inhibition of chemotaxis by Lyn inhibitors (siRNA or Bafetinib) was much stronger in stellate CP cells than in normal stellate cells, consistent with differential Lyn kinase activity. Our findings support the concept that drug targeting of Lyn in CP patients can be exploited for therapeutic purposes to block chemotaxis of activated myofibroblast-like stellate cells, and thus reduce fibrosis and other deleterious effects of chronic inflammation.
Tu1891 Feasibility of Non-Invasive Method of Diagnosing Pancreatic Fibrosis Using Shear Wave Elastography Takamichi Kuwahara, Yoshiki Hirooka, Hiroki Kawashima, Eizaburo Ohno, Hiroyuki Sugimoto, Daijuro Hayashi, Tomomasa Morishima, Manabu Kawai, Hiroki Suhara, Tomoaki Takeyama, Takeshi Yamamura, Kazuhiro Furukawa, Kohei Funasaka, Masanao Nakamura, Ryoji Miyahara, Hidemi Goto (BACKGROUND AND AIM) Estimation of the pancreatic fibrosis is not easy due to difficulty in obtaining sufficient pancreas tissue. Shear wave elastography (SW) is a potential technology for diagnosing stage of chronic pancreatitis, because SW can calculate the tissue elasticity as correlation value of Young's modulus. The aim of this study was to evaluate the accuracy of diagnosing pancreatic parenchymal fibrosis in both downstream and upstream of tumors by SW. (PATIENTS AND METHODS) From September 2012 to October 2014, 155 patients who underwent SW estimating pancreatic parenchymal fibrosis were enrolled in this study. All patients were assessed for elastic modulus using SW with iU22 (Philips, Amsterdam, Netherlands). We performed SW measurements more than five times at the same area. Elastic modulus was expressed kPa and presented as mean value of 5 measurements. We checked measurement success rate (number of success measurement / number of all measurement * 100), and the patients whose success rate was less than 60% were excluded from this study. 120 patients (normal pancreas: NP group) underwent SW (head: 32, body: 70, tail: 18). 35 patients had pancreas tumors and underwent pancreatectomy (resected pancreas: RP group). First, these patients underwent SW at both head side and tail side of the tumor second, their elastic modulus of head and tail side of the tumor were compared with degree of histopathological fibrosis. Degree of pancreatic fibrosis (DPF) was classified to the 13 score (DPFS) using the scoring system reported previously (Klo˝ppel, et al. Pancreas 1991).Evaluated points were as follows: 1) Diagnosis accuracy of SW in NP group.2) Correlation elastic modulus with degree of pancreatic fibrosis in RP group. (RESULTS) 1) In NP group, failure of SW was in 4 patients, and 12 patients were unreliable cases. 104 patients (86.7%) were available for this study. The mean value of elastic modulus and success rate were 3.73±1.84kPa and 79.4%, respectively, and these values in each pancreas lesion were head:3.74kPa/84.1%, body:3.67kPa/78.1% and tail:3.98kPa/77.1%, respectively,: no significant difference (elastic modulus; P =0.93, success rate; P =0.17) .2) In RP group, the mean value of DPFS and elastic modulus were 4.26±4.43 and 7.18±5.50kPa. DPFS was significantly correlated with elastic modulus (r =0.724, P <0.001). In ROC analysis, the AUCs of elastic modulus for the diagnosis of mild grade fibrosis (DPFS >3), moderate grade fibrosis (DPFS >6), and severe grade fibrosis (DPFS >10) were 0.920, 0.843 and
Tu1894 Chronic Pancreatitis Occurs in Obese Rats and Is Reduced After Bariatric Surgery Vinciane Rebours, Marion Lavigne, Jean-Baptiste Cavin, Konstantinos Arapis, Véronique Albano, Jean-Pierre Marmuse, Andre Bado, Philippe Ruszniewski, Maude Le Gall, Anne Couvelard Introduction Obesity is a well-known risk factor for pancreatitis. Mechanisms involved are multiple, including pro-inflammatory cytokines secreted by the adipose tissue, insulin resistance and hyperinsulinism. Pancreatic stellate cells are activated and increase the extra
cellular matrix synthesis. Bariatric surgery is one of the more efficient treatments of morbid obesity. Aim- To assess pancreatic endocrine and exocrine lesions in obese rats (high fat diet, HFD) and to analyze consequences of bariatric surgery on these lesions. Methods 32 male Wistar rats were included: obese (after 4 months HFD) or not (ND, normal diet). A part of HFD rats underwent bariatric surgery: by pass (BP) or sleeve gastrectomy (SG). Four groups were constituted: HFD (n=8); HFD-BP (n=11); HFD-SG (n=5); ND (n=8). After rat sacrifice (D14 or D40 after surgery), weight, % of weight loss and glycoregulation were analyzed. Each entire pancreas specimen was analyzed by pathological exam: number of adipocytes, islets, fibrosis, acinar-ductal metaplasia, abnormality of Langerhans islets (HHF: hypertrophy, hypervascularisation, fibrosis), and hemosiderin deposits in acinar or endocrine location. Results A first comparison between HFD and ND rats showed an increase of HHF and hemosiderin deposits in HFD rats (p=0.0012 and p=0.0078, respectively). The increase of the weight in HFD rats was associated with glycoregulation abnormalities (r= 0.44, p= 0.08). In the 4 groups, HHF lesions were associated with hemosiderin deposits (r=0.88, p<0.0001). A second analysis was performed in HFD rats after bariatric surgery. In rats operated on, a decrease in number of HHF lesions (p=0.001), hemosiderin deposits (p= 0.0006) and amount of adipocytes (p=0.01) was observed, which was more important in HFD by-pass rats compared to those with HFD-sleeve gastrectomy. In HFD-BP rats, HHF lesions and hemosiderin deposits were associated with acinar-ductal metaplasia lesions (p= 0.08 and p=0.02 respectively). The number of acinar-ductal metaplasia lesions was higher in rats with higher weight before sacrifice (p=0.03). Moreover, number of HHF lesions was associated with decreased blood glucose after by-pass procedure (r=-0.76, p=0.02) but with increased blood glucose after sleeve gastrectomy (r=0.95, p=0.05). Conclusion Obese rats develop chronic pancreatitis with acinar-ductal metaplasia and fibrosis in acinar location and at the endocrine-exocrine interface. Abundant hemosiderin deposits suggest angiogenesis-related mechanism due to vascular lesions associated with the activation of the pancreatic stellate cells and fibrogenesis.
BACH2+/CD4+ T lymphocytes in Chronic Pancreatitis Tu1896 Acinar-Immune Interactions in Human Acute Pancreatitis Ramaiah Jangala, Aparna Jakkampudi, Ratnakar R. Bynigeri, Mitnala Sasikala, Nageshwar D. Reddy, Rupjyoti Talukdar Introduction: Persistent early systemic inflammatory response syndrome (SIRS) in acute pancreatitis (AP) is associated with adverse outcomes. Even though studies have demonstrated an elevation of circulating cytokines it is unclear how human acinar injury leads to SIRS. We explore the mechanisms of acinar injury in human AP and its relationship to SIRS. Methods: Experiments were conducted on healthy pancreata obtained from Whipple's surgery performed for nonpancreatic indications. Harvested tissues were minced into fine bits as described earlier and exposed to TLCS, FAEE, cerulein and LPS. Trypsin and cathepsin B activities were estimated in subcellular fractions of tissue homogenates. Cell injury was evaluated by H&E, IHC and transmission electron microscopy (TEM). Cytokines were evaluated by flowcytometry in the acinar media which was subsequently used to activate autologous PBMCs. Normal pancreata was further exposed to conditioned media from LPS activated PBMCs and cytokine release by acini was assessed. AR42J cell lines were subjected to inhibition of trypsin and calcium to study the mechanistic link between early acinar injury and IL-6 expression with Q-PCR . Blood samples from consecutive patients with documented biliary & alcoholic AP was assessed for cytokines within the first week of illness. Results: Trypsin and cathepsin B activities revealed significant redistribution cathepsin B to zymogen containing organelles at 15mins and 30mins after TLCS and FAEE treatment respectively. H&E showed significant acinar injury in the treated specimens with a time dependent increase. IHC for LC3 revealed autophagy in TLCS treated specimens after 2hrs exposure, while it was absent with FAEE. TEM confirmed presence of autophagolysosomes in TLCS treated specimen. Flowcytometry of FAEE and TLCS treated acinar tissue media showed time-dependent increase in cytokines. IL-6 concentration was maximum at 24hrs in FAEE and 3hrs in TLCS treated specimens. IL-6 concentration was markedly higher with FAEE. PBMCs activated with acinar conditioned media showed increased secretion of pro and anti-inflammatory cytokines. Treatment of normal pancreata with conditioned media from LPS activated PBMCs also induced cytokine secretion even in the absence of FAEE & TLCS exposure. IL-6 secretion was observed in TLCS treated AR42J cells. Inhibition of trypsin was not associated with any reduction in IL-6 expression by these cells. IL-6 concentration was significantly higher in alcoholic AP, which was in tune with the cytokine patterns in the experimental studies. Conclusion: Human acinar cells secrete cytokines in reponse to TLCS and FAEE which can activate circulating PBMCs. Cytokines secreted by the activated PBMCs can cause further acinar injury and propagate systemic inflammation. Secretion of cytokines by the acinar cells appear to be independent of trypsin activation.
Tu1895 Bach2 Repression Enhances Th17 Cell Axis in Chronic Pancreatitis Mitnala Sasikala, Murali M. Kuruva, Sandhya Singh, Pavan K. Pondugula, Mamatha R. Kuncharam, Rupjyoti Talukdar, venkat Rao Guduru, Pradeep Rebala, Nageshwar D. Reddy Background and Aim:Chronic pancreatitis(CP) is a progressive inflammatory disorder and adaptive immune system is implicated in its chronicity. Although CD4+,CD8+Tcell recruitment into pancreatitis lesions has been reported, only one study described the functional phenotype in comparison to pancreatic cancer.Considering the regulatory role of BACH2(BTB and CNC homology basic leucine zipper transcription factor2)in the homeostasis of FOXP3+ regulatory T cells to suppress effector Tcells and inflammation,we investigated its role in CP. Methods: Pancreatic tissues, peripheral blood from CP patients(n=22) and controls without any CP(n=20)undergoing pancreatic resections were obtained with informed consent. mRNA of BACH2,other Tcell transcription factors along with interleukin23 receptor (IL23R;marker of pathogenic Th17cells)were evaluated by qPCR and BACH2 protein by western blot. BACH2+/CD4+T lymphocytes were enumerated on flowcytometer and confirmed by immunofluorescence.The effect of BACH2 repression on T helper cell differentiation was assessed by its invitro silencing through transduction of BACH2 shRNA lentiviral particles in CD4+T lymphocytes.Further, control and BACH2 silenced CD4+T lymphocytes were exposed to polarizing conditions(TGFβ, IL6)and cell free crude CP and normal pancreatic tissue extract. Results:BACH2 mRNA was significantly decreased (P<.001)while that of Tbet,RORγt,BLIMP1 were significantly increased in CD4+T lymphocytes (P<.001)in circulation and pancreatic tissue of CP patients.Despite significant increase in CD4+T lymphocytes(P<.01), percentage of BACH2+/CD4+T lymphocytes was significantly decreased(P<.01) in CP.IL23R expression was significantly high in pancreatic tissue of CP patients(P<.0001).In comparison to normal CD4+T lymphocytes,BACH2silenced CD4+T lymphocytes showed enhanced differentiation of Th1,Th2,Th17 cells(1.18,1.88,1.70 folds respectively)and exposure of BACH2 silenced cells to Th17polarizing conditions(TGFβ, IL6)resulted in higher differentiation of Th17cells(10.20 fold).Further, normal CD4+T lymphocytes exposed to cell free crude tissue extract from pancreatic tissues of CP patients showed 5.32 fold increased Th17 differentiation than normal pancreatic tissue exposure. Comparisons drawn between BACH2 silenced CD4+ T lymphocytes with and without exposure to polarizing conditions showed 3.0 fold increase in Th17 under polarizing conditions, whereas normal CD4+T lymphocytes exposed to CP tissue extract compared with BACH2 silenced Tcells showed 1.91 fold greater differentiation of Th17 cells. Taken together, these results suggest Th17cell differentiation to be greater under BACH2 repressed state along with Th17polarizing conditions of pancreatic tissue in CP. Conclusion:BACH2 repression in CP promotes Th17cell differentiation which acquires pathogenic phenotype upon infiltration into pancreatic tissue.
Tu1897 Pharmacologic Attenuation of NF-κB Activity Reduces the Severity of Acute Pancreatitis Mark Wells, Christopher Arkfeld, Katrina Schneider, Amarnath Natarajan, Shailender Singh, Dahn Clemens Background and Aims: Acute pancreatitis is a necro-inflammatory disease of the pancreas. This disease is characterized by tissue damage with concomitant local and systemic inflammation that is mediated by inflammatory cytokines and chemokines. Currently, there is no specific pharmacotherapy for acute pancreatitis. It has been shown that the inflammation resulting from the prolonged activation of the transcriptional regulator NF-κB mediates much of the tissue damage associated with acute pancreatitis. Because of this important role of NF-κB in the pathobiology of acute pancreatitis, we have investigated the effects of attenuating NF-κB activity in an animal model of acute pancreatitis. Methods: Using a coxsackievirus-induced (CVB3) model of acute pancreatitis, we investigated the efficacy of 13-197 and TPCA-1, two inhibitors of IKK-β, the upstream activator of the NF-κB pathway in attenuating the severity of acute pancreatitis. Mice were treated with either 13-197 or TPCA-1, for two days prior to infection with CVB3. Four days after infection, mice were euthanized, their pancreata harvested, and analyzed for the severity of necrosis and NF-κB activation. Results: Histological results demonstrated that both 13-197 and TPCA-1 reduced necrosis and inflammation in the pancreata of mice infected with CVB3 (Figure 1: Top). Immunohistochemical analysis of nuclear RelA/p65 indicated that NF-κB activation was reduced approximately 5-fold in the pancreata of mice treated with IKK-β inhibitors (Figure 1: Bottom). Additionally, immunoblot analysis of nuclear extracts from the pancreata of CVB3 infected, 13-197 treated mice revealed a greater than 10-fold reduction in nuclear NF-κB when compared with untreated mice (Figure 2). Conclusions: These findings indicate