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Sa1812 Application of the Targeted Next Generation Sequencing to the Comprehensive Analysis of Pancreatitis Susceptibility Genes
Sa1814 Leukotriene B4 Is Enriched in Human Pancreatic Post Inflammatory Collections and Its Generation May Mediate Unsaturated Fatty Acid Toxicity in Acute Pancreatitis Pawan Noel, Krutika Patel, Ram Trivedi, Cristiane de Oliveira, Kenneth Lee, Randall Brand, Jennifer Chennat, Adam Slivka, Georgios I. Papachristou, Asif Khalid, Dhiraj Yadav, Faris Murad, Sarah Navina, Vijay P. Singh BACKGROUND: We have recently shown that unsaturated fatty acids (UFA) enriched in human pancreatic post inflammatory collections (PIC) worsen severity of acute pancreatitis (AP). Linoleic acid (LA) is enriched in these collections and is a precursor of arachidonic acid (AA), from which various pro and anti-inflammatory metabolites may be formed. Proinflammatory metabolites include leukotriene B4 (LTB4) formed via 5-lipoxygenase (5-LOX) and anti-inflammatory ones include lipoxins formed from 15-lipoxygenase (15-LOX). We therefore went on to measure metabolites of these UFAs in human PICs and studied the roles these may have in exocrine pancreatic injury. METHODS: 5-LOX and 15-LOX metabolites, LTB4 and 15-(S)-hydroxy eicosatetraenoic acid [15-(S)-HETE] were measured in human PICs (necrotic collections; n=13, pseudocysts; n=11) and pancreatic cystic neoplasms (PCNs; n=10). Mice pancreatic acinar cells were treated with LA alone or in the presence of the LOX inhibitor caffeic acid (CA; 50 micromolar), anti-oxidants n-acetyl L-cysteine (NAC; 1 millimolar) or Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP; 200 micromolar) and cell injury (LDH leakage) was measured. LA induced formation of LTB4 and 15-(S)-HETE in the presence and absence of CA were also measured. RESULTS: Both LTB4 and 15-(S)-HETE were > 5 fold increased in PICs vs. PCNs (p<0.001). LA induced a steady increase in both these metabolites, which increased by 8-10 folds over 4 hours. LA also induced a similar and significant 8-10 fold increase in acinar cell death. CA but not NAC or MnTBAP significantly reduced LA induced cell death by 21% (p<0.05). CA also significantly reduced LA induced LTB4 production, while minimally affecting 15-(S)HETE production. CONCLUSIONS: Acinar cells can metabolize LA via both the 5- and 15- LOX pathways, the products of which are increased in human PICs, and which respectively may have both pro- and anti-inflammatory roles. The deleterious effects of UFAs are more likely to be mediated by 5-LOX products including LTB4 and not their oxidative metabolites.
Sa1812 Application of the Targeted Next Generation Sequencing to the Comprehensive Analysis of Pancreatitis Susceptibility Genes Eriko Nakano, Atsushi Masamune, Tetsuya Niihori, Kiyoshi Kume, Shin Hamada, Yoko Aoki, Tooru Shimosegawa Backgrounds: Since the identification of mutations in the cationic trypsinogen (PRSS1) gene as a cause of hereditary pancreatitis in 1996, several pancreatitis susceptibility genes have been identified including the serine protease inhibitor Kazal type 1 (SPINK1), trypsindegrading enzyme chymotrypsin C (CTRC), cystic fibrosis transmembrane conductance regulator (CFTR) and carboxypeptidase A1 (CPA1) genes. Direct DNA sequencing of polymerase chain reaction-amplified gene segments is a first-line method for the genetic analysis. But, in the case of genes with large size and many exons such as CFTR, this is a tedious and labor-intensive endeavor. A new approach that uses massive parallel sequencing called next generation sequencing (NGS) is becoming standardized. Targeted capture of selected regions of interest followed by NGS provides an efficient and cost-effective option. We here report a comprehensive analysis of pancreatitis susceptibility genes by targeted NGS. Methods: One hundred ninety three patients with chronic pancreatitis (CP) (121 idiopathic, 46 alcoholic, 17 hereditary, 9 familial) were enrolled in this study. We designed the HaloPlex Target Enrichment System (Agilent Technologies) targeting all of the exons and adjacent intronic regions of the known pancreatitis susceptibility genes. All samples were sequenced on the Illumina Miseq platform with paired-end 151-bp reads. SIFT (Sorting Intolerant From Tolerant) and PolyPhen-2 were used to predict whether an amino acid substitution would affect the structure and function of a protein. This study was approved the Ethics Committee of Tohoku University School of Medicine. Results: All of the exons of the known pancreatitis susceptibility genes could be successfully analyzed with a high resolution capability. In the case of CFTR, 98.8%, 97.0%, 95.1%, and 91.6 % of the coding regions were covered by at least one, 5, 10 , and 20 sequence reads, respectively. We could identify 12 non-synonymous variants including 3 novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and 7 synonymous variants including 3 novel ones in the exonic regions. Among the 193 patients with CP, 29 patients had pancreatitisassociated variants in the PRSS1, SPINK1, CTRC, or CPA1 genes. Among them, 9 patients had the non-synonymous CFTR variants, which are probably damaging based on the SIFT and/or the PolyPhen-2 prediction Conclusions: The targeted NGS allowed us to perform rapid screening of the known susceptibility genes simultaneously and gave an overview of potentially pathogenic variants in patients with pancreatitis.
Sa1815 p21-Activated Kinase, PAK2, Is an Important Mediator of Numerous Signaling Cascades Mediating CCK`S Physiological and Pathophysiological Effects in Pancreatic Acinar Cells Bernardo Nuche-Berenguer, Paola Moreno, Robert T. Jensen Introduction: The p21-activated kinases (PAKs) are highly conserved serine/threonine protein kinases and are important effectors of the small GTP-ases: cdc42 and Rac1. PAKs comprise six different members divided into group I (PAK 1, 2, 3) and group II (PAK 4,5, 6). We previously demonstrated rat pancreatic acinar cells possess only PAK2 of the group I PAKs and it is activated by a number of growth factors and gastrointestinal hormones/ neurotransmitters. However, the role of PAK2 in the activation of possible downstream signaling cascades, shown in previous studies to be important in mediating physiological and pathophysiological effects in pancreatic acini has not been studied. Aim: To investigate the role of PAK2 in mediating the CCK- and TPA- induced activation of signaling cascades in rat pancreatic acinar cells. Methods: Dispersed rat pancreatic acini were prepared. PAK2 modulation of signaling cascades was assessed using a specific small allosteric inhibitor that targets the regulatory interface where the small GTP-ases -Cdc42 and Rac1- bind PAKs. The inactive IPA-3 analogue, Pir 3,5 was used as a control. Phospho-specific antibodies were used to investigate signaling pathways activation activation with Western blotting. Results: CCK-8 (0.3, 100nM) and TPA (1mM) activated PAK2, Pyk2, p125FAK, paxillin, p130CAS, Mek 1/2, ERK1/2, JNK, p85PI3K, Src, PKD and MARCKS, decreased the activation of Akt and had no effect on p38 and GSK-3-b. The CCK activation of PAK2, Pyk2 , p125FAK, paxillin, p130CAS and p85PI3K was inhibited by IPA3, as was activation of Mek1/2, activation of ERK1/2 and activation of JNK . IPA3 reversed the CCK- and TPA-induced inhibition of Akt and this effect was tranfered downstream to p70s6k. Neither basal nor CCK- or TPAmediated PKD, Src or MARCKS activation was affected by IPA3. The incubation with the inactive analogue Pir 3,5 did not have any effect in any of the studied cascades. Conclusions: In rat pancreatic acini, PAK2 activation is important for CCK- and TPA-mediated activation of a number of important key signaling cascades including focal adhesion kinases (Pyk2, p125FAK), the adapter proteins (paxillin, p130CAS) and mitogen-activated kinases (Erk1/2 and JNK), whereas it does not affect PKC or Src activation. PAK2 is also important in modulating activation of the PI3K/Akt mediating the downstream inhibition of Akt by CCK and TPA, possibly by induced phosphatase activity, as shown in other tissues. These results show PAK2 has a central role in activating a number of important cellular signaling cascades activated by CCK and demonstrated to have important roles in mediating CCK's numerous physiological and pathophysiological responses important in pancreatic acini such as growth, secretion, apoptosis, cell division and cytoskeletal integrity.
Sa1813 Differential Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression on Pancreatic Duct Epithelial Cells (PDEC) From Proximal and Distal Human Pancreatic Ducts Supports a Two-Step Mechanism for Bicarbonate Secretion by Pancreatic Ducts Toan D. Nguyen Introduction: Bicarbonate (HCO3) secretion by pancreatic ducts displays i) inverse relationship between HCO3 and chloride (Cl) concentrations in stimulated secretion, ii) luminal HCO3 concentration of up to 145 mM in humans, and iii) key function for the cAMPstimulated cystic fibrosis conductance regulator (CFTR) chloride channel. Current models for HCO3 secretion by PDEC, coupling Cl secretion with Cl/HCO3 exchange on its apical membrane (AM), cannot fully account for an intraluminal concentration of HCO3 of 145 mM. A two-step model, with upstream Cl and HCO3 secretion in the proximal pancreatic duct followed by downstream Cl reabsorption in the distal duct may fulfill all three characteristics of pancreatic ductal HCO3 secretion. We previously observed that polarized PDEC derived from the main pancreatic duct of dogs express functional CFTR on both AM and basolateral membrane (BLM), allowing these cells to transepithelially reabsorb Cl (Gastroenterol. 144, 5: S68, 2013). We now report that, in human pancreas, while PDEC from proximal centroacinar and intralobular pancreatic ducts express CFTR on the AM, PDEC from the distal main ducts express CFTR on both AM and BLM. Methods: Formalin fixed, paraffin embedded, human pancreas samples, from a UTHSC Pathology Dpt. tissue bank, were sectioned (5 μm), reconstituted, and incubated with antibodies against CFTR (R&D Systems), subsequently detected with fluorescent goat anti-mouse secondary antibodies (Invitrogen). Antibodies against sodium-potassium ATPase (Na,K-ATPase, Santa Cruz), detected with donkey anti-rabbit secondary antibodies (Invitrogen), served to delineate the BLM. Fluorescence microscopy was used to determine the cellular expression of CFTR and Na,K-ATPase on PDEC. Results: CFTR was localized to the AM of PDEC from centroacinar and intralobular pancreatic ducts. It was sparsely expressed in interlobular pancreatic ducts. In contrast, on PDEC from large pancreatic ducts, CFTR was expressed not only on the AM, but also on the BLM. Conclusion: We previously reported that PDEC monolayers derived from the main pancreatic duct of dogs expressed CFTR on both AM and BLM, a configuration that allowed them to mediate transepithelial Cl absorption, from the luminal compartment into the serosal compartment. We now provide the histologic correlate that, in humans, while CFTR are localized to the AM of PDEC from proximal centroacinar and intralobular ducts, they are expressed on both AM and BLM of PDEC from the distal main pancreatic duct. Following secretion of Cl and HCO3 from the proximal pancreatic ducts, human distal pancreatic ducts can therefore reabsorb Cl into the serosal compartment in exchange for further luminal secretion of HCO3, generating the high HCO3 concentration of 145 mM found in pancreatic juice. This research was funded by a VA Merit Review and by institutional UTHSC support.
Sa1816 Association of Genetic Mutations With Pediatric Acute Recurrent and Chronic Pancreatitis Joseph J. Palermo, Tom K. Lin, Kimberly Jackson, Lindsey Hornung, Lin Fei, Maisam Abu-El-Haija BACKGROUND: Genetic associations have been reported in 18% of adults with acute recurrent pancreatitis (ARP). The most common genes involved are cationic trypsinogen (PRSS1), serine protease inhibitor, Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR), chymotrypsinogen C (CTRC) and calcium sensing receptor (CASR). It is not fully known to what extent genetic mutations play a role in the pathogenesis of pancreatitis in children. AIM: We sought to better understand the role of genetic mutations in pediatric patients with ARP and determine if patients with chronic pancreatitis (CP) were
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AGA Abstracts
AGA Abstracts
and HPDE-Kras. Autophagy has the same protective role in AA-depleted HPDE cells; however, in AA-depleted cells with oncogenic Kras, i.e., CAPAN-2, PANC-1 and HPDE-Kras, autophagy inhibition is without effect, and the OCR and proliferation remain significantly decreased. Thus, the non-canonical autophagy operating in cells with oncogenic Kras fails to maintain ATP production sufficient to sustain cancer cell growth in conditions of AA depletion. Conclusions: Pancreatic cells harboring oncogenic Kras lose nutrient sensitivity of the mTORC1/ULK1 pathway, and instead activate non-canonical autophagy. However, the noncanonical autophagy fails to sustain growth of nutrient-deprived PaCa cells. The results suggest AA depletion as a promising therapeutic approach in Kras-driven pancreatic cancer.
Sa1812 Application of the Targeted Next Generation Sequencing to the Comprehensive Analysis of Pancreatitis Susceptibility Genes Eriko Nakano, Atsushi Masamune, Tetsuya Niihori, Kiyoshi Kume, Shin Hamada, Yoko Aoki, Tooru Shimosegawa Backgrounds: Since the identification of mutations in the cationic trypsinogen (PRSS1) gene as a cause of hereditary pancreatitis in 1996, several pancreatitis susceptibility genes have been identified including the serine protease inhibitor Kazal type 1 (SPINK1), trypsindegrading enzyme chymotrypsin C (CTRC), cystic fibrosis transmembrane conductance regulator (CFTR) and carboxypeptidase A1 (CPA1) genes. Direct DNA sequencing of polymerase chain reaction-amplified gene segments is a first-line method for the genetic analysis. But, in the case of genes with large size and many exons such as CFTR, this is a tedious and labor-intensive endeavor. A new approach that uses massive parallel sequencing called next generation sequencing (NGS) is becoming standardized. Targeted capture of selected regions of interest followed by NGS provides an efficient and cost-effective option. We here report a comprehensive analysis of pancreatitis susceptibility genes by targeted NGS. Methods: One hundred ninety three patients with chronic pancreatitis (CP) (121 idiopathic, 46 alcoholic, 17 hereditary, 9 familial) were enrolled in this study. We designed the HaloPlex Target Enrichment System (Agilent Technologies) targeting all of the exons and adjacent intronic regions of the known pancreatitis susceptibility genes. All samples were sequenced on the Illumina Miseq platform with paired-end 151-bp reads. SIFT (Sorting Intolerant From Tolerant) and PolyPhen-2 were used to predict whether an amino acid substitution would affect the structure and function of a protein. This study was approved the Ethics Committee of Tohoku University School of Medicine. Results: All of the exons of the known pancreatitis susceptibility genes could be successfully analyzed with a high resolution capability. In the case of CFTR, 98.8%, 97.0%, 95.1%, and 91.6 % of the coding regions were covered by at least one, 5, 10 , and 20 sequence reads, respectively. We could identify 12 non-synonymous variants including 3 novel ones [c.A1231G (p.K411E), c.1753G>T (p.E585X) and c.2869delC (p.L957fs)] and 7 synonymous variants including 3 novel ones in the exonic regions. Among the 193 patients with CP, 29 patients had pancreatitisassociated variants in the PRSS1, SPINK1, CTRC, or CPA1 genes. Among them, 9 patients had the non-synonymous CFTR variants, which are probably damaging based on the SIFT and/or the PolyPhen-2 prediction Conclusions: The targeted NGS allowed us to perform rapid screening of the known susceptibility genes simultaneously and gave an overview of potentially pathogenic variants in patients with pancreatitis.
Sa1815 p21-Activated Kinase, PAK2, Is an Important Mediator of Numerous Signaling Cascades Mediating CCK`S Physiological and Pathophysiological Effects in Pancreatic Acinar Cells Bernardo Nuche-Berenguer, Paola Moreno, Robert T. Jensen Introduction: The p21-activated kinases (PAKs) are highly conserved serine/threonine protein kinases and are important effectors of the small GTP-ases: cdc42 and Rac1. PAKs comprise six different members divided into group I (PAK 1, 2, 3) and group II (PAK 4,5, 6). We previously demonstrated rat pancreatic acinar cells possess only PAK2 of the group I PAKs and it is activated by a number of growth factors and gastrointestinal hormones/ neurotransmitters. However, the role of PAK2 in the activation of possible downstream signaling cascades, shown in previous studies to be important in mediating physiological and pathophysiological effects in pancreatic acini has not been studied. Aim: To investigate the role of PAK2 in mediating the CCK- and TPA- induced activation of signaling cascades in rat pancreatic acinar cells. Methods: Dispersed rat pancreatic acini were prepared. PAK2 modulation of signaling cascades was assessed using a specific small allosteric inhibitor that targets the regulatory interface where the small GTP-ases -Cdc42 and Rac1- bind PAKs. The inactive IPA-3 analogue, Pir 3,5 was used as a control. Phospho-specific antibodies were used to investigate signaling pathways activation activation with Western blotting. Results: CCK-8 (0.3, 100nM) and TPA (1mM) activated PAK2, Pyk2, p125FAK, paxillin, p130CAS, Mek 1/2, ERK1/2, JNK, p85PI3K, Src, PKD and MARCKS, decreased the activation of Akt and had no effect on p38 and GSK-3-b. The CCK activation of PAK2, Pyk2 , p125FAK, paxillin, p130CAS and p85PI3K was inhibited by IPA3, as was activation of Mek1/2, activation of ERK1/2 and activation of JNK . IPA3 reversed the CCK- and TPA-induced inhibition of Akt and this effect was tranfered downstream to p70s6k. Neither basal nor CCK- or TPAmediated PKD, Src or MARCKS activation was affected by IPA3. The incubation with the inactive analogue Pir 3,5 did not have any effect in any of the studied cascades. Conclusions: In rat pancreatic acini, PAK2 activation is important for CCK- and TPA-mediated activation of a number of important key signaling cascades including focal adhesion kinases (Pyk2, p125FAK), the adapter proteins (paxillin, p130CAS) and mitogen-activated kinases (Erk1/2 and JNK), whereas it does not affect PKC or Src activation. PAK2 is also important in modulating activation of the PI3K/Akt mediating the downstream inhibition of Akt by CCK and TPA, possibly by induced phosphatase activity, as shown in other tissues. These results show PAK2 has a central role in activating a number of important cellular signaling cascades activated by CCK and demonstrated to have important roles in mediating CCK's numerous physiological and pathophysiological responses important in pancreatic acini such as growth, secretion, apoptosis, cell division and cytoskeletal integrity.
Sa1813 Differential Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Expression on Pancreatic Duct Epithelial Cells (PDEC) From Proximal and Distal Human Pancreatic Ducts Supports a Two-Step Mechanism for Bicarbonate Secretion by Pancreatic Ducts Toan D. Nguyen Introduction: Bicarbonate (HCO3) secretion by pancreatic ducts displays i) inverse relationship between HCO3 and chloride (Cl) concentrations in stimulated secretion, ii) luminal HCO3 concentration of up to 145 mM in humans, and iii) key function for the cAMPstimulated cystic fibrosis conductance regulator (CFTR) chloride channel. Current models for HCO3 secretion by PDEC, coupling Cl secretion with Cl/HCO3 exchange on its apical membrane (AM), cannot fully account for an intraluminal concentration of HCO3 of 145 mM. A two-step model, with upstream Cl and HCO3 secretion in the proximal pancreatic duct followed by downstream Cl reabsorption in the distal duct may fulfill all three characteristics of pancreatic ductal HCO3 secretion. We previously observed that polarized PDEC derived from the main pancreatic duct of dogs express functional CFTR on both AM and basolateral membrane (BLM), allowing these cells to transepithelially reabsorb Cl (Gastroenterol. 144, 5: S68, 2013). We now report that, in human pancreas, while PDEC from proximal centroacinar and intralobular pancreatic ducts express CFTR on the AM, PDEC from the distal main ducts express CFTR on both AM and BLM. Methods: Formalin fixed, paraffin embedded, human pancreas samples, from a UTHSC Pathology Dpt. tissue bank, were sectioned (5 μm), reconstituted, and incubated with antibodies against CFTR (R&D Systems), subsequently detected with fluorescent goat anti-mouse secondary antibodies (Invitrogen). Antibodies against sodium-potassium ATPase (Na,K-ATPase, Santa Cruz), detected with donkey anti-rabbit secondary antibodies (Invitrogen), served to delineate the BLM. Fluorescence microscopy was used to determine the cellular expression of CFTR and Na,K-ATPase on PDEC. Results: CFTR was localized to the AM of PDEC from centroacinar and intralobular pancreatic ducts. It was sparsely expressed in interlobular pancreatic ducts. In contrast, on PDEC from large pancreatic ducts, CFTR was expressed not only on the AM, but also on the BLM. Conclusion: We previously reported that PDEC monolayers derived from the main pancreatic duct of dogs expressed CFTR on both AM and BLM, a configuration that allowed them to mediate transepithelial Cl absorption, from the luminal compartment into the serosal compartment. We now provide the histologic correlate that, in humans, while CFTR are localized to the AM of PDEC from proximal centroacinar and intralobular ducts, they are expressed on both AM and BLM of PDEC from the distal main pancreatic duct. Following secretion of Cl and HCO3 from the proximal pancreatic ducts, human distal pancreatic ducts can therefore reabsorb Cl into the serosal compartment in exchange for further luminal secretion of HCO3, generating the high HCO3 concentration of 145 mM found in pancreatic juice. This research was funded by a VA Merit Review and by institutional UTHSC support.
Sa1816 Association of Genetic Mutations With Pediatric Acute Recurrent and Chronic Pancreatitis Joseph J. Palermo, Tom K. Lin, Kimberly Jackson, Lindsey Hornung, Lin Fei, Maisam Abu-El-Haija BACKGROUND: Genetic associations have been reported in 18% of adults with acute recurrent pancreatitis (ARP). The most common genes involved are cationic trypsinogen (PRSS1), serine protease inhibitor, Kazal type 1 (SPINK1), cystic fibrosis transmembrane conductance regulator (CFTR), chymotrypsinogen C (CTRC) and calcium sensing receptor (CASR). It is not fully known to what extent genetic mutations play a role in the pathogenesis of pancreatitis in children. AIM: We sought to better understand the role of genetic mutations in pediatric patients with ARP and determine if patients with chronic pancreatitis (CP) were
S-339
AGA Abstracts
AGA Abstracts
and HPDE-Kras. Autophagy has the same protective role in AA-depleted HPDE cells; however, in AA-depleted cells with oncogenic Kras, i.e., CAPAN-2, PANC-1 and HPDE-Kras, autophagy inhibition is without effect, and the OCR and proliferation remain significantly decreased. Thus, the non-canonical autophagy operating in cells with oncogenic Kras fails to maintain ATP production sufficient to sustain cancer cell growth in conditions of AA depletion. Conclusions: Pancreatic cells harboring oncogenic Kras lose nutrient sensitivity of the mTORC1/ULK1 pathway, and instead activate non-canonical autophagy. However, the noncanonical autophagy fails to sustain growth of nutrient-deprived PaCa cells. The results suggest AA depletion as a promising therapeutic approach in Kras-driven pancreatic cancer.