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Acute pancreatitis conditioned mesenteric lymph can cause cardiac dysfunction
Abstracts / Pancreatology 14 (2014) S1eS129
profiles measured. EAP severity was assessed by standard biochemical parameters and blinded histopathology. Results: GSK-7975A inhibited calcium influx and necrotic pathway activation in murine (at 3, 10 or 30 mM, p<0.05) and human (at 10, 30 or 100 mM, p<0.05) PACs after toxin administration. TRPC3 inhibition did not further increase inhibition of calcium influx. PK confirmed GSK-6288B consistently achieved drug levels of GSK-7975A (circa 4.2 and 13.32 mM in blood or 8.87 and 49.31mM in pancreas at the dose of 28 and 110mg/kg/h, respectively). GSK-6288B significantly reduced all EAP severity parameters in both models (all p<0.05). Conclusion: Orai1 plays a major role in PAC Ca2+ entry and injury in response to toxins. This work confirms the role of calcium overload in EAP and suggests Orai1 inhibition has potential in the treatment of AP.
F-048. Does the genetic strain influence severity of acute necrotizing pancreatitis and SIRS in a murine model? Gabriel Seifert, Karoline Sander, Sabine Richter, Ulrich Hopt, Uwe Wittel University Clinic Freiburg, Germany Background: To date, it remains unclear which impact the genetic background has on the severity of acute pancreatitis concerning local and systemic parameters. Aims: To evaluate the impact of genetic background on the severity of acute necrotizing pancreatitis and systemic inflammatory response in mice. Materials & methods: We examined 9 strains of male mice: Balb/CB4J, C3H/HEJ, NOD/SHILT, A/J, AKR/J, C57BI/6J, DBA/2J, FVB/NJ, 129S1/SVlm. 10 animals per strain were randomly allotted to even groups of 5 animals. Sterile necrotizing pancreatitis was induced by retrograde injection of 4% taurocholate in the common bile duct, or saline was injected in the same way. After 24 hours, serum, organs, and bronchoalveolar fluid were examined. Statistical significance was assumed for p<0,05. Results: Histologically, taurocholate treated animals scored significantly higher than control animals (C). Concordantly, serum lipase and amylase were significantly elevated in the taurocholate groups across all strains. The degree of elevation was strain-specific. NOD/SHILT and AKR/J mice showed significantly higher lipase (4502±1455 and 2828±1161 U/l respectively) and amylase (57180±17863 and 74608±23200 U/l respectively) levels than C57BI/6J, DBA/2J, and FVB/NJ mice (lipase: 444±198, 436±71, 776±433 U/l respectively; amylase: 14236±5080, 9540±1496, 16292±8286 U/l respectively). Remarkably, C57BL/6J, C3H/HeJ, and 129S1/ SVlm mice showed no significant IL-6 elevation (C: 11,14 ± 6,11 vs AP:3,28±2,03 pg/ml; C: 0,23±021 vs AP: 3,78±3,09 pg/ml; C: 160,11±111,8 vs AP: 685,6±415,95 pg/ml) compared to all other strains. Pulmonary damage didn't vary significantly. Conclusion: Our results indicate that the genetic background is an important determinant of the severity of acute pancreatitis and systemic inflammatory response in a murine model.
F-049. A protective role for immune suppressive macrophages in acute pancreatitis Edward Alabraba a, Fiona Campbell b, Diane Lataweic a, John Neoptolemos a, Robert Sutton a a NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK, United Kingdom b Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom
Background: Acute pancreatitis (AP) triggers inflammatory immune responses. M2 macrophages express anti-inflammatory cytokines, promote wound healing, polarize T cells to anti-inflammatory (Th2) phenotype and dampen immune responses.
S101
Aims: We tested if adoptively transferred M2 macrophages could attenuate pancreatic injury in experimental AP. Materials & methods: AP was induced by 7 intra-peritoneal caerulein injections (50micrograms/Kg/hr), either with or without M2 macrophages injected intravenously into the tail vein of mice 12 hours before the first caerulein injection. M2 macrophages were generated by conditioning bone marrow-monocyte-derived macrophages (BMDM) with IL13 for 24hrs. BMDM were obtained by flushing femurs of syngeneic mice induced with AP, purifying the resulting suspension using density gradient media, then positively selecting monocytes with antiCD11b magnetic beads, followed by culture with colony-stimulating factor for 10-14 days. Pancreatic enzymes, cytokines, immune cells and histopathology (blinded) were assessed at 6, 12, and 24 hours (n¼6 all groups). Results: Histopathological severity of pancreatic injury, amylase and myeloperoxidase levels were significantly (p<0.05) reduced at all time points in mice with AP and adoptively transferred M2 macrophages compared to mice with AP alone. These mice also showed Th2 polarized CD4 T cells with downregulated expression of chemokine receptors CCR2 and CCR5. In addition serum MCP-1, IL-6, and TNF-alpha were significantly (p<0.05) reduced while IL10 was significantly increased (p<0.05). Conclusion: BMDM conditioned to anti-inflammatory M2 phenotype by treatment with IL13 can attenuate pancreatic necrosis and inflammatory cell infiltrate, reduce effector leukocyte activation, and skew the circulating cytokine profile in favour of anti-inflammatory cytokines when adoptively transferred to mice before inducing AP with caerulein.
F-050. Acute pancreatitis conditioned mesenteric lymph can cause cardiac dysfunction Anthony Phillips, Satya Shanbhag, Bernard Choong, John Windsor University of Auckland, New Zealand Background: Acute pancreatitis (AP) is associated with cardiac and pulmonary dysfunction. The ‘gut-lymph hypothesis’ states this is due to the release of toxic factors from the intestine into mesenteric lymph (ML). Aims: The aim of this study was to determine the effect of AP conditioned ML on cardiac function and the effect of therapeutic external drainage of ML. Materials & methods: Groups (n¼8/group) of normal rats and those with taurocholate induced severe AP, had either no lymphatic intervention or thoracic duct (TD) ligation with ML drainage (to protect the hearts from exposure to ML). After 6h the hearts underwent ex vivo functional assessment, including cardiac output and ventricular contractility measures (+/-dP/dt). In a separate experiment ML from normal rats and with established taurocholate AP was infused into ex vivo normal perfused working hearts to assess the impact on cardiac function. Results: Significant cardiac dysfunction was found in the hearts removed from rats with established AP alone (no lymphatic intervention) compared to the control group (p<0.05). Strikingly this dysfunction did not occur in rats with established AP and TD ligation with ML drainage. In the second experiment infusion of AP conditioned ML resulted in an immediate, similar reduction of cardiac output (p< 0.05) with corresponding reductions in cardiac contractility and relaxation (p<0.05). Conclusion: AP conditioned ML causes significant cardiac dysfunction which can be prevented by TD ligation and external ML drainage. Further research is now required to study the impact of conditioned ML in AP on the function of other vital organs in MODS.
F-051. Predictors of organ failure and pancreatic infection in acute pancreatitis: A systematic review Catherine Yang, Joseph Chen, Anthony Phillips, John Windsor, Max Petrov University of Auckland, New Zealand
profiles measured. EAP severity was assessed by standard biochemical parameters and blinded histopathology. Results: GSK-7975A inhibited calcium influx and necrotic pathway activation in murine (at 3, 10 or 30 mM, p<0.05) and human (at 10, 30 or 100 mM, p<0.05) PACs after toxin administration. TRPC3 inhibition did not further increase inhibition of calcium influx. PK confirmed GSK-6288B consistently achieved drug levels of GSK-7975A (circa 4.2 and 13.32 mM in blood or 8.87 and 49.31mM in pancreas at the dose of 28 and 110mg/kg/h, respectively). GSK-6288B significantly reduced all EAP severity parameters in both models (all p<0.05). Conclusion: Orai1 plays a major role in PAC Ca2+ entry and injury in response to toxins. This work confirms the role of calcium overload in EAP and suggests Orai1 inhibition has potential in the treatment of AP.
F-048. Does the genetic strain influence severity of acute necrotizing pancreatitis and SIRS in a murine model? Gabriel Seifert, Karoline Sander, Sabine Richter, Ulrich Hopt, Uwe Wittel University Clinic Freiburg, Germany Background: To date, it remains unclear which impact the genetic background has on the severity of acute pancreatitis concerning local and systemic parameters. Aims: To evaluate the impact of genetic background on the severity of acute necrotizing pancreatitis and systemic inflammatory response in mice. Materials & methods: We examined 9 strains of male mice: Balb/CB4J, C3H/HEJ, NOD/SHILT, A/J, AKR/J, C57BI/6J, DBA/2J, FVB/NJ, 129S1/SVlm. 10 animals per strain were randomly allotted to even groups of 5 animals. Sterile necrotizing pancreatitis was induced by retrograde injection of 4% taurocholate in the common bile duct, or saline was injected in the same way. After 24 hours, serum, organs, and bronchoalveolar fluid were examined. Statistical significance was assumed for p<0,05. Results: Histologically, taurocholate treated animals scored significantly higher than control animals (C). Concordantly, serum lipase and amylase were significantly elevated in the taurocholate groups across all strains. The degree of elevation was strain-specific. NOD/SHILT and AKR/J mice showed significantly higher lipase (4502±1455 and 2828±1161 U/l respectively) and amylase (57180±17863 and 74608±23200 U/l respectively) levels than C57BI/6J, DBA/2J, and FVB/NJ mice (lipase: 444±198, 436±71, 776±433 U/l respectively; amylase: 14236±5080, 9540±1496, 16292±8286 U/l respectively). Remarkably, C57BL/6J, C3H/HeJ, and 129S1/ SVlm mice showed no significant IL-6 elevation (C: 11,14 ± 6,11 vs AP:3,28±2,03 pg/ml; C: 0,23±021 vs AP: 3,78±3,09 pg/ml; C: 160,11±111,8 vs AP: 685,6±415,95 pg/ml) compared to all other strains. Pulmonary damage didn't vary significantly. Conclusion: Our results indicate that the genetic background is an important determinant of the severity of acute pancreatitis and systemic inflammatory response in a murine model.
F-049. A protective role for immune suppressive macrophages in acute pancreatitis Edward Alabraba a, Fiona Campbell b, Diane Lataweic a, John Neoptolemos a, Robert Sutton a a NIHR Liverpool Pancreas Biomedical Research Unit, Royal Liverpool University Hospital, Liverpool, UK, United Kingdom b Department of Molecular and Clinical Cancer Medicine, University of Liverpool, Liverpool, United Kingdom
Background: Acute pancreatitis (AP) triggers inflammatory immune responses. M2 macrophages express anti-inflammatory cytokines, promote wound healing, polarize T cells to anti-inflammatory (Th2) phenotype and dampen immune responses.
S101
Aims: We tested if adoptively transferred M2 macrophages could attenuate pancreatic injury in experimental AP. Materials & methods: AP was induced by 7 intra-peritoneal caerulein injections (50micrograms/Kg/hr), either with or without M2 macrophages injected intravenously into the tail vein of mice 12 hours before the first caerulein injection. M2 macrophages were generated by conditioning bone marrow-monocyte-derived macrophages (BMDM) with IL13 for 24hrs. BMDM were obtained by flushing femurs of syngeneic mice induced with AP, purifying the resulting suspension using density gradient media, then positively selecting monocytes with antiCD11b magnetic beads, followed by culture with colony-stimulating factor for 10-14 days. Pancreatic enzymes, cytokines, immune cells and histopathology (blinded) were assessed at 6, 12, and 24 hours (n¼6 all groups). Results: Histopathological severity of pancreatic injury, amylase and myeloperoxidase levels were significantly (p<0.05) reduced at all time points in mice with AP and adoptively transferred M2 macrophages compared to mice with AP alone. These mice also showed Th2 polarized CD4 T cells with downregulated expression of chemokine receptors CCR2 and CCR5. In addition serum MCP-1, IL-6, and TNF-alpha were significantly (p<0.05) reduced while IL10 was significantly increased (p<0.05). Conclusion: BMDM conditioned to anti-inflammatory M2 phenotype by treatment with IL13 can attenuate pancreatic necrosis and inflammatory cell infiltrate, reduce effector leukocyte activation, and skew the circulating cytokine profile in favour of anti-inflammatory cytokines when adoptively transferred to mice before inducing AP with caerulein.
F-050. Acute pancreatitis conditioned mesenteric lymph can cause cardiac dysfunction Anthony Phillips, Satya Shanbhag, Bernard Choong, John Windsor University of Auckland, New Zealand Background: Acute pancreatitis (AP) is associated with cardiac and pulmonary dysfunction. The ‘gut-lymph hypothesis’ states this is due to the release of toxic factors from the intestine into mesenteric lymph (ML). Aims: The aim of this study was to determine the effect of AP conditioned ML on cardiac function and the effect of therapeutic external drainage of ML. Materials & methods: Groups (n¼8/group) of normal rats and those with taurocholate induced severe AP, had either no lymphatic intervention or thoracic duct (TD) ligation with ML drainage (to protect the hearts from exposure to ML). After 6h the hearts underwent ex vivo functional assessment, including cardiac output and ventricular contractility measures (+/-dP/dt). In a separate experiment ML from normal rats and with established taurocholate AP was infused into ex vivo normal perfused working hearts to assess the impact on cardiac function. Results: Significant cardiac dysfunction was found in the hearts removed from rats with established AP alone (no lymphatic intervention) compared to the control group (p<0.05). Strikingly this dysfunction did not occur in rats with established AP and TD ligation with ML drainage. In the second experiment infusion of AP conditioned ML resulted in an immediate, similar reduction of cardiac output (p< 0.05) with corresponding reductions in cardiac contractility and relaxation (p<0.05). Conclusion: AP conditioned ML causes significant cardiac dysfunction which can be prevented by TD ligation and external ML drainage. Further research is now required to study the impact of conditioned ML in AP on the function of other vital organs in MODS.
F-051. Predictors of organ failure and pancreatic infection in acute pancreatitis: A systematic review Catherine Yang, Joseph Chen, Anthony Phillips, John Windsor, Max Petrov University of Auckland, New Zealand