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Characterisation of rat mesenteric afferent responses to ligands selective at protease-activated receptor (PAR)-1, -2 and -4
12 IU/1.), and LD (4,840 -+ 695 IU/L) were significantly lower than those of Cr (0.55 -+ 0 0 4 mg/dL), GOT (1,457 '- 403 IU/L), GPT (412 _+ 104 IU/L), and LD (9,417 _+ 2,016 1UFL) in Group A, respectively. No sigmficant differences were observed in other blood parameters (amylase, Ht, etc.) between two groups. There was no significant difference in volume of ascitic fluid between two groups. These results suggest that VEGF may function as not a vascular permeability factor, but a protective factor against the organ injuries in rat experimental severe acute pancreatitis. Further investigations are necessary to elucidate the role of VEGF in this disease.
180 150.
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--.a-- Parl --.or--p at2
r-, 90 9 S1781
~
Degradation and Inactivation Of Plasma TNF~ By Pancreatic Proteases In Experimental Acute Pancreatitis Guido Alsfasser, Bozena Antoniu, Andrew L. Warshaw, Carlos Fernandez-Del-castillo
0 -30 9
50 75 tO0 Time course (a) Fig. I. Meaent~,.'icaft'wentdischargesin responmeto PAR1, 2 md 4-AP.
Hypothesis: TNFa is thought to play an important role in mediating systemic effects and progression of acute pancreatitis. However, TNFc~ levels in plasma of patients with acute pancreatitis (AP) are often unmeasurabfy low and many studies show contradictory findings. We hypothesize that TNFcr in plasma is susceptible to catabolism by circulating pancreatic proteases. Materials and Methods: In Vivo: Male Spragne-Dawley rats were divided into four groups: Healthy controls, sham operated controls, mild and severe pancreatitls. AP was induced by Cerulein hyperstimulation preceded by intraductal infusion of saline (mild) or glycodeoxychofic acid (severe). Plasma TNFc~ levels were measured at 0.5, 1, 2, 6, 12 and 24 h after induction of AP using a competitive EL1SA. In Vitro: i) Recombinant rat or human TNFa was incubated at 37~ with clinically relevant graded concentrations of trypsin, elastase and chymotrypsin for 30 rain and 24 h; 15% SDS PAGE gel-electrophoresis was performed to visualize TNF degradation. 2) TNF bioactivity was evaluated with a cytotoxicity assay using SYTOX Green stain to detect TNF induced cell death in mouse fibroblast-derived WEHI 13var cells. Results: In Vivo: TNFa level in healthy animals was 4.8+/-2.4 ng/ml. At 0.5 h levels were 10.53+/-1.34 ng/ml, 7.26+/-1.75 ng/ml and 3.95+/-1.56 in sham operated controls, mild and severe pancreatitis, respectively. The levels in severe pancreatitis were significantly lower than in sham operated controls (p = 0.0327). This difference persisted up to 6 hours (4.01 +/-0.18 ng/ml vs. 7.54 +/-0.84 ng/ml, p = 0.0145). No difference was seen at other time points. In Vitro: 1) Undamaged TNFa formed a band of 78 kD, indicating a trimefic configuration. This band vanished after incubation with each of the proteofytic enzymes at all concentrations tested. 2) The ECs0 (dose required to induce death in 50% of cells) was 3ng for human TNFa, and 0.3 ng for rat TNFa. 30 min incubation of TNFa with 1 Izg/ml trypsin decreased bioactivity by 50% and 24 h incubation abolished it completely. Conclusion: TNFcr released during acute pancreatitis is probably degraded rapidly by circulating pancreatic proteases. The bioactivity of this degraded TNFa is likely to be significantly diminished or abolished. Whatever the local effect of TNFa in acute pancreatitis, we suggest that its enzymatic degradation limits any substantial role in systemic pathophysiology.
The Role of LBP in Animal Model of Severe Acute Pancreatitis Xing-Peng Wang, Li-Ying Wu Background/Aims: It is well known LPS was eligible to induce the transition from edematous pancreatitis to necrotizing pancreatitis. In order to elucidate the pathogenic effect ofendotoxin in severe acute pancreatitis, anti-LBP antibody was applied and the therapeutic effects were investigated in this study. Methods: Thirty-six BALB/c mice were randomly divided into SAP group and anti-LBP antibody group. SAP was induced by admmistration of seven intraperitoneal injections of cemlein (50 microg/kg) at hourly intervals. Lipopolysaccharide (LPS) was injected 6 hours after the first cerulein injection. Treatment with the anti-LBF antibody was started 15 minutes before the LPS injection. Serum levels of amylase and lactate dehydrogenase (LDH) were measured at the end of the experiments. The severity of pancreatitis was evaluated by histological sconng using a semi-quantitative analysis of hematoxylin and eosin-stained sections of the pancreas. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the intrapancreatic TNF-alpha IL-l beta ICAM-1 and E-selectin mRNA levels. The expressions of nuclear factor NF-kB and polymorphonuclear leukocyte (PMN)-specific antigen Mac-1 were investigated by the method of immunohistochemistry and western blotting. The activity of PMN myeloperoxidase (MPO) was determined by zymohistochemistryResults: Seven times cerulein rejection induced edematous pancreatins, however when LPS was added, necrotizing pancreatitis developed. At 9, 12, 24 hours after the first cerulein injection, the blood and specimens of pancreas were harvested. A marked elevation of serum amylase at 12 hours and LDH at 24 hours was observed in anti-LBF group. Histologically, treatment of anti-LBP group increased the severity of pancreatic injury including edema at 9 and 12 hours, inflammatory cell infiltration at 9,12 and 24 hours and necrosis at 9,12 and 24 hours. These changes were accompanied by the up-regulation of tumor necrosis factor (TNF)-alpha, interleukin-lbeta (IL-lbeta), ICAM-1 and E-selectin mRNA expressions in the pancreas compared with SAP group. There was significant increase in MPO activity at 12 and 24 hours in anti-LBP antibody group. Immunohistochemistry and western blotting showed up-regulation of nuclear factor NF-kB and Mac-1 at 9 hours in anti-LBP antibody group. Conclusions: The aggravating effect of the anti-LBP antibody was mediated, at least in part, through promoting endotoxin signal pathway.
Characterisation of Rat Mesenteric Afferent Responses to Ligands Selective at Protease-Activated Receptor (PAR)-1, -2 and -4 Wen Jiang, Anthony J. Kirkup, Nigel Bunnett, David Grundy Introduction: Protease-activated receptors (PAR) are expressed throughout the rat gastrointestinal tract (Vergnofle, et al. Trends Pharmacol Sci. 22:146-52, 2001). The relative role of different PAR receptors in visceral hyperalgesia has not been determined. We aimed to utilized the selectivity of synthetic PAR activating peptides (PAR-AD TFLLRN-NH2, SLIGRLNH2 and AYPGKF-NH2 to investigate the effect of PAR1, 2 and 4-AP on rat small intestinal motor function and mesenteric afferent sensitivity. Methods: Extracellular recordings of jejunal afferent activity and intraluminal pressure were obtained from pentobarbitone sodium anaesthetized (60 mg/kg, i.p.) male Wistar rats. For dose response studies PAR-AP were administered iv into separated groups of animals at 1, 10, 30, 100, 300, and 10001xg/kg (at 5 rain interval). In other experiments, single doses of the PAR-AP were administered (lmg/kg, iv) aflowmg full recovery before proceeding to the next PAR ligand (min. lOmin interval). Data are the mean_+ SEM from 6-7 animals and were analyzed by unpaired Students' t-test or one-way ANOVA as appropriate. Results: All 3 PAR-AP evoked dose-dependent increase in afferent discharge (AD) with threshold between 30 to 100p,g/kg but different response profile (Fig. 1). Peak afferent firings to PAR1-AP are significantly higher than that to PAR2 and 4-AP (176_+20 vs 107_+ 12 and117_+ 16 spikes/s) whereas the duration of PAR2-AP afferent response is longer than that of PAR1- and 4-AP (207 _+21 vs. 67-+ 9 and 62 _+ 12s). PAR1 and 2-AP evoked a dose-dependent increases in intrafuminal pressure (LP), which in the case of PAR1-AP was preceded by a transient relaxation. At lmg/kg the increase m LP evoked by PAR1-AP was smaller and briefer comparing with the same dose of PAR2AP (1.9 _+03 vs. 5.7 _+1 3mmHg, 103 -+ 14 vs. 332 -- 81s). PAR4-AP did not significantly influence LP. Conclusion: PAR1, 2 and 4-AP are powerful stimuli for mesenteric afferent discharge with similar potency but different magmtude and ume-course of response and have different effects on intestinal motor activity. Supported by UK BBSRC and NIH DK57840.
Abstracts
25
$1783
$1782
AGA
60 30.
S1784 Platelet Function in Ischemia-reperfusion induced acute Pancreatitis Thilo Hackert, Jens Wemer, Wemer Hartwig, Dagmar Pfeil, Martha-Maria Gebhard, Markus W. Buechler, Waldemar Uhl Background: Post-transplantation pancreatitis is an important complication of pancreatic transplantation, lschemia-reperfusion associated microcirculatory changes play a major role in the pathogenesis of this complication. The pathophysiological role of platelets in theses events are unknown. Therefore, the aim of our study was to examine the role of platelet adhesion and function during early reperfusion in experimental complete iscbemia of the pancreas. Methods: 12 Wistar rats were subjected to warm pancreatic iscbem~a by crossclamping of the pancreatic vessels for one hour. Following one hour reperfusion, intravital microscopy was performed in 6 animals. Platelet-endothelium interaction was evaluated after platelet separation and staining with rhodamin 6G by flourescence microscopy regarding 3 capillary fields and 3 postcapillary venules. In the other 6 animals blood samples and pancreatic tissue were taken after 24 h of reperfusion to evaluate serum amylase and histological changes (edema, inflammation, necrosis). 12 additional animals served as controis, following the identical protocol vathout induction of ischemia. Results: 24 h after ischemia-reperfusion, animals showed a mild pancreatitls. Serum amylase was sigmficantly (p<0.05) higher than in control animals. As evaluated by intravital microscopy, adherent platelets in capillary fields and venules were significantly increased (p<0.01) and. platelet velocity in capillaries was siginficantly lower after ischemia-reperfusinn compared to control animals (p<0.05). Moreover, histological analysis showed significantly more edema formation and inflammation in these animals (p<0.05). Conclusion: Following lfi of warm ischemia and 24hrs of reperfusion, a mild pancreatins was observed in our model. Pancreatic microcirculation was characterized by pronounced platelet-endothelium interaction in capillaries and postcapiUary venules. These results show that platelet activation plays an important role in ischemia-reperfusion induced acute pancreatitis.
A-266
180 150.
~Tt~.
--.a-- Parl --.or--p at2
r-, 90 9 S1781
~
Degradation and Inactivation Of Plasma TNF~ By Pancreatic Proteases In Experimental Acute Pancreatitis Guido Alsfasser, Bozena Antoniu, Andrew L. Warshaw, Carlos Fernandez-Del-castillo
0 -30 9
50 75 tO0 Time course (a) Fig. I. Meaent~,.'icaft'wentdischargesin responmeto PAR1, 2 md 4-AP.
Hypothesis: TNFa is thought to play an important role in mediating systemic effects and progression of acute pancreatitis. However, TNFc~ levels in plasma of patients with acute pancreatitis (AP) are often unmeasurabfy low and many studies show contradictory findings. We hypothesize that TNFcr in plasma is susceptible to catabolism by circulating pancreatic proteases. Materials and Methods: In Vivo: Male Spragne-Dawley rats were divided into four groups: Healthy controls, sham operated controls, mild and severe pancreatitls. AP was induced by Cerulein hyperstimulation preceded by intraductal infusion of saline (mild) or glycodeoxychofic acid (severe). Plasma TNFc~ levels were measured at 0.5, 1, 2, 6, 12 and 24 h after induction of AP using a competitive EL1SA. In Vitro: i) Recombinant rat or human TNFa was incubated at 37~ with clinically relevant graded concentrations of trypsin, elastase and chymotrypsin for 30 rain and 24 h; 15% SDS PAGE gel-electrophoresis was performed to visualize TNF degradation. 2) TNF bioactivity was evaluated with a cytotoxicity assay using SYTOX Green stain to detect TNF induced cell death in mouse fibroblast-derived WEHI 13var cells. Results: In Vivo: TNFa level in healthy animals was 4.8+/-2.4 ng/ml. At 0.5 h levels were 10.53+/-1.34 ng/ml, 7.26+/-1.75 ng/ml and 3.95+/-1.56 in sham operated controls, mild and severe pancreatitis, respectively. The levels in severe pancreatitis were significantly lower than in sham operated controls (p = 0.0327). This difference persisted up to 6 hours (4.01 +/-0.18 ng/ml vs. 7.54 +/-0.84 ng/ml, p = 0.0145). No difference was seen at other time points. In Vitro: 1) Undamaged TNFa formed a band of 78 kD, indicating a trimefic configuration. This band vanished after incubation with each of the proteofytic enzymes at all concentrations tested. 2) The ECs0 (dose required to induce death in 50% of cells) was 3ng for human TNFa, and 0.3 ng for rat TNFa. 30 min incubation of TNFa with 1 Izg/ml trypsin decreased bioactivity by 50% and 24 h incubation abolished it completely. Conclusion: TNFcr released during acute pancreatitis is probably degraded rapidly by circulating pancreatic proteases. The bioactivity of this degraded TNFa is likely to be significantly diminished or abolished. Whatever the local effect of TNFa in acute pancreatitis, we suggest that its enzymatic degradation limits any substantial role in systemic pathophysiology.
The Role of LBP in Animal Model of Severe Acute Pancreatitis Xing-Peng Wang, Li-Ying Wu Background/Aims: It is well known LPS was eligible to induce the transition from edematous pancreatitis to necrotizing pancreatitis. In order to elucidate the pathogenic effect ofendotoxin in severe acute pancreatitis, anti-LBP antibody was applied and the therapeutic effects were investigated in this study. Methods: Thirty-six BALB/c mice were randomly divided into SAP group and anti-LBP antibody group. SAP was induced by admmistration of seven intraperitoneal injections of cemlein (50 microg/kg) at hourly intervals. Lipopolysaccharide (LPS) was injected 6 hours after the first cerulein injection. Treatment with the anti-LBF antibody was started 15 minutes before the LPS injection. Serum levels of amylase and lactate dehydrogenase (LDH) were measured at the end of the experiments. The severity of pancreatitis was evaluated by histological sconng using a semi-quantitative analysis of hematoxylin and eosin-stained sections of the pancreas. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed to quantify the intrapancreatic TNF-alpha IL-l beta ICAM-1 and E-selectin mRNA levels. The expressions of nuclear factor NF-kB and polymorphonuclear leukocyte (PMN)-specific antigen Mac-1 were investigated by the method of immunohistochemistry and western blotting. The activity of PMN myeloperoxidase (MPO) was determined by zymohistochemistryResults: Seven times cerulein rejection induced edematous pancreatins, however when LPS was added, necrotizing pancreatitis developed. At 9, 12, 24 hours after the first cerulein injection, the blood and specimens of pancreas were harvested. A marked elevation of serum amylase at 12 hours and LDH at 24 hours was observed in anti-LBF group. Histologically, treatment of anti-LBP group increased the severity of pancreatic injury including edema at 9 and 12 hours, inflammatory cell infiltration at 9,12 and 24 hours and necrosis at 9,12 and 24 hours. These changes were accompanied by the up-regulation of tumor necrosis factor (TNF)-alpha, interleukin-lbeta (IL-lbeta), ICAM-1 and E-selectin mRNA expressions in the pancreas compared with SAP group. There was significant increase in MPO activity at 12 and 24 hours in anti-LBP antibody group. Immunohistochemistry and western blotting showed up-regulation of nuclear factor NF-kB and Mac-1 at 9 hours in anti-LBP antibody group. Conclusions: The aggravating effect of the anti-LBP antibody was mediated, at least in part, through promoting endotoxin signal pathway.
Characterisation of Rat Mesenteric Afferent Responses to Ligands Selective at Protease-Activated Receptor (PAR)-1, -2 and -4 Wen Jiang, Anthony J. Kirkup, Nigel Bunnett, David Grundy Introduction: Protease-activated receptors (PAR) are expressed throughout the rat gastrointestinal tract (Vergnofle, et al. Trends Pharmacol Sci. 22:146-52, 2001). The relative role of different PAR receptors in visceral hyperalgesia has not been determined. We aimed to utilized the selectivity of synthetic PAR activating peptides (PAR-AD TFLLRN-NH2, SLIGRLNH2 and AYPGKF-NH2 to investigate the effect of PAR1, 2 and 4-AP on rat small intestinal motor function and mesenteric afferent sensitivity. Methods: Extracellular recordings of jejunal afferent activity and intraluminal pressure were obtained from pentobarbitone sodium anaesthetized (60 mg/kg, i.p.) male Wistar rats. For dose response studies PAR-AP were administered iv into separated groups of animals at 1, 10, 30, 100, 300, and 10001xg/kg (at 5 rain interval). In other experiments, single doses of the PAR-AP were administered (lmg/kg, iv) aflowmg full recovery before proceeding to the next PAR ligand (min. lOmin interval). Data are the mean_+ SEM from 6-7 animals and were analyzed by unpaired Students' t-test or one-way ANOVA as appropriate. Results: All 3 PAR-AP evoked dose-dependent increase in afferent discharge (AD) with threshold between 30 to 100p,g/kg but different response profile (Fig. 1). Peak afferent firings to PAR1-AP are significantly higher than that to PAR2 and 4-AP (176_+20 vs 107_+ 12 and117_+ 16 spikes/s) whereas the duration of PAR2-AP afferent response is longer than that of PAR1- and 4-AP (207 _+21 vs. 67-+ 9 and 62 _+ 12s). PAR1 and 2-AP evoked a dose-dependent increases in intrafuminal pressure (LP), which in the case of PAR1-AP was preceded by a transient relaxation. At lmg/kg the increase m LP evoked by PAR1-AP was smaller and briefer comparing with the same dose of PAR2AP (1.9 _+03 vs. 5.7 _+1 3mmHg, 103 -+ 14 vs. 332 -- 81s). PAR4-AP did not significantly influence LP. Conclusion: PAR1, 2 and 4-AP are powerful stimuli for mesenteric afferent discharge with similar potency but different magmtude and ume-course of response and have different effects on intestinal motor activity. Supported by UK BBSRC and NIH DK57840.
Abstracts
25
$1783
$1782
AGA
60 30.
S1784 Platelet Function in Ischemia-reperfusion induced acute Pancreatitis Thilo Hackert, Jens Wemer, Wemer Hartwig, Dagmar Pfeil, Martha-Maria Gebhard, Markus W. Buechler, Waldemar Uhl Background: Post-transplantation pancreatitis is an important complication of pancreatic transplantation, lschemia-reperfusion associated microcirculatory changes play a major role in the pathogenesis of this complication. The pathophysiological role of platelets in theses events are unknown. Therefore, the aim of our study was to examine the role of platelet adhesion and function during early reperfusion in experimental complete iscbemia of the pancreas. Methods: 12 Wistar rats were subjected to warm pancreatic iscbem~a by crossclamping of the pancreatic vessels for one hour. Following one hour reperfusion, intravital microscopy was performed in 6 animals. Platelet-endothelium interaction was evaluated after platelet separation and staining with rhodamin 6G by flourescence microscopy regarding 3 capillary fields and 3 postcapillary venules. In the other 6 animals blood samples and pancreatic tissue were taken after 24 h of reperfusion to evaluate serum amylase and histological changes (edema, inflammation, necrosis). 12 additional animals served as controis, following the identical protocol vathout induction of ischemia. Results: 24 h after ischemia-reperfusion, animals showed a mild pancreatitls. Serum amylase was sigmficantly (p<0.05) higher than in control animals. As evaluated by intravital microscopy, adherent platelets in capillary fields and venules were significantly increased (p<0.01) and. platelet velocity in capillaries was siginficantly lower after ischemia-reperfusinn compared to control animals (p<0.05). Moreover, histological analysis showed significantly more edema formation and inflammation in these animals (p<0.05). Conclusion: Following lfi of warm ischemia and 24hrs of reperfusion, a mild pancreatins was observed in our model. Pancreatic microcirculation was characterized by pronounced platelet-endothelium interaction in capillaries and postcapiUary venules. These results show that platelet activation plays an important role in ischemia-reperfusion induced acute pancreatitis.
A-266