Human pancreatitis and the role of cathepsin B.
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Acute Disease↗
Biomedical subjects
W Halangk
Publications and source records attributed to W Halangk.
Enzymatic and histological alterations in the isolated perfused rat pancreas under conditions of oxidative stress.
BACKGROUND: Oxidative stress is a relevant event in the pathogenesis of acute pancreatitis. Investigations in vivo are limited because of the complexity of the organism and the short half-life of free radicals. The isolated perfused rat pancreas could be useful for investigations in the early phase of acute pancreatitis especially under conditions of oxidative stress. METHODS: External perfusions of the pancreatic glands of Wistar rats were carried out using a modified Krebs-Ringer buffer including an additive of the detergents Triton X-100 and a perfusion including hydrogen peroxide (0.0012%) or tert-butylhydroperoxide (0.0042%) or xanthine oxidase (0.1 U/ml). Changes in amylase, lipase, LDH in the portal outflow fluid and for histological alterations were analyzed. RESULTS: Damage to pancreatic parenchyma using Triton X-100 was indicated by increased levels of pancreatic enzymes in the perfusion medium. During perfusion with hydrogen peroxide or tert-butylhydroperoxide we found no changes in pancreatic enzymes in the portal outflow. In contrast, perfusion with xanthine oxidase induced a significant elevation in lipase and amylase in the effusion fluid after 30 min. We found a significant increase in edema in the hydrogen peroxide and in the xanthine oxidase group. Focal necroses of the pancreatic parenchyma were detected in all groups of oxidative stress. CONCLUSIONS: The isolated perfused rat pancreas is a valuable experimental model for investigating the early phase of pathophysiology in acute pancreatitis, for instance, the effect of oxidative stress as an early event in acute pancreatitis. Using hydrogen peroxide tert-butylhydroperoxide or xanthine oxidase, only xanthine oxidase was able to induce a typical elevation in the pancreas enzymes in the effusion fluid.
Acute Disease↗
Prevention and treatment of peritoneal carcinomatosis in experimental investigations with CPT-11 and oxaliplatin.
Following surgical resection of colorectal carcinoma, local recurrence in the tumor bed or in the mesentery remains a frequently encountered problem. Currently there are no recognized standard therapy protocols for the prevention of local recurrence or the treatment of peritoneal carcinomatosis. The aim of our trial was to investigate whether CPT-11 and oxaliplatin could decrease i.p. tumor growth in a basic experimental animal model. Experiments were performed on three groups of animals plus controls. In the first group, the cytostatic agents were applied directly following tumor cell implantation into the peritoneal cavity. In the second group, early postoperative i.p. chemotherapy (days 5, 10 and 15 following surgery) was administered. In the third group, late i.p. chemotherapy (days 15, 20 and 25 after tumor cell transfer) was administered with the intention of reducing a manifest peritoneal carcinomatosis. The trial also set out to describe any side effects observed following i.p. administration. The results indicated that CPT-11 and oxaliplatin were highly effective in reducing i.p. tumor spread after direct i.p intraoperative application. Intraperitoneal administration of CPT-11 or oxaliplatin also decreased i.p. tumor growth after early i.p. chemotherapy. CPT-11 was a little more effective with lower side effects. However, it was clear that it was not possible to treat a manifest peritoneal carcinomatosis in this way.
Anesthesia↗
Mutation in the SPINK1 trypsin inhibitor gene, alcohol use, and chronic pancreatitis.
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Alcoholism↗
A proteinase inhibitor decreases tumor growth in a laparoscopic rat model.
BACKGROUND: The balance between proteolysis and protease inhibition in the formation and breakdown processes of the extracellular matrix plays a major role in tumor cell invasion. An understanding of this relationship gave rise to the therapeutic concept of lowering tumor cell invasion by inhibiting protease activity. Phosphoramidon is an unspecific proteinase inhibitor. This experimental study investigated the effect of intraperitoneal phosphoramidon administration on tumor growth in a laparoscopic rat model. METHODS: In the first phase of the study, we investigated the influence of phosphoramidon on tumor cell invasion in a collagen matrix gel chamber in vitro. In a second experiment, a suspension of colon carcinoma cells (CC531) was introduced into the peritoneal cavity of male WAG rats. Prior to laparoscopy (at 6 mmHg for 20 min), the animals were randomized to two groups. At the start of laparoscopy, the test substance was applied intraperitoneally (group 1: controls, 1 ml 0.9% NaCl; group 2: 250 mg phosphoramidon in 1 ml 0.9% NaCl). Three weeks after the injection of tumor cells, the animals were autopsied and the tumor mass determined. RESULTS: In comparison with the control group (tumor weight 7.42 +/- 1.01 g), intraperitoneal tumor growth in the experimental group was significantly (p < 0.001) reduced by the application of phosphoramidon (tumor weight, 3.22 +/- 1.06 g). Phosphoramidon also significantly (p < 0.05) reduced tumor cell invasion through the matrix gel. CONCLUSION: The proteinase inhibitor phosphoramidon reduced tumor cell invasion in vitro and tumor cell growth in vivo in this laparoscopic rat model.
Animals↗
Prophylaxis of peritoneal carcinomatosis in experimental investigations.
BACKGROUND AND AIMS: We compared the effectiveness and side effects of various cytostatic agents for use in perioperative intraperitoneal irrigation to prevent peritoneal carcinomatosis. METHODS: The adenocarcinoma cell line CC-531 was implanted during laparotomy at the mesenterial trunk of anesthetized male WAG rats. Direct perioperative intraperitoneal chemotherapy was performed after 5 min with either 5-fluorouracil, cisplatin, or mitomycin; controls received only tumor cells. The animals were inspected daily over 30 days for side effects. They were then killed, and the greater omentum and mesentery were resected, the tumor mass was examined for the presence of peritoneal carcinomatosis, and tumor nodules in the greater omentum and mesentery were counted. RESULTS: All the animals in the control group developed histologically confirmed peritoneal carcinomatosis. Animals receiving cisplatin or mitomycin by direct intraperitoneal perioperative chemotherapy showed no macroscopic or histological evidence of tumor growth. Two animals in the fluorouracil group had macroscopically and histologically manifest tumor growth; another animal showed only histological evidence of malignancy. Substantial side effects were noted in the cisplatin group, with all animals experiencing bleeding in the peritoneum and toxic necrotic reactions of the colon; two animals died of these side effects. CONCLUSION: Direct intraperitoneal chemotherapy with cisplatin or mitomycin prevents peritoneal carcinomatosis in experimental investigations.
Adenocarcinoma↗
Differential oxidative injury in extrapancreatic tissues during experimental pancreatitis: modification of lung proteins by 4-hydroxynonenal.
Oxidative stress is considered to be a pathogenic factor for multisystem organ failure during acute pancreatitis. Infusion of 3% and 5% sodium taurocholate into the pancreatic duct of rats resulted in a 24-hr lethality of 8% and 82%, respectively. Kidney tissue showed a long-lasting significant elevation of malondialdehyde (lipid peroxidation). Only small amounts of this aldehyde were formed in the liver. In the lung malondialdehyde was increased during the first 6 hr after pancreatitis induction. Malondialdehyde levels were not different for pancreatitis initiated by 3% or 5% taurocholate. Protein-bound carbonyls (protein oxidation) in the tissues were not significantly changed at any time point. However, after infusion of 5% taurocholate, lung proteins were oxidatively modified by the product of lipid peroxidation, 4-hydroxynonenal. Another parameter characteristic for pancreatitis with high lethality was the high number of neutrophils in the lungs. We conclude that oxidative stress is important for the injury of extrapancreatic tissues during pancreatitis, but survival is determined by the degree of systemic inflammation.
Aldehydes↗
[Enzymatic and histological alterations in the isolated perfused rat pancreas in the taurocholate and cerulein model of acute pancreatitis].
METHODS: The pancreas of 24 male Wistar rats was perfused extracoporally by modified Krebs-Ringer-buffer for 80 minutes (including a 20 minutes equilibration period). To verify any organ damage we measured the activity of pancreatic enzymes like amylase, lipase and lactatdehydrogenase in the portal effluent. Furthermore histological changes were analysed after perfusion. Organ damage was induced by adding cerulein in a physiological dose (10(-10) M, n = 6) and in a supramaximal dose (10(-8) M, n = 6) and by intraductal injection of taurocholate (3.5 %, n = 6). RESULTS: Already 10 minutes after stimulation with cerulein (10(-8) M) and after intraductal injection of taurocholate increased activities (p < 0.01) of amylase and lipase were measured in the portal effluent compared to the group without any treatment. Lactatdehydrogenase levels did not changed. Apart from marked oedema in both groups considerable zones of necrosis could be noticed especially in the taurocholate group. CONCLUSION: Our data suggest that the isolated perfused rat pancreas (IPRP) is a valuable experimental tool to verify pathophysiological changes in the early phase of acute pancreatitis (AP). Various established models of AP such as by cerulein hyperstimulation or intraductal injection of taurocholate, could be applied to the IPRP. We conclude that this method enlarges the spectrum of established experimental models of acute pancreatitis.
Acute Disease↗
[The isolated perfused rat pancreas - an experimental model for investigation the early events in the pathogenesis of acute pancreatitis].
METHODS: External perfusions of the pancreatic glands of Wistar-rats were done, using a modified Krebs-Ringer-Buffer (KRB). We looked for an elevation of amylase, lipase and lactate-dehydrogenase in the effusion fluid (portal outflow fluid). We investigated a normal perfusion (KRB, 60 minutes), a long term perfusion (KRB, 240 minutes) and a perfusion (60 minutes) including an additive of the detergents triton x-100 or the cholecystokinin analogue ceruletid (10(-8) M). RESULTS: An isolated external perfusion of a rat pancreas is possible without inducing any increase of parameters of damage such as amylase, lipase or lactate-dehydrogenase in the outflow medium. The perfusion time should be limited to 80 minutes including a 20 minutes equilibration period. A damage of pancreatic parenchyma is indicated by increased levels of pancreatic enzymes in the perfusion medium. Such damage can be induced by various noxious substances like detergents or cerulein, which has a significance in the pathophysiology of experimental acute pancreatitis. A significant increase (p < 0.01) of lactate-dehydrogenase, lipase and amylase was found 10, 20 and 30 minutes after an application of triton x-100. During a perfusion with the cholecystokinin analogue ceruletid (10(-8) M) we found an increase of lipase (p < 0.05) after 30 minutes and an increase of amylase (p < 0.05) after 50 minutes perfusion. CONCLUSIONS: The isolated perfused rat pancreas is a valuable experimental model to investigate the early phase of pathophysiology in acute pancreatitis.
Acute Disease↗
Real-time assessment of intraperitoneal tumor growth in a rat model using CEA immunoscintigraphy.
BACKGROUND: In most preclinical models, assessment of intraperitoneal tumor location and size require killing the animal. The dynamics of postoperative intraperitoneal tumor implantation and growth remain unclear. A noninvasive method allowing reliable in vivo, real-time assessment of tumor growth is desirable. METHODS: An intraperitoneal tumor homograft using cultured CC531 colorectal cells was created by laparotomy in 24 Wistar Albino Glaxo rats. Eight additional rats were used as controls. Then, 10 MBq technetium 99m-labeled anticarcinoembryonic antigen (anti-CEA) monoclonal antibodies were administrated intravenously and radioactivity uptake was measured by using extracorporeal gamma counting at various time points. Subsequently, the animals were killed for tumor weighting. In 2 more groups of 8 animals, real-time, repeated measures were performed. RESULTS: Correlation between gamma counting and tumor weight was highly significant (P <.001). The regression equation obtained by using the least squares method was: tumor weight (g) = 2.422 + 0.267 x counts. It was possible to obtain real-time tumor growth curves when repeated measurements of radioactivity were performed. At day 25, the predicted tumor weight was 8.49 +/- 0.76 g, the measured weight was 8.16 +/- 0.99 g. CONCLUSIONS: Immunoscintigraphic measurements with technetium 99m anti-CEA antibodies are highly correlated with tumor weight in this model. As opposed to other tumor graft models based on autopsy findings, real-time monitoring is possible. This will allow dynamic studies of intraperitoneal tumor implantation and growth and will reduce the number of animals used in further studies.
Animals↗
Randomized, placebo-controlled trial of lazaroid effects on severe acute pancreatitis in rats.
OBJECTIVES: To evaluate the therapeutic potential of lazaroids in severe necrotizing acute pancreatitis and to investigate the association between oxidative stress, protease activation, and local production of proinflammatory cytokines and the severity and lethality of the disease. BACKGROUND: Oxidative stress is a crucial factor in the pathophysiology of acute pancreatitis and its systemic complications. Treatment with antioxidants, however, failed to improve survival in most studies performed so far. Lazaroids are a novel class of antioxidants that potently protect pancreatic acinar cells against oxidant attack in vitro. DESIGN: Prospective, randomized, controlled experimental study. SETTING: University research laboratory. SUBJECTS: Seventy-five anesthetized male Wistar rats (300-350 g). INTERVENTIONS: Severe acute pancreatitis was induced by retrograde injection of 3.5% taurocholate-sodium into the common bile-pancreatic duct. Interventions were performed to mimic the clinical situation, including continuous intravenous fluid substitution and administration of lazaroids in a therapeutic protocol. Therapy was started 1 hr after injection of the bile salt by using three different lazaroids, lactated Ringer's solution (placebo), and methylprednisolone as a corticosteroid control (n = 15 in each group). All the substances were given by continuous intravenous infusion throughout the 20-hr trial period. MEASUREMENTS AND MAIN RESULTS: Pancreatic homogenates and ascites were analyzed for indicators of oxidative stress, antioxidants, proteases, and proinflammatory cytokines. Pancreatic edema, morphologic pancreatitis severity, and pancreatic histopathology also were assessed. All three lazaroids and methylprednisolone diminished pancreatic tumor necrosis factor-alpha concentrations. Lethality was 33% in the placebo group. Neither the lazaroids nor methylprednisolone influenced survival. The local pancreatic and peritoneal concentrations of lipid peroxidation products, antioxidants, and proteases did not differ among the five groups. Nonsurviving rats, however, had a higher total protease activity in the pancreas and higher concentrations of trypsinogen activation peptide in ascites, as compared with surviving animals. There were no differences between survivors and nonsurvivors with regard to variables of oxidative stress and cytokines. CONCLUSIONS: Lazaroid application under clinically relevant conditions (i.e., after induction of fulminant acute pancreatitis) does not influence lethality or biochemical variables relevant to this disease. Protease activation rather than oxidative stress or local pancreatic cytokine production is an important determinant of disease severity and survival in acute pancreatitis. In experimental studies evaluating novel therapeutics, the same strict criteria should be applied as in the human setting.
Animals↗
Adaptation of protein carbonyl detection to the requirements of proteome analysis demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria.
The key technique in proteome analysis is high-resolution two-dimensional (2D) electrophoretic separation of proteins from biological samples. This method combines isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Derivatization of protein carbonyls with 2, 4-dinitrophenylhydrazine (DNPH) and subsequent anti-dinitrophenyl (DNP) immunoblotting is widely used for the detection of oxidatively modified proteins. In previous studies on adapting this method to 2D electrophoresis the derivatization step was carried out before and after the 2D procedure, resulting in an altered spot pattern and high background staining, respectively. The aim of the present experiments was to develop a method for protein derivatization with DNPH between the IEF and the SDS-PAGE steps. Mitochondria were exposed to 10 min hypoxia and 5 min reoxygenation. After IEF using immobilized pH gradients the gel strips were incubated in DNPH/trifluoroacetic acid/SDS for 20 min and neutralized, and SDS-PAGE was performed. Proteins were either stained with Coomassie dye or subjected to Western blotting using anti-DNP IgG. Gels and blots were scanned and matched to a master gel, and the relative carbonyl content of each spot was calculated and compared for five experiments. Importantly, the spot patterns in DNPH-treated and untreated gels were not different. Protein carbonyls could be detected in 59 of the 125 matched spots. Although there was no significant increase in the total protein carbonyl content after hypoxia/reoxygenation, eighteen 2D spots exhibited an increase in carbonyl content. However, most protein spots did not show a change or even a decline (4 spots) in protein carbonyls.
Animals↗
The role of cysteine proteases in intracellular pancreatic serine protease activation.
Autodigestion by proteolytic enzymes is thought to represent the critical mechanism by which acute pancreatitis is initiated. Where and why pancreatic proteases, which are physiologically stored and secreted as inactive precursor zymogens, are activated within the pancreas has remained controversial. Here we present data which indicate that: the lysosomal protease cathepsin B can activate trypsinogen in vitro in a manner that is similar to trypsinogen activation by enterokinase; that cathepsin B colocalizes with trypsinogen in the secretory compartment of the rat pancreas and of the human pancreas; that trypsinogen activation begins in a secretory compartment that is distinct from mature zymogen granules; and that the inhibition of cathepsin B can either increase or decrease premature trypsinogen activation depending on the concentration of the inhibitor, its specificity and its site of action in the pancreatic acinar cell. These observations elucidate some of the complex relations between cysteine and serine proteases in the pancreas with respect to their mechanisms of activation, their subcellular sites of action, and their possible role in the onset of pancreatitis.
Acute Disease↗
Oxidative stress in lung tissue induced by CO(2) pneumoperitoneum in the rat.
BACKGROUND: Clinical trials have found that the pneumoperitoneum has potentially hazardous side effects. The biochemical basis of organ injury induced by pneumoperitoneum is, however, not well defined. Since oxidative stress is believed to play an important role in many pathological conditions, we set out to examine oxidative stress markers in the lung, liver, kidney, and pancreas by using a rat model of laparoscopy with CO(2) pneumoperitoneum and comparing it to a group with gasless laparoscopy. METHODS: Malondialdehyde (for lipid peroxidation), protein-bound carbonyls (for protein oxidation), reduced and oxidized glutathione, and the neutrophil marker myeloperoxidase were evaluated in tissue homogenates at 2 h, 6 h, and 18 h after laparoscopy. Immunoblotting was used to analyze the modification of lung proteins by 4-hydroxynonenal at 6 h. RESULTS: Significant lipid peroxidation was found selectively in lungs at 2 h and 6 h after CO(2) pneumoperitoneum. This was accompanied by a loss of glutathione but only minor protein oxidation. Further, lung proteins were clearly modified by the aldehydic product of lipid peroxidation 4-hydroxynonenal. Myeloperoxidase in lungs increased continuously up to 18 h in both experimental groups, but there were higher levels in the group with pneumoperitoneum. CONCLUSION: Oxidative stress is likely to contribute to the impairment of pulmonary function after laparoscopic operations using a CO(2) pneumoperitoneum.
Analysis of Variance↗
Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis.
Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb(-/-) animals was more than 80% lower than in ctsb(+/+) animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb(-/-) animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.
Acute Disease↗
Induction of permeability transition in pancreatic mitochondria by cerulein in rats.
Hyperstimulation with cholecystokinin analogue cerulein induces a mild edematous pancreatitis in rats. There is evidence for a diminished energy metabolism of acinar cells in this experimental model. The aim of this study was to demonstrate permeability transition of the mitochondrial inner membrane as an early change in mitochondrial function and morphology. As functional parameters, the respiration and membrane potential of mitochondria isolated from control and cerulein-treated animals were measured, and changes in volume and morphology were investigated by swelling experiments and electron microscopy. Five hours after the first injection of cerulein, the leak respiration was nearly doubled and the resting membrane potential was decreased by about 17 mV. These alterations were reversed by extramitochondrial ADP or did not occur when cyclosporin A was added to the mitochondrial incubation. A considerable portion of the mitochondria isolated from cerulein-treated animals was swollen and showed dramatic changes in morphology such as a wrinkled outer membrane and the loss of a distinct cristae structure. These data provide evidence for the opening of the mitochondrial permeability transition pore at an early stage of cerulein induced pancreatitis. This suggests that the permeability transition is an initiating event for lysis of individual mitochondria and the initiation of apoptosis and/or necrosis, as had been shown to occur in this experimental model.
Animals↗
Evaluation of UVA-mediated oxidative damage to proteins and lipids in extracorporeal photoimmunotherapy.
The combination of UVA and 8-methoxypsoralen (8-MOP) is known for the ability to produce reactive oxygen species (ROS) that react subsequently with DNA, lipids and proteins. In most studies concerned with UVA effects mediated by free radicals, UVA doses higher than those exhibiting beneficial clinical results in extracorporeal photoimmunotherapy (ECPI) were used. The present study was undertaken to determine markers of oxidative stress in plasma and cells from the buffy coat using conditions relevant for ECPI (cumulative UVA dose at the sample level < or = 2 J/cm2). Plasma exposed to UVA of 20 J/cm2 resulted in protein oxidation as well in crosslinking and fragmentation revealed by electrophoresis. Exposure of the buffy coat and plasma to considerably lower doses of UVA (up to 2 J/cm2) combined with various 8-MOP concentrations resulted neither in an increase of malondialdehyde as a marker of lipid peroxidation nor in a changed electrophoretic protein pattern. In these same experiments the total antioxidative capacity decreased to 65% of the initial value, suggesting that the antioxidative defense of plasma is able to cope with oxidative stress under ECPI conditions. These results were confirmed by data from 10 patients with scleroderma or cutaneous T-cell lymphoma during ECPI treatment. The present results suggest that, although ROS are formed during ECPI, gross oxidative damage does not occur. It is, however, possible, that specific effects mediated by oxygen radicals may co-trigger the photoimmunomodulatory effects of ECPI.
Blood Proteins↗
[Peritonitis models in the rat with special reference to laparoscopic therapy options].
With ongoing development of minimally invasive surgery the laparoscopic techniques are increasingly used also in patients with intra-abdominal inflammatory diseases. The systemic sequelae of laparoscopy are not yet clear. In this study, we investigated three experimental models of peritonitis in rats (cecal ligation and puncture n = 14, cecal stent peritonitis n = 14, implantation of a standardized fecal inoculum n = 18) concerning the therapeutic potential of laparoscopy, reproducibility of the results and clinical relevance (elimination of the inflammatory focus, intraperitoneal modulation of inflammation). The peritonitis model of implantation of a standardized fecal inoculum was shown to be the experimental model with the closest clinical relevance. In this technically simple, reproducible model, the primary inflammatory focus can be eliminated by laparoscopic lavage. A significant difference (p < 0.002) of survival between the groups with and without laparoscopic lavage was found. In addition, the intraperitoneal application of drugs is possible in order to modulate peritoneal and systemic inflammation.
Animals↗