An in vitro study of the influence of plasma protease inhibitors and aprotinin on trypsin-induced C3 cleavage in human serum.

Cleavage of native C3 in human serum following the addition of increasing amounts of human cationic trypsin was studied using an in vitro model. The cleavage was correlated with the degree of saturation of the plasma protease inhibitors alpha 2-macroglobulin and alpha 1-antitrypsin, and also with varying amounts of aprotinin (Trasylol). When alpha 2-macroglobulin reached 70% saturation, there was a prompt cleavage of most of the native C3 in spite of 90% free alpha 1-antitrypsin. The consumption of alpha 2-macroglobulin, and especially of alpha 1-antitrypsin, decreased with increasing concentrations of aprotinin. Aprotinin was needed in a concentration of 60 microM to block trypsin-induced C3 cleavage almost completely. This concentration is about 20 times higher than ever used clinically. One possible explanation of the poor clinical effect of aprotinin to date in human pancreatitis is the need for this high concentration.

Cultured blood T-cell responses predict anti-TNF therapy response in patients with ulcerative colitis.

BACKGROUND: Anti-tumour necrosis factor (TNF) therapy is used for treatment of ulcerative colitis (UC). As approximately 30% of patients with UC do not benefit from the treatment, it is of clinical interest to identify biomarkers of response before therapy is initiated. AIM: To identify prognostic biomarkers of anti-TNF therapy response in anti-TNF therapy-naïve patients with UC. METHODS: Peripheral blood cells were obtained from 56 patients with UC before therapy started. Thirty-four patients were included in an exploratory cohort and 22 patients in a validation cohort. Blood cells were stimulated in vitro with influenza vaccine with and without anti-TNF. T-cell surface receptor expression and cytokine release were determined (in total 17 variables). Treatment response was evaluated using the Mayo score 12-14 weeks after the first infusion. RESULTS: In the exploratory cohort, blood cells from the patients showed stronger anti-TNF-dependent suppression of T-cell surface receptor expression and cytokine secretion among therapy responders than nonresponders. In particular, anti-TNF suppressed the expression of CD25 on T cells and secretion of interleukin 5, to a higher degree in responders than in nonresponders. These variables were used to a create model to predict therapy outcome, which was confirmed in the validation cohort. Correct classification of future therapy response was achieved in 91% of the cases in the validation cohort. CONCLUSION: The effects of anti-TNF on cultured blood T cells, obtained before therapy started, predict treatment outcome in patients with UC.

The involvement of multiple protease-antiprotease systems and gut origin sepsis in zymosan-associated endothelial barrier injury and multiple organ dysfunction in rats.

Multiple organ dysfunction syndrome is a dominant cause of mortality in the intensive care unit. Experimentally, a condition similar to the multiple organ dysfunction syndrome can be induced by the intraperitoneal injection of sterile zymosan. In the present study we investigate potential alterations in multiple organ functions, endothelial permeability, and antiproteinases after intraperitoneal injection of zymosan at various doses. Zymosan-induced generalized inflammation lead to endothelial barrier injury in multiple organs/tissues, a decrease in systemic arterial pressure, impaired organ function and gut defence function, and consumption of protease inhibitors, particularly the consumption of alpha2 antiplasmin. Endothelial barrier injury appears to present a dose- and organ-dependent pattern in multiple organs/tissues, and the increase in endothelial barrier permeability occurred prior to organ dysfunction. Zymosan induced the development of multiple organ dysfunction syndrome, probably initiating multiple protease-antiprotease systems, particularly the fibrinolytic system, leading to endothelial barrier injury, tissue edema, parenchymal cell damage, and eventual organ dysfunction, potentially augmented by a secondary bacterial infection.

The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.

Intestinal ischemia and reperfusion injury (I/R) is probably involved in the pathogenesis of intestinal barrier dysfunction, associated with the concomitant translocation of enteric bacteria and toxins and the potential development of multiple organ failure. The intestinal endothelial and epithelial layers play a major role preventing the entry of toxic substances from the gut, but the influence of protease-antiprotease systemic balance on these barrier functions and the relationship between epithelial DNA synthesis, apoptosis, and endothelial and epithelial barrier macromolecule permeability are not fully investigated. Endothelial and epithelial barrier macromolecular permeability, epithelial DNA synthesis, the endothelial and epithelial plasma membrane system, apoptosis and oncosis, plasma levels of proteinase inhibitors, and proenzymes were measured in rats subjected to 20 and 40 min intestinal ischemia and 1, 3, 6, or 12 h reperfusion. Endothelial permeability increased after both 20 and 40 min intestinal ischemia. Epithelial permeability significantly increased during 1-6 h reperfusion after 20 min ischemia and during 1-12 h reperfusion after 40 min ischemia. Epithelial DNA synthesis increased in animals with 20 min ischemia followed by 12 h reperfusion. Plasma levels of prekallikrein, C1-esterase inhibitor, and alpha1-macroglobulin were significantly lower following both 20 and 40 min ischemia from 3 h reperfusion and on. Apoptotic epithelial cells significantly increased in animals subjected to 20 min ischemia followed by 12 h reperfusion. The severity of reperfusion injury in the intestinal endothelial and epithelial barrier seems to correlate with the period of ischemia and the pathway of cell damage and death, together with proteinase-antiproteinase imbalance.

Proteases and protease inhibitor balance in peritonitis with different causes.

Protease activation and protease-antiprotease interactions were sequentially studied in two different groups of patients with peritonitis. The biochemical changes were related to the cause of the disease and to the clinical course. Protease activation and protease inhibitor consumption were most pronounced in the peritoneal fluid, especially in bacterial peritonitis. Plasma changes also indicated activation of the complement, kinin, and fibrinolytic systems and protease inhibitor consumption, especially of alpha 2-macroglobulin and antithrombin III. There was no significant difference between chemical and bacterial peritonitis regarding these plasma changes. Immunohistologic studies showed evidence of active uptake of protease-antiprotease complexes in macrophage-like cells in the peritoneum in both groups. It is concluded that peritonitis results in protease activation and protease inhibitor consumption, especially in the peritoneal fluid. The peritoneum has an active role in the clearance of protease-antiprotease complexes. The intensity, not the type, of the intra-abdominal challenge determines the biochemical changes in peritonitis.

Ipsilateral multiple groin hernias.

BACKGROUND: Recurrence rates after surgical repair of groin hernia vary between 3% and 20%. One possible reason for recurrent hernias are ipsilateral multiple hernias, which might have been overlooked at the primary operation. METHODS: In the present series 1010 patients with unclear groin pain underwent herniography. RESULTS: A total of 314 patients had hernias, and seventy-one (23%) of these had multiple hernias. Ipsilateral multiple hernias were found in 18 (6%) patients. Ipsilateral multiple hernias were present in 9 (6%) of 144 patients with an indirect hernia, in 17 (12%) of 144 patients with a direct hernia, in 5 (21%) of 24 patients with a femoral hernia, and in 3 (23%) of 13 patients with an obturator hernia. The hernias were of indirect, direct, femoral, and obturator types. CONCLUSIONS: The frequency of ipsilateral multiple hernias is much higher than the frequency reported during herniorrhaphy. Such overlooked ipsilateral multiple groin hernias may account for some of the so-called recurrences after herniorrhaphy. Therefore a careful exploration of the groin is mandatory. Preoperative herniography may also prove to be useful in patients with recurrent groin symptoms after herniorrhaphy.

Consumptive coagulopathy, fibrinolysis and protease-antiprotease interactions during acute human pancreatitis.

Twenty-seven attacks of acute human pancreatitis of different severity were analysed concerning clinical outcome and activation of the coagulation and fibrinolytic systems. Consumptive coagulopathy was suggested by decreased platelet counts, decreased prothrombin values and consumption of fibrinogen during the first days in severe attacks. Factor X was slightly decreased the first 5 days in all attacks. Increased fibrinolysis was suggested by decreased plasminogen values in severe attacks. Fibrinogen degradation products were seen in 40% of the patients in blood and in 100% of the patients in the peritoneal fluid. The four main protease inhibitors of the two systems all showed protease-antiprotease complexation and lower functional than quantitative values. Plasma levels of antithrombin III and alpha 2-macroglobulin were low, while the levels of C1-inhibitor and alpha 2-antiplasmin were high. Functional levels of all the four protease inhibitors were almost zero in the peritoneal fluid in severe attacks. It is concluded that severe acute pancreatitis results in both consumptive coagulopathy and in increased fibrinolysis. A local antiprotease deficiency is seen in the peritoneal cavity and high levels of protease-antiprotease complexes are also seen in plasma. All these changes are closely correlated to the severity of the disease and may probably determine the clinical outcome of the acute attack.

Changes in the kallikrein kinin system during acute pancreatitis in man.

Changes in the kallikrein-kinin system were analysed in 19 attacks of acute pancreatitis in man and correlated to the severity and clinical course of the disease. Prekallikrein, kininogen and kallikrein inhibition were significantly lower in blood in severe attacks than in moderate or mild attacks. These changes were even more pronounced in peritoneal fluid, where kallikrein activity was above normal, while kininogen and kallikrein inhibition were nil in severe attacks. Both high and low molecular weight kininogen were decreased, denoting an activation also by kininogenases other than plasma kallikrein. These changes indicate an activation of the kallikreinkinin system in acute severe pancreatitis in man, especially in the abdominal cavity. Although there is a great deal of evidence for activation of the kinin system in experimental shock states in animals (1-4), there are to date rather few studies showing that such an activation takes place in human acute pancreatitis. This lack of clinical studies is mainly explained by the difficulty in measuring activated components of the system, especially since these components are rapidly inactivated in different ways in vivo (5).

Serum levels of immunoreactive PSTI in acute abdominal disorders, with special reference to a possible extrapancreatic PSTI production.

The concentration of the pancreatic secretory trypsin inhibitor (PSTI) was measured in serum from 360 patients with acute abdominal diseases. Elevated levels were found in acute pancreatitis, cholangitis, choledocholithiasis, acute cholecystitis, pancreatic pseudocyst, malignancy, renal failure and in several different inflammatory conditions not connected with the pancreas. The results suggest the possibility of an extra-pancreatic production of PSTI, especially since the changes seen over time do not favour leakage or reabsorption as the cause of the high PSTI levels seen in acute pancreatitis. PSTI rather behaves as an acute phase reactant, as judged from the parallelism in the reaction pattern with antichymotrypsin.

Pre-operative plasma levels of C-reactive protein, albumin and various plasma protease inhibitors for the pre-operative assessment of operability and recurrence in cancer surgery.

Pre-operative levels of the acute phase protein C-reactive protein (CRP), albumin (assessing nutritional status), the tumour marker CEA and three plasma protease inhibitors, i.e. C1-esterase inhibitor, alpha-2-macroglobulin and antithrombin III, were prospectively studied in 183 patients with various solid cancers. First, the predictive value of abnormal levels for operability at the primary operation was studied. Secondly, the predictive value of abnormal levels for cancer recurrence and metastases was evaluated during 2 years of follow-up. The results show that malignancy induces increased CRP and C1-esterase inhibitor levels and decreased albumin levels in serum. These changes, as well as raised alkaline phosphatase and lowered haemoglobin levels, also correlate to the 'overall' tumour burden. The most important conclusion is, that increased pre-operative CRP levels (CRP > or = 10 mg/l; sensitivity, 79%; specificity, 71%) and/or low albumin levels (albumin <37 g/l; sensitivity, 94%; specificity, 54%) are seen in inoperable cancer patients compared with patients having operable cancers. The second main important conclusion is, that high pre-operative C1-esterase inhibitor levels (C1-esterase inhibitor >152%; sensitivity, 45%; specificity, 90%), and in some patients a high alkaline phosphatase level, are seen in patients exhibiting early cancer recurrence (within 2 years post-operatively).

Gut barrier permeability, reticuloendothelial system function and protease inhibitor levels following intestinal ischaemia and reperfusion--effects of pretreatment with N-acetyl-L-cysteine and indomethacin.

BACKGROUND: Pathophysiological mechanisms and ways to intervene on intestinal barrier dysfunction following small intestinal ischaemia and prolonged reperfusion are still not fully clarified. AIMS: To evaluate the effect of oxygen free radical and prostaglandin inhibition on intestinal barrier injury following intestinal ischaemia/reperfusion. METHODS: Endothelial and epithelial barrier permeability was evaluated by clearance of radiolabelled albumin. Parameters included 125I-Escherichia coli uptake rate index, host reticuloendothelial system function and organ distribution, as well as protease inhibitor and proenzyme activities in rats subjected to small intestinal ischaemia for 40 minutes followed by 12 hours reperfusion (ischaemia/reperfusion), pretreated with N-acetyl-L-cysteine or indomethacin. RESULTS: Following ischaemia/reperfusion, endothelial and epithelial permeability increased, reticuloendothelial system activation occurred and plasma protease inhibitors were consumed. N-acetyl-L-cysteine pretreatment resulted in improved endothelial and epithelial barrier integrity, a decrease in protease inhibitor consumption and less pronounced reticuloendothelial system activation. Pretreatment with indomethacin was not effective. CONCLUSION: Oxygen free radicals seem to play an important role in the development of intestinal barrier impairment following ischaemia/reperfusion. N-acetyl-L-cystine may be a potential agent for preventing ischaemia/reperfusion damage.

Femoral hernia: clinical significance of radiologic diagnosis.

UNLABELLED: A retrospective study of 18 patients with femoral hernia assessed by herniography is presented. Although a palpable lump was present in 11 patients (61%), the diagnosis of a femoral hernia was not made before herniography. Surgical exploration was performed in 12 patients and a femoral hernia was found and repaired with beneficial outcome in 9 of them. IN CONCLUSION: herniography is of value for the diagnosis of a femoral hernia in patients with obscure groin pain.

Kinin activation and protease inhibitors in acute pancreatitis in man.

The changes in the protease inhibitors and in the kallikrein-kinin system were analysed in 19 attacks of acute pancreatitis in man and correlated to the severity and clinical course of the disease. Functional alpha 2-macroglobulin was 5% in peritoneal fluid and 32% in blood in severe attacks. Functional Cl inactivator was zero in the peritoneal fluid, while values were normal in blood. High levels of complexes between alpha 1-proteinase inhibitor and trypsin were found during the first 6 days of illness in severe attacks, especially in the peritoneal fluid. Prekallikrein, kininogen and kallikrein inhibition were significantly lower in blood in severe attacks than in moderate or mild attacks. These changes were even more pronounced in the peritoneal fluid, where kallikrein-like activity was above normal, while kininogen and kallikrein inhibition were zero in severe attacks. Both high and low molecular weight kininogen were decreased, denoting an activation also by kininogenases other than plasma kallikrein. In conclusion, kinin activation was demonstrated in acute pancreatitis, especially in the peritoneal fluid. Kinin activation and protease inhibitory activity were closely correlated to the severity and clinical course of the disease. Tryptic activation of the kinin system seems probable at the low alpha 2-macroglobulin levels found in severe attacks, according to our earlier in vitro studies.

On the potential role of trypsin and trypsin inhibitors in acute pancreatitis.

The protective role of alpha 2-macroglobulin, alpha 1-antitrypsin and Aprotinin against trypsin-induced effects on C3 and kininogen was studied in a human in vitro model. When human cationic trypsin was added to human serum or plasma, there was a gradual saturation of alpha 2-macroglobulin and later of alpha 1-antitrypsin. When alpha 2-macroglobulin was 70% saturated, there was a prompt cleavage of both C3 and kininogen, in spite of 80% free and active alpha 1-antitrypsin. These biochemical changes and antiprotease levels are identical to our findings in patients with acute pancreatitis, especially in their peritoneal exudate. Very high concentrations of Aprotinin, 5-15 times higher than ever used clinically, blocked the cleavage of both C3 and kininogen, while doses commonly used clinically were without significant effect. The clinical implications are: A trypsin-induced activation of both the complement and kinin system with clinical consequence is possible in patients with acute pancreatitis because of very low alpha 2-macroglobulin levels. Aprotinin in adequate doses, 5-15 times higher than ever used clinically, seems to protect against activation of two systems.

Acute pancreatitis in man. A clinical and biochemical study of pathophysiology and treatment.

The correlation between clinical course, protease inhibitors, complement and kinin activation was studied in vivo in 27 attacks of acute pancreatitis and also in vitro. The value of peritoneal lavage was retrospectively analyzed in 73 pancreatitis attacks. The effect of aprotinin was studied in vitro. Complement and kinin activation occurred in vitro when alpha-macroglobulin (alpha 2-M) concentration was below 35%, in spite of 90% free and reactive alpha 1-protease inhibitor. These low alpha 2-M levels were found in the general circulation in severe attacks. Functional alpha 2-M levels were lower than electroimmunoassay values during the acute disease. Complement and kinin activation was present both in blood and in peritoneal fluid. The extent of activation was closely correlated to the severity of the pancreatitis attack. Classical as well as alternative complement activation occurred. Kinin activation was caused by plasma kallikrein as well as by other kininogenases. Functional kallikrein inhibition was lower than electroimmunoassay values for the C1 inactivator. High levels of activated trypsin was found in complex with alpha 1-protease inhibitor in severe attacks. These findings together with the low functional alpha 2-M and a possible functional deficiency also of the C1 inactivator make trypsin-induced activation of the complement as well as the kinin system possible in acute pancreatitis. Changes in proteases and protease inhibitors were most pronounced in the peritoneal fluid, functional alpha 2-M and C1 inactivator being zero, indicating that the primary event occurs locally in and around the pancreas. Peritoneal lavage thus ought to be beneficial. The good results achieved with peritoneal lavage in the retrospective analysis of the 73 clinically severe attacks seem to verify this conclusion. The biochemical changes seen in vivo indicate alpha 2-M and C1 inactivator to be most important against proteolytic activation of the complement and kinin systems. Treatment with purified protease inhibitors might be beneficial. This also seems to be true of aprotinin, according to the in vitro results, provided very high doses are used. All antiprotease therapy seems to be most important intraperitoneally.

Disseminated intravascular coagulation and antiprotease activity in acute human pancreatitis.

The coagulation and fibrinolytic systems were analysed parallel to the clinical evaluation in 27 attacks of acute human pancreatitis of different severity. Consumptive coagulopathy was evident from decreased platelet counts, decreased prothrombin values and consumption of fibrinogen during the first days in severe attacks. Fibrinolysis was suggested by decreased plasminogen values and the presence of fibrinogen degradation products. All main protease inhibitors of the two systems showed protease-antiprotease complexation and lower functional than quantitative values. Functional levels of the protease inhibitors were almost zero in the peritoneal fluid in severe attacks. It is concluded that severe acute pancreatitis results in consumptive coagulopathy and fibrinolysis together with a local antiprotease deficiency. All the changes are closely correlated to the severity of the disease.