Expression and localisation of kinin receptors in colorectal polyps.

Kinins increase vascular permeability as well as mitogenesis and proliferation, hence they have a potential to promote neoplasmatic transformation. In the present study we investigated the expression profile and localization of kinin B1 and B2 receptors in colorectal polyps. The biopsy samples from various polyps were obtained during endoscopy in tubular (n=18), villous (n=15) and hyperplasic polyps (n=15). The expression of genes encoding B1 and B2 was estimated by QRT-PCR TaqMan analysis. In second series B1 and B2 receptors were visualized by immunohistochemical staining in tissue specimens from colonic polyps and adjacent normal tissue. We found the highest expression of gene encoding B1 in tubular adenomas (1891 number of copies mRNA/microg total RNA+/-312 SE) which is significantly higher as compared with controls (683+/-197 SE, p<0.013). In contrast, the expression of gene for B2 was significantly increased in hyperplastic polyps (3852+/-936 SE) as compared with controls (843+/-263 SE, p<0.0016). In normal colon a well as in hyperplasic polyps B1 and B2 receptors were immunohistochemically localized in enterocytes, however in hyperplastic polyps the intensity of staining was more prominent for B2 comparing to the control group. In contrast, in tubular adenomas staining reaction for B1 was more intense than in control samples. Increased level of B1 in adenoma suggests that kinins may play a role in abnormal cellular transformation; whereas higher B2 level in hyperplasic polyp suggests its protective role. Our data may indicate that the overall effect of kinins on cellular proliferation depends on the relative level of B1 and B2 receptor expression.

Concentration profile of mRNA of kinine B1 and B2 receptors in the evaluation of colon pathologies.

Colon pathologies involve the activation of a number of inflammation mediators: cytokines, prostanoids and kinines. The expression patterns of the genes associated with their activation may provide a very sensitive marker of the physiological condition of the cells. This paper demonstrates certain statistically significant differences (p = 0.008) between the expression patterns of kinine B1 and B2 receptor encoding genes for colitis ulcerosa and a control group. The ratio of kinine B1/B2 concentrations changes significantly, on the average from 1.3 for a tissue assessed as healthy to 6.6 for colitis ulcerosa.

Expression of the histone H3 gene in the evaluation of cellular proliferation in colitis ulcerosa.

A comparison of the number of mRNA molecules of histone H3 in 1 microg total RNA extracted from colon sections sampled during colonoscopy was used to evaluate cellular proliferation activity in the rectum and sigmoid, in normal tissue and in colitis ulcerosa. Samples with similar intensity of the disease were selected for the study. Statistically significant differences between both groups of rectal sections were found in the expression of histone H3 encoding genes. The statistically significant result (p = 0.0485) indicates a more active division of cells in the healthy rectum, with no statistically significant differences in the sigmoid (p=0.9575).

Kininogens are antithrombotic proteins In vivo.

Kininogens have recently been shown to possess antiadhesive, anticoagulant, and profibrinolytic properties and can inhibit platelet activation at low thrombin concentrations. To test whether kininogens have antithrombotic properties in vivo, we devised a model of limited arterial injury confined to removal of the endothelium. Brown-Norway Katholiek strain rats with an absence of low- and high-molecular-weight kininogen due to a single point mutation, A163T, were compared in the thrombosis model to the wild-type animals, which were otherwise genetically identical. Despite an equivalent vascular injury, the mean time (+/-SEM) for a 90% decrease in flow measured by laser Doppler was 38.4+/-17 minutes in the kininogen-deficient rats compared with 194+/-29 minutes in the wild-type animals (P<0.002). The degree of vascular injury was the same. No evidence for disseminated intravascular coagulation (decrease in factor V, antithrombin, or fibrinogen) or excessive fibrinolysis (elevation of fibrinogen degradation products) was found in either group of animals. The results suggest that kininogens have antithrombotic properties at low concentrations of thrombin and that inhibitory peptides derived from kininogen may constitute a new antithrombotic strategy.

Kallikrein-kininogen system activation and bradykinin (B2) receptors in indomethacin induced enterocolitis in genetically susceptible Lewis rats.

BACKGROUND: The plasma kallikrein-kinin (K-K) system is activated in acute and chronic relapsing intestinal inflammation induced in Lewis rats by intramural injection of exogenous bacterial components. AIMS: To determine whether this effect is model specific, K-K system activation was investigated in a modified indomethacin induced enterocolitis model, as well as bradykinin 2 (B2) receptor distribution in the normal and acutely inflamed intestine. METHODS: Lewis rats injected with daily sublethal doses of indomethacin for two days developed acute (two days) and chronic (14 days) intestinal inflammation. Plasma prekallikrein (amidolytic), high molecular weight kininogen (HK, coagulant) and cleavage of HK (western blot) were assayed to detect K-K activation. RESULTS: Liver and spleen weights were significantly higher, and body weights and haematocrit values were significantly lower in the indomethacin group than in the control group. During both acute and chronic phases, rats displayed K-K system activation manifested by a significant decrease in plasma prekallikrein and HK functional levels, and by HK cleavage. Plasma T kininogen (a major acute phase protein) was significantly elevated. B2 receptors were identified in both normal and inflammatory intestine with more prominent specific immunohistochemical staining in the acutely inflamed tissue. CONCLUSIONS: K-K system activation occurs in association with both acute and chronic phases of intestinal injury, regardless of the triggering agent, suggesting that activation of this system is integrally involved in intestinal inflammation in genetically susceptible hosts. Localisation of B2 receptors across intestinal layers provides a structural basis for the kinin function in the intestine.

Localization and secretion of tissue kallikrein in peptidoglycan-induced enterocolitis in Lewis rats.

The plasma kallikrein-kinin system is a mediator of intestinal inflammation induced by peptidoglycan-polysaccharide from group A streptococci (PG-APS) in rats. In this study we investigated the participation of intestinal tissue kallikrein (ITK). Lewis rats were injected intramurally with PG-APS. ITK was visualized by immunohistochemical staining. Cecal ITK concentration was measured by radioimmunoassay, and gene expression was evaluated by RNase protection assay. Kallikrein-binding protein (KBP) was evaluated in plasma by ELISA. Tissue kallikrein was identified in cecal goblet cells in both control and PG-APS-injected rats and in macrophages forming granulomas in inflamed tissues. Cecal ITK was significantly lower in acute and chronic phases of inflammation and in supernatant from in vitro cultures of inflamed cecum. ITK mRNA levels were not significantly different. Plasma KBP levels were significantly reduced in inflamed rats. The presence of tissue kallikrein in macrophages suggests participation in experimental colitis. The decrease of ITK in the inflamed intestine associated with unchanged mRNA levels suggests ITK release during intestinal inflammation.

Recombinant tumor necrosis factor receptor p75 fusion protein (TNFR:Fc) alters endotoxin-induced activation of the kinin, fibrinolytic, and coagulation systems in normal humans.

The effects of inhibition of tumor necrosis factor (TNF) on cell and protease activation were evaluated in 18 normal volunteers given endotoxin (4 ng/kg, i.v.) after an infusion of low (10 mg/m2 i.v., n = 6) or high dose (60 mg/m2 i.v., n = 6) recombinant human dimeric TNF receptor protein (TNFR:Fc) or its vehicle (placebo n = 6). Activation of the coagulation system occurred by 2 h in the TNFR:Fc vehicle-placebo group manifested by decreased prekallikrein functional levels and increased levels of prothrombin F1+2 fragments (p < 0.0001). High or low dose TNFR:Fc delayed the fall in prekallikrein functional levels by 1 h and 4 h, respectively (p < 0.0002), but did not inhibit the increase in circulating levels of prothrombin F1+2 fragments. In contrast, endothelium activation, characterized by increased levels of tissue plasminogen activator, plasminogen activator inhibitor-1, and von Willebrand Factor antigen was blunted by both low and high dose TNFR:Fc (p < 0.001). While the endotoxin-associated decrease in platelet number was not altered, platelet-derived beta-thromboglobulin peak levels were blunted and delayed by TNFR:Fc (p < 0.02). Increased levels of neutrophil elastase were attenuated by low and high dose TNFR:Fc (p < 0.001). These results suggest that although TNF is functionally linked to the activation of endothelium, neutrophils, coagulation, and fibrinolysis, alternative pathways are present in vivo that result in activation of the kallikrein-kinin system after endotoxin-induced TNF release. These alternative pathways may limit some of the anti-inflammatory effects of TNFR:Fc.

Specific inhibition of plasma kallikrein modulates chronic granulomatous intestinal and systemic inflammation in genetically susceptible rats.

The kallikrein-kinin (K-K) (contact) system is activated during acute and chronic relapsing phases of enterocolitis induced in genetically susceptible Lewis rats by intramural injection of peptidoglycan-polysaccharide (PG-APS). Using the selective plasma kallikrein inhibitor P8720, we investigate whether activation of the K-K system plays a primary role in chronic granulomatous intestinal and systemic inflammation in this model. Group I (negative control) received human serum albumin intramurally. Group II (treatment) received PG-APS intramurally and P8720 orally. Group III (positive control) received PG-APS intramurally and albumin orally. P8720 attenuated the consumption of the contact proteins, high molecular weight kininogen (P<0.03), and factor XI (P<0.04) in group II vs. group III. P8720 decreased chronic intestinal inflammation measured by blinded gross (P<0.01) and histologic (P<0.0005) scores as well as systemic complications (arthritis, splenomegaly, hepatomegaly, leukocytosis, and acute-phase reaction) (P<0.01) in group II as compared with group III. We conclude that relapsing chronic enterocolitis and systemic complications are in part due to plasma K-K system activation, and that inhibition of this pathway is a potential therapeutic approach to human inflammatory bowel disease and associated extraintestinal manifestations.

Activation of plasma contact and coagulation systems and neutrophils in the active phase of ulcerative colitis.

We have shown that the contact (kallikrein-kinin) system is involved in the pathogenesis of experimental enterocolitis. We now investigate activation of the contact and coagulation pathways, platelets, and neutrophils in active and inactive ulcerative colitis patients as compared to normal controls. In active ulcerative colitis patients, a significant decrease of plasma prekallikrein, high molecular weight kininogen, and C1 inhibitor levels was observed as compared with controls, as well as prekallikrein activation on western blots. Significant elevation of prothrombin fragment (F1 + 2), which indicates thrombin generation, and elastase-alpha 1-antitrypsin complexes, reflecting neutrophil activation, were found in patients with active disease. Plasma beta-thromboglobulin, a marker of platelet activation, was elevated in both active and inactive disease and appears to be a feature of ulcerative colitis. Activation of contact and coagulation pathways, as well as neutrophils, may mediate inflammation in the active phase of ulcerative colitis.

Activation of the kallikrein-kinin system in arthritis and enterocolitis in genetically susceptible rats: modulation by a selective plasma kallikrein inhibitor.

We have developed models of acute and chronic inflammatory arthritis and enterocolitis using peptidoglycan-polysaccharide injected intraperitoneally or subserosally (intramurally) into the distal ileum and cecum. Acute inflammation occurs in both Buffalo and Lewis rats, characterized by inflammation of the injected areas of the intestine. However, only the genetically susceptible Lewis rat develops chronic synovitis and joint erosion or adhesions and granulomatous enterocolitis. In the Lewis rat but not the Buffalo rat, these changes are accompanied by a decrease in plasma prekallikrein and high-molecular-weight kininogen, reflecting activation of the kallikrein-kinin system. Pretreatment with a specific plasma kallikrein inhibitor modulates the acute and chronic arthritis. The same inhibitor partially abrogates the acute changes characteristic of enterocolitis, and preliminary data suggest similar results in the chronic model. The results of these studies indicate that the kallikrein-kinin system plays an important role in arthritis and enterocolitis induced by bacterial products and that kallikrein inhibitors are potential therapeutic agents for inflammatory arthritis and inflammatory bowel disease.

[Peripheral blood neutrophils in patients with internal endometriosis in light of enzymatic tests]. / Granulocyty obojetnochlonne krwi obwodowej u chorych z endometrioza wewnetrzna w swietle badan enzymatycznych.

The diagnosis of the internal endometriosis was verified in histopathological investigation in 16 patients. The following indices were evaluated in this group: 1) the ability of reduction of nitroblue tetrazolium (NBT) by neutrophils with the calculation of indices of spontaneous and stimulating reduction by latex of this dye, 2) activity of myeloperoxidase (MPO) in neutrophils with the semiquantitative measurement determined in blood smears, 3) activity of alkaline phosphatase (FA) in neutrophils with the semiquantitative measurement determined in blood smears, 4) lysozyme activity in blood serum by turbidimetric method. The obtained results were compared with control values obtained in 20 healthy women. The following statistically significant differences were shown-the increase of spontaneous and stimulated reduction of NBT, the decrease of myeloperoxidase activity, the increase of alkaline phosphatase activity and the increase of the activity of lysozyme in diluted and undiluted blood serum. On this basis the following conclusion is suggested: The neutrophils of the peripheral blood in patients with internal endometriosis are characterized by metabolic disturbances and the change of the function of cell structures. The indices of these changes are myeloperoxidase and alkaline phosphatase. The above changes may have an important influence on defensive function of these cells in the case of real infectious conditions.

Activation of the contact and fibrinolytic systems after intravenous administration of endotoxin to normal human volunteers: correlation with the cytokine profile.

Severe progressive failure of multiple organ systems has emerged during the past three decades as a common cause of death among patients in intensive care units. Sepsis is now better defined as systemic inflammatory response syndrome (SIRS), but its mortality rate has not changed and it continues to be a major health problem. Endotoxin interacts with plasma proteins and contributes to the pathophysiology of SIRS. Information is limited on the effect of endotoxin on human coagulation and fibrinolytic proteins in vivo, as well as on the cell response involved in the cytokine cascade. For this reason we performed quantitative assays to establish the sequence of events that occurs in vivo in the regulation of the contact and fibrinolytic pathways as well as in the cytokine cascade as a response to a single dose administration of endotoxin to normal non-smoking human volunteers.

Selective plasma kallikrein inhibitor attenuates acute intestinal inflammation in Lewis rat.

A specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroArg (P8720), was used to define the relationship between the kallikrein-kinin (K-K) system and acute intestinal inflammation induced by bacterial peptidoglycan-polysaccharide (PG-APS) in Lewis rats. Group I received human serum albumin (HSA) intramurally in the intestine and was treated with HSA. Group II received PG-APS and was treated with P8720. Group III received PG-APS and was treated with HSA. P8720 attenuated the decrease of high-molecular-weight kininogen and factor XI activity (group II vs group III, P < 0.01). P8720 therapy significantly but modestly decreased acute intestinal inflammation measured by gross gut score (P < 0.01) and more dramatically reduced the tissue myeloperoxidase activity (P < 0.05), a measure of granulocyte recruitment, in group II compared with group III. We conclude that the K-K system is directly involved in the pathogenesis of the acute phase of experimental acute inflammation. A specific inhibitor may modulate inflammatory bowel disease.

Selective kallikrein-kinin system activation in inbred rats differentially susceptible to granulomatous enterocolitis.

BACKGROUND & AIMS: Crohn's disease is characterized by unrestrained inflammation with a genetic component. Genetic susceptibility and activation of the kalli-krein-kinin (contact) system were investigated in experimental enterocolitis and extraintestinal inflammation induced by bacterial polymers. METHODS: Kinetics of inflammation in inbred Lewis and Buffalo rats injected subserosally with peptidoglycan-polysaccharide polymers were correlated with in vivo and in vitro activation of the contact system. RESULTS: Lewis rats had a biphasic course of enterocolitis. Acute inflammation peaked 1 day after injection, gradually decreasing until day 14 when intestinal inflammation spontaneously reactivated and persisted for 16 weeks, accompanied by arthritis, granulomatous hepatitis, anemia, and leukocytosis. Self-limited acute enterocolitis in Buffalo rats resolved by 24 days without extraintestinal involvement. Consumption of the precursor proteins prekalli-krein and high-molecular-weight kininogen indicated activation of the plasma contact system in Lewis rats and closely correlated with chronic intestinal inflammation. Contact system activation did not occur in Buffalo rats, even during acute inflammation. In vitro studies showed a decreased rate of kininogen cleavage in Buffalo plasma. CONCLUSIONS: Selective in vivo and in vitro activation of the contact system in susceptible Lewis rats suggests that this pathway is one determinant of genetic susceptibility to granulomatous enterocolitis and systemic complications.

Inhibition of plasma kallikrein prevents peptidoglycan-induced arthritis in the Lewis rat.

We investigate whether the previously shown contact system activation plays a pathogenetic role in a rat model of acute inflammation induced by peptidoglycan-polysaccharide (PG-APS) using a new specific plasma kallikrein inhibitor, Bz-Pro-Phe-boroArg-OH (P8720). Group I (control) received neither PG-APS nor inhibitor. Group II (disease-treated) received PG-APS intraperitoneally (IP) and P8720 orally. Group III (disease-untreated) received PG-APS IP. Anemia was evident at 49 h in group III but was not present (P < 0.01) in groups I and II. Spleen weight was significantly decreased in group II compared to group III. Acute arthritis progressively developed in group III from 27 to 49 h, but P8720 decreased the joint swelling in group II by 61% (P < 0.0005). We observed a significant fall in prekallikrein and factor XI (P < 0.01) in groups II and III but not in group I. The decrease in the functional levels of high molecular weight kininogen (P < 0.05) observed in group III were prevented by P8720 in group II. The changes in T-kininogen and alpha 1-inhibitor 3 acute-phase proteins were partially prevented by P8720. We conclude that the inflammatory reactions leading to arthritis and anemia, as well as the acute-phase reaction, are due in part to contact activation, and that specific kallikrein inhibitors may have therapeutic potential.

Factor XIII subunits in relation to some other hemostatic parameters in ulcerative colitis.

The hemostatic parameters, particularly with respect to F.XIII subunits, were examined in 48 untreated UC patients (22 at active and 26 at quiescent stage). UC active patients showed a significant decrease of F.XIII subunit "a," compared with healthy subjects, as well as in UC patients in remission. In contrast, the level of F.XIII subunit "b" in each group was similar. Compared with normal subjects, UC active patients revealed a significant decrease in AT III concentration, prolonged ELT, and elevated fibrinogen level. In addition, the elevated titer of SDPS test for SFMC appeared in approximately 40% of those patients. However, no strict relationship was found between the presence of positive SDPS and diminution of AT III, as well as of F.XIIII subunit "a" in active UC state. In patients in remission, AT III level and ELT were similar to those as in the control group, but fibrinogen concentration was elevated. Such constellation of hemostatic parameters may indicate a tendency to blood hypercoagulability in UC active patients, whereas, in general, these changes are not associated with the stage of remission. The present data may also suggest that F.XIII behavior pattern should be taken into account in the clinical management of UC.