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J Wojta

Publications and source records attributed to J Wojta.

At least 73 records · Page 4Linked to original sources
PubMed1990

Bartonella bacilliformis stimulates endothelial cells in vitro and is angiogenic in vivo.

Bartonellosis, a biphasic disease caused by motile intracellular bacteria, produces in its tissue phase a characteristic dermal eruption (Verruga peruana) resulting from a pronounced endothelial cell proliferation. Bacteria are found in the interstitium and within the cytoplasm of endothelial cells (Rocha-Lima inclusion). The aim of this study was to determine if Bartonella bacilliformis produce a substance(s) that might be responsible for the vascular proliferation seen in the Verruga. This was assessed in an in vitro system using human endothelial cells and measuring proliferation as well as production of tissue type plasminogen activator after exposure to the endothelial cultures to B. bacilliformis extracts. Our results indicate that B. bacilliformis possess an activity that stimulates endothelial cell proliferation up to three times that of control. The factor(s) is specific for endothelial cells, heat sensitive, larger than 12 to 14 kd, not enhanced by heparin, has no affinity for heparin, and is precipitated by 45% ammonium sulfate. In addition, the B. bacilliformis extracts stimulate production of t-PA antigen in a concentration-dependent fashion. This activity is also heat sensitive and not lost after dialysis (12 to 14 kd). B. bacilliformis extracts, however, do not increase the production of plasminogen activator inhibitor. It was also determined that B. bacilliformis extracts stimulate the formation of new blood vessels in an in vivo model for angiogenesis. These results describe a bacterial factor(s) that stimulates two important steps in the development of new blood vessels in vitro, as well as the formation of new blood vessels in vivo. Determining the mechanism of action, combined with a complete characterization of this factor(s), may help in understanding the pathogenesis not only of the Verruga and angiogenesis in general but also the recently described Cat-Scratch-associated epithelioid hemangiomas in patients with AIDS and Kaposi sarcoma.

Animals↗
PubMed1989

Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo.

Protein C inhibitor (PCI) and plasminogen activator inhibitor 3 (PAI-3; urinary urokinase inhibitor) are immunologically identical. The role of PCI for urokinase (uPA) inhibition in vivo was investigated. We therefore developed an enzyme-linked immunosorbent assay (ELISA) specific for uPA-PCI complexes: Rabbit anti-PCI IgG was immobilized on a microtiter plate and following incubation with uPA-PCI complex-containing samples, bound uPA-PCI complexes were quantified with a horseradish-peroxidase-linked monoclonal antibody (MoAb) to uPA. Using this assay, time, dose, and heparin-dependent complexes were detected when uPA was incubated with normal plasma or purified urinary PCI, whereas no complexes were measurable using PCI-immunodepleted plasma. Plasma samples (containing 20 mmol/L benzamidine to prevent complex formation ex vivo) from patients undergoing systemic urokinase therapy (1 x 10(6) IU/60 min intravenously [IV]) after myocardial infarction were also studied. uPA present in these plasma samples (up to 1,200 ng/mL) had only 43% to 70% of the specific activity of purified 2-chain uPA, suggesting that a major portion of uPA is complexed to inhibitors. In these plasma samples uPA-PCI complexes were present in a concentration corresponding to 21% to 25% of inactive uPA antigen. These data suggest that at high uPA concentrations, such as during uPA therapy, plasma PCI might contribute significantly to uPA inhibition in vivo.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

Functional characterization of monoclonal antibodies directed against fibrin binding domains of tissue-type plasminogen activator.

Fibrin interacts with tissue-type plasminogen activator (tPA) via the finger and the kringle 2 domains. Three monoclonal antibodies against tPA, designated MPW3VPA, MPW6VPA, and MPW7VPA, which react with epitopes in the tPA molecule involved in fibrin binding, were characterized. The IgM monoclonal antibody MPW6VPA, directed against an epitope close to the finger and epidermal growth factor domains, stimulated plasminogen activation only in the absence of CNBr-fibrinogen fragments by increasing kcat in a dose-dependent fashion, an effect which was not restricted to the intact molecule. These results suggest that MPW6VPA mimics the initial effect of fibrin bound to the tPA molecule, which results in a change of kcat values. The MPW6VPA effect was reversed by another antibody, MPW3VPA, also directed against epidermal growth factor and finger domains. The latter antibody also inhibited plasminogen activation by tPA in the presence of CNBr-fibrinogen fragments in a dose-dependent, apparently noncompetitive way. No effect of MPW3VPA was seen in the absence of CNBr-fibrinogen fragments. MPW7VPA directed against kringle 2 of tPA inhibited plasminogen activation by tPA only when CNBr-fibrinogen fragments were present. This inhibition was apparently competitive and dose-dependent. These data suggest that MPW3VPA interferes with the first phase of fibrin binding to tPA, whereas MPW7VPA interferes with the second phase of fibrin binding to the tPA molecule via kringle 2, resulting in Km changes.

Antibodies, Monoclonal↗
PubMed1989

Evaluation of fibrinolytic capacity in plasma during thrombolytic therapy with single (scu-PA) or two-chain urokinase type plasminogen activator (tcu-PA) by a combined assay system for urokinase type plasminogen activator antigen and function.

A combined assay for urokinase type plasminogen activator (u-PA) activity and antigen determination in plasma samples is described. This assay is based on binding of u-PA to an antibody immobilized on a microtiter plate followed by determination of the enzymatic activity of the bound u-PA. Thereafter bound u-PA antigen can be quantified by means of a specific peroxidase labelled monoclonal antibody against u-PA. By use of this assay system u-PA activity and antigen can be determined with lower detection limits of 0.08 IU/ml and 1.0 ng/ml, respectively, and intraassay as well as interassay coefficients of variation of 10% and 12% for activity and 5% and 7% for antigen determinations, respectively. Normal plasma levels of u-PA antigen could be determined to be 1.88 ng/ml +/- 0.61. Furthermore, this assay system allows specific quantification of u-PA antigen and activity during thrombolytic therapy.

Antigens↗
PubMed1989

Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator.

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.

Cells, Cultured↗
PubMed1989

Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line.

Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-PA in its active form and expresses high-affinity u-PA receptors with a Kd of 5.2 x 10(-10) M and 2.8 x 10(4) binding sites per cell. Approximately 70% of the u-PA receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal anti-u-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of [3H]thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. In addition, diisopropyl fluorophosphate-inactivated u-PA, in a concentration 50-fold higher than the concentration necessary to saturate the u-PA receptor (250 pM), decreased [3H]thymidine incorporation similarly to the specific antibody, proving that active u-PA is required for the mitogenic effect. Inhibition of endogenous u-PA production by cycloheximide reduced [3H]thymidine incorporation significantly; after addition of exogenous u-PA, [3H]thymidine incorporation increased again in the cycloheximide-treated cells. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.

Antibodies, Monoclonal↗
PubMed1988

Mitogenic effect of urokinase on malignant and unaffected adjacent human renal cells.

Primary cultures of renal cell carcinomas and of the corresponding normal adjacent kidney tissue from 6 patients were analyzed for the effects of exogenously added urokinase-type plasminogen activator on cell proliferation as compared to the effects of tissue type plasminogen activator, plasmin and dihydrocortisone. Cell proliferation was studied over a period of up to 5 days by measuring 3H-thymidine incorporation as well as cell viability and cell count; conditioned media of the cultures were also analyzed for their plasminogen activator and plasminogen activator inhibitor content. Addition of urokinase stimulated cell proliferation in a time and dose dependent fashion; after 3 days 3H-thymidine incorporation was significantly increased in malignant renal cells (188.3 +/- 28.7%), while it reached in normal renal cells approximately 130% of the 3H-thymidine incorporation of untreated cultures. Tissue-type plasminogen activator had no effect and plasmin decreased cell proliferation slightly while dihydrocortisone inhibited cell proliferation significantly (34.1 +/- 4.9%) in malignant cells. It is concluded that urokinase-type plasminogen activator itself exhibits a mitogenic effect also on primary cultures of renal cell carcinomas.

Adult↗
PubMed1988

Plasminogen activators and plasminogen activator inhibitor in malignant and non-malignant ascitic fluid.

Ascitic fluid from tumour patients (hepatoma, gastric cancer, gallbladder cancer, colorectal cancer, ovarian cancer) and from non-malignant diseases (liver cirrhosis, congestive heart failure) were compared with respect to their content of determinants of the fibrinolytic system, tissue-type plasminogen activator antigen (t-PAag) and activity (t-PAact), urokinase-type plasminogen activator antigen (u-PA) and plasminogen activator inhibitor activity (PAI). Furthermore, SDS-polyacrylamide slab-gel electrophoresis (SDS-PAGE) was performed to evaluate molecular weight distribution of the detectable fibrinolytic parameters. In malignant ascites, PAI activity was three to four times higher, and increased complex formation of PAI with t-PA could be demonstrated, compared with non-malignant ascitic fluid. Tissue-type plasminogen activator antigen and activity showed a similar concentration in ascites of both study groups. Urokinase-type plasminogen activator antigen was detectable neither in ascites of malignant nor in ascites of non-malignant origin. It is concluded that t-PA is the physiological plasminogen activator in ascites and that increased PAI levels followed by increased complex formation between t-PA and PAI might reflect a reaction of the peritoneum.

Adult↗
PubMed1988

Comparison of fibrinolytic activities of human and bovine endothelial cells.

Human aortic (HAE), human umbilical vein (HUVE), and bovine aortic (BAE) endothelial cells were compared in their synthesis and release of fibrinolytic components during culturing. After isolation, the cultures were grown to confluency and then studied under identical conditions for release of tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor (PAI) into serum-free medium. HAE cells released 10 times more t-PA antigen than HUVE cells, and the respective cell lysates also contained comparably higher values. Free PAI capacity was found in the conditioned media of both HAE and HUVE cells. BAE cell t-PA release was much lower than that of the HAE cells, and free inhibitor capacity was not found in the conditioned medium. BAE cells contained significant amounts of PA activity in cell membrane-bound form. This PA activity on the cell surface was not stimulated by addition of CNBr fibrinogen fragments but could be partially inhibited by activated bovine PAI and antibodies against human t-PA and urokinase PA, respectively.

Animals↗
PubMed1988

Effect of anti plasminogen monoclonal antibodies on whole blood clot lysis.

Several monoclonal antibodies (MABs) against specific parts of the plasminogen molecule were developed. One of them, MPW1PG, recognizes only the native form Glu-plasminogen, while the other one, MPW2PG, reacts equally well with Glu-plasminogen and its proteolytically degraded derivative Lys-plasminogen as confirmed by immunoblotting experiments. Both MABs do not alter the cleavage of the low molecular weight substrate S-2251 by plasmin in a purified system but rather increase the rate of plasmin formation by urokinase and t-PA in a system without fibrin. In the presence of fibrin MPW1PG has no effect on plasmin formation by t-PA, while MPW2PG exhibits mixed type inhibition. To investigate whether this effect can also be seen in a whole blood clot lysis system, the Chandler loop was used (PVC tubes, 4 mm inner diameter, 28 mm length), 125I-fibrinogen was added to citrated whole blood (0.36% sodium citrate) to about 5,000 cpm/10 microliter sample. 1.5 ml of the mixture were pipetted into each tube, the tubes were closed, put on a rotary plate, tilted to an angle of 23 degrees, and rotated at 16 rpm. 10 microliter samples were taken for radioactive counting before clotting by addition of 10 microliter 3.02M CaCl2 (100%-value), 45 min after clotting (0%-value), and 30, 60, 120, 180, 360 minutes after addition of activators. Lysis was started one hour after recalcification. Different amounts of MABs were added either 30 min before recalcification (MAB endogen) or together with the activator (MAB exogen) Percent lysis was calculated from the radioactivity released into the fluid phase. Double determinations were done in all experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Reduction in pO2 decreases the fibrinolytic potential of cultured bovine endothelial cells derived from pulmonary arteries and lung microvasculature.

The effect of anoxia on the fibrinolytic potential of cultured endothelial cells derived from bovine pulmonary artery and bovine lung microvasculature was studied. Both cell types reacted with an increase in plasminogen activator inhibitor (PAI) activity and a decrease in the plasminogen activator (PA) activity in the media after incubation under anoxic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin autography and reverse fibrin autography indicated that the change in fibrinolytic potential was due to an impaired release of PA and not an increase in the production of PAI. Although anoxia did not affect the viability of the cells as judged by 51Cr release, their metabolism was influenced, which is reflected by increases in the levels of lactate in cell lysates and media. Furthermore, the effect of short-term anoxia on PA and PAI could not be reversed by reoxygenation for 24 hours. The results are discussed in terms of helping to explain the tendency of reocclusion after successful thrombolytic therapy, the development of pulmonary hypertension, and the thrombotic tendency of areas with an impaired circulatory supply.

Animals↗
PubMed1987

Effect of urokinase on the proliferation of primary cultures of human prostatic cells.

The effects of exogenously added urokinase type plasminogen activator, tissue type plasminogen activator, plasmin and thrombin on the proliferation of primary cultures of cells derived from prostatic hyperplasia or prostatic carcinomas were investigated by measuring the incorporation of 3H-thymidine into the cultures. Addition of urokinase type plasminogen activator (1.35 x 10(-9) M) or thrombin (10(-7) M) to the culture medium caused a two-fold increase of 3H-thymidine incorporation, regardless of the origin of the prostatic cells. Tissue type plasminogen activator did not alter the rate of 3H-thymidine incorporation, whereas plasmin caused a 25% decrease of 3H-thymidine incorporation in all cultures.

Cell Division↗
PubMed1987

Effect of the cyanogen-bromide-2 fragment of fibrinogen on plasminogen activation by single-chain urokinase-type plasminogen activator.

Activation of Glu-plasminogen by single-chain urokinase-type plasminogen activator (sc-uPA), isolated from human urine, was studied in a purified system in the absence and presence of the cyanogen bromide fibrinogen fragment, FCB 2, and compared to plasminogen activation by two-chain high-Mr urokinase. Plasminogen activation by sc-uPA was significantly increased by the FCB-2 fibrinogen fragment, an effect brought about by decrease of apparent Km and increase of apparent kcat. During the course of plasminogen activation by scu-PA, two-chain urokinase was formed from 125I-sc-uPA to a significant degree only when a concentration of 30 nM plasmin was reached in the incubation mixture; this was only the case in the system stimulated by FCB-2 fibrinogen fragment and only after 30 min. Formation of two-chain urokinase was not, however, related to the increase in the rate of plasmin formation induced by the FCB-2 fibrinogen fragment.

Cyanogen Bromide↗
PubMed1987

Monoclonal antibodies against plasminogen and alpha-2-antiplasmin: binding to native and modified antigens.

Two monoclonal antibodies against plasminogen and two against alpha-2-antiplasmin were characterized regarding their binding properties towards their respective native and modified antigens and towards the plasmin-alpha-2-antiplasmin complex. Using an ELISA system, the two monoclonal antibodies against plasminogen (MPW1PG, MPW2PG), reacted equally well with purified Glu-plasminogen; binding of one (MPW1PG) to the purified plasmin-alpha-2-antiplasmin complex was, however, more than three orders of magnitude weaker than that of the other. Similarly, both antibodies against alpha-2-antiplasmin (MPW2AP, MPW3AP) reacted equally well with native alpha-2-antiplasmin but binding of MPW3AP to the plasmin-alpha-2-antiplasmin complexes was more than one order of magnitude weaker than that of the other. These results were confirmed by immunoblotting by which it was made evident that the antiplasminogen antibody MPW1PG reacted only with the free or complexed Glu-form of plasminogen or plasmin, while MPW2PG reacted equally well with the Glu- and Lys-form. The alpha-2-antiplasmin antibody MPW2AP reacted with native and complexed alpha-2-antiplasmin while MPW3AP reacted only with the native antigen and with a 8,000 dalton cleavage product of the complex.

Antibodies, Monoclonal↗
PubMed1987

Enhanced fibrinolysis caused by tissue plasminogen activator release in hemodialysis.

The effect of hemodialysis on components of the fibrinolytic system was measured in 22 patients. Plasma levels of t-PA antigen, t-PA activity, u-PA antigen and plasminogen activator inhibitor were determined by means of immunological and functional assays. During hemodialysis, 11 patients exhibited an increase in t-PA antigen within the first hour to about three times the starting values (P less than 0.05), followed by a decrease to about double of the initial values until the end of the treatment. Eleven patients showed a continuous increase up to 200% of the starting values until the end of hemodialysis. u-PA levels did not change significantly during the time of investigation (P greater than 0.5).

Adolescent↗
PubMed1987

Proliferation of a human epidermal tumor cell line stimulated by urokinase.

Several tumor cells secrete significantly increased amounts of the plasminogen activator urokinase, a trypsinlike serine protease, whose biological function in tumor biology is unclear. In this study we report that cells of the human epidermal tumor cell line CCL 20.2 express about 80,000 high-affinity urokinase receptors per cell that bind active as well as diisopropylfluorophosphate-treated high-molecular-weight (HMW) urokinase. Low-molecular-weight (LMW) urokinase is not bound to the receptor. Occupation of these receptors by active HMW urokinase stimulates cell proliferation independently in the presence of plasminogen in the culture medium. LMW urokinase has again no effect on cell proliferation. Calculated on a molar basis, this effect is about 28% of that of epidermal growth factor. Active HMW urokinase might therefore provide an autocrine receptor-mediated growth-promoting mechanism for tumor cells similar to those described for other growth factors.

Cell Division↗
PubMed1987

Evaluation of fibrinolytic capacity by a combined assay system for tissue-type plasminogen activator antigen and function using monoclonal anti-tissue-type plasminogen activator antibodies.

An assay system has been developed that allows consecutive quantification of tissue-type plasminogen activator (t-PA) activity and t-PA antigen in the same plasma sample. In the first step t-PA is bound to an immobilized IgM monoclonal anti-t-PA antibody and functional activity of bound t-PA is quantified by its plasminogen-activating activity. In the second step the amount of bound t-PA antigen is determined by using a different peroxidase-labeled monoclonal anti-t-PA antibody. In this combined assay system t-PA functional activity was found to depend not only on the amount of t-PA antigen but also on the amount of plasminogen activator inhibitor (PAI), whereas in the t-PA antigen assay PAI did not affect the results. In plasma samples obtained from normal controls t-PA activity was detected only in post-venous occlusion plasma (3.7 +/- 2.5 IU/ml), whereas 2.7 +/- 0.5 ng/ml t-PA antigen was found before and 12.6 +/- 4.4 ng/ml after venous occlusion. Using this combined assay system to study plasma samples from patients who did not respond to venous occlusion with shortening of the euglobulin clot lysis time (ECLT), it was possible not only to confirm that in none of these patients could t-PA activity be detected in the postocclusion plasma samples but also to subdivide that group of patients into a group of about 39% not reacting with normal t-PA antigen release to venous occlusion and into a second group of about 61% that reacted with normal t-PA antigen release.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗