Effects of ethylenediaminetetraacetic acid and 1,10-phenanthroline on cell proliferation and DNA synthesis of Ehrlich ascites cells.

The interactions of ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline with Ehrlich cells have been studied. Both compounds inhibit proliferation of cells in culture. After long-term incubation of cells with these metal-chelating agents under conditions in which cell viability is not impaired but proliferation is retarded, the rate of DNA synthesis of EDTA-exposed cells is the same as that of untreated controls, whereas the rate of DNA synthesis of 1,10-phenanthroline-exposed cells is markedly reduced. This is in agreement with the inhibition of short-term DNA synthesis by similar concentrations of 1,10-phenanthroline as well as the lack of effect of EDTA upon DNA synthesis. The rapid direct effect of 1,10-phenanthroline upon cells in S phase is consistent with the finding that a large fraction of this metal-binding ligand but not of EDTA can be readily taken up by cells. These results differ in significant ways from the response of Ehrlich cells in vivo to host zinc deficiency. Finally, titration of ligand-treated cell cultures with zinc reverses the inhibition of DNA synthesis and cell proliferation in a linear manner, suggesting that the removal of ligand by complexation and the addition of a previous unavailable essential metal is occurring in these reactions.

Binding of interleukin 2 to gangliosides.

Exogenous gangliosides inhibit interleukin 2 (IL2)-dependent growth of a T cell line, AKIL -1.E8. IL2 activity is retained by columns of ganglioside covalently linked to poly(L-lysine)-agarose and is not eluted with ethylene glycol but is completely recovered by elution with 1% SDS. The ability of gangliosides to inhibit IL2 activity is directly related to the complexity of their carbohydrate portion, and related ceramide derivatives at similar concentrations do not inhibit IL2 activity. We conclude that IL2 bound to exogenous gangliosides is inactive and that the carbohydrate portion of the ganglioside is crucial to its interaction with IL2.

Comparative cytotoxic and biochemical effects of ligands and metal complexes of alpha-N-heterocyclic carboxaldehyde thiosemicarbazones.

Several alpha-N-heterocyclic carboxaldehyde thiosemicarbazones and their iron and copper complexes have been tested for their cytotoxicity and inhibiting activity against DNA synthesis under controlled metal conditions. No ligands show cytotoxicity against Ehrlich cells at the concentrations tested, while some iron and copper complexes are active. In contrast, the ligands inhibit DNA synthesis at much lower concentrations than used above. Similarly, the metal complexes are effective inhibitors at concentrations much below those necessary to demonstrate cytotoxicity. In addition, the iron complexes of 1-formylisoquinoline thiosemicarbazone, 2-formylpyridine thiosemicarbazone, and 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone were shown to be three- to sixfold more active than their free ligands as inhibitors of partially purified ribonucleotide reductase to which no iron has been added. The copper complex of 2-formylpyridine thiosemicarbazone was slightly more active than the free ligand against the reductase.

Effect of plasminogen activator inhibitor-1 on tissue-type plasminogen activator-induced fibrinolysis.

The effect of fibrin on the interaction of human recombinant single-chain tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in normal rabbit plasma and in plasma with high levels of native PAI-1. t-PA was added to diluted plasma containing calcium (10 mM) and 125I-fibrinogen at 37 degrees C. Clotting was initiated with human thrombin, and lysis was monitored both turbidimetrically and by release of 125I-fibrin degradation products (fdp). The activity of t-PA (50 IU/ml) was rapidly reduced to 15% of the initial value in plasma containing PAI-1 (23 AU/ml). When thrombin and t-PA were added simultaneously to the plasma, more than 70% of the activity was retained through incorporation of t-PA into the fibrin clot. t-PA-induced fibrinolysis in PAI-1 enriched plasma was further delayed when the temperature was reduced from 37 to 25 degrees C. Turbidimetric and 125I-fdp release data provided complementary information. The former technique traced fiber dissolution, while the latter reflected network integrity. These results indicate that t-PA-induced fibrinolysis in PAI-1 enriched plasma is modulated by the presence of fibrin and by temperature.

Differential effects of beta- and gamma-interferons on natural killer cell-mediated lysis of lung carcinoma cells.

Peripheral blood lymphocytes from healthy donors (PBL) poorly lyse lung carcinoma cell lines A-549, A-427 and SK- MES-1 when tested in a short-term chromium release assay. When PBL are preincubated with human beta-interferon (IFN-beta), these cell lines are lysed with an efficacy comparable to that of erythroleukemia K-562 cells, the standard targets used in natural killer cell assays. However, when PBL are preincubated with gamma-interferon (IFN-gamma) instead, lysis of the lung carcinoma lines is little augmented. Unlabeled lung carcinoma A-549 cells block chromium release from labeled K-562 cells with non-boosted and IFN-gamma or IFN-beta-boosted effector cells. Also with the IFN-beta treated effectors, chromium release from A-549 targets is inhibited by unlabeled K-562 cells. Therefore, cells that lyse K-562 cells must be able to recognize A-549 cells, and, in the case of IFN-beta pretreated effectors, cause the killing of these cells as well. Data obtained with effector cells separated on discontinuous Percoll gradients also indicate that the same cells that lyse A-549 cells are responsible for lysis of K-562 cells. We conclude that in response to IFN-beta, effector cells previously able to lyse K-562, but unable to lyse A-549 targets, mature into fully competent killer cells capable of lysing tumor cells from lymphoid as well as from lung cancer origin. This effect is not elicited by IFN-gamma, indicating that killer cells respond differently to both interferon types.

Plasminogen activator and plasminogen activator inhibitor activities in a reference population.

The influence of age, gender, and aspirin ingestion on plasma levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) activities was studied in a reference population of 35 men and 35 women between the ages of 20 and 65 years. The t-PA values (mean +/- SD) in the women before and after 5 minutes of venous occlusion were 3.8 +/- 1.4 and 7.8 +/- 4.4 micrograms/L, respectively; in men these values were 3.3 +/- 1.2 and 8.8 +/- 8.9 micrograms/L. Men had higher mean PAI levels than did women (5.0 vs. 2.5 kU/L). T-PA showed an inverse relationship to PAI in both sexes. There was a negative correlation of t-PA levels with age, whereas PAI levels were positively correlated. The ingestion of a single dose of aspirin (650 mg) did not alter PAI or t-PA activities. This study indicates that factors such as age and sex may need to be considered when reference populations are developed for clinical studies of fibrinolysis.

Diagnosis of heparin-induced thrombocytopenia in the vascular surgery patient.

BACKGROUND: Recent studies have shown that patients with heparin-induced thrombocytopenia (HIT) form immunoglobulin G (IgG) and/or IgM antibodies directed against a complex of platelet factor 4 (PF4) and heparin. This recognition has resulted in the development of enzyme-linked immunosorbent assays (ELISAs) that use the heparin/PF4 complex as the antigen. This study describes the use of a standardized ELISA to assess antibody formation in five patients suspected of having HIT. METHODS: Five patients received heparin for treatment of arterial or venous thrombotic disorders. All patients had the ELISA performed to detect IgG or IgM antibodies directed against heparin-PF4, as well as the 14C serotonin release assay, when HIT was clinically suspected. RESULTS: HIT was diagnosed in four patients and ruled out in a fifth by using the ELISA. All patients had a 40% decrease in platelet count that returned to normal after heparin cessation. Only one of the four patients who tested positive by ELISA for IgG antibodies also tested positive by the 14C serotonin release assay. Treatment was significantly altered by the ELISA results in all five patients. CONCLUSIONS: It is likely that the ELISA is more sensitive in the diagnosis of HIT than the more traditional aggregation tests, and it may emerge as a new gold standard. Prospective studies in which serial laboratory testing is combined with measurement of clinical outcomes are needed and will eventually provide a greater understanding of the full spectrum of HIT and the clinical settings that precipitate thrombosis in the vascular surgery patient.

Stimulation of plasminogen activator inhibitor activity in human monocytes infected with dengue virus.

Human monocytes, in an essentially serum-free culture medium, were infected with dengue 2 virus in the presence of sub-neutralizing concentrations of antibody. Changes in procoagulant activity (PCA), in the plasminogen activator urokinase (UK), and the plasminogen activator inhibitor-2 (PAI-2) were quantitated. One day after exposure to dengue virus, the cell-associated PAI-2 activity in the infected monocytes was 562 +/- 9 mU/10(6) cells (mean +/- SE) compared to 206 +/- 56 mU/10(6) cells for uninfected monocytes. Supernatants of the infected cells also showed greater than 2-fold increase in PAI-2 activity. This increase in cell-associated and supernatant PAI-2 activity was maintained during 4 days of culture. UK activity was not detected in control and infected cells nor in their supernatants. PCA activity was the same in control and dengue virus infected monocytes when measured during 4 days of culture. These data suggest that dengue infected monocytes may affect fibrinolysis at a localized level through increased production of PAI-2.

Mechanisms of hemorrhage in dengue without circulatory collapse.

To characterize the molecular basis for the hemostatic defects of dengue infections, a study was conducted in Bangkok, Thailand. Febrile children (n = 68) hospitalized with suspected dengue were enrolled before their clinical syndromes were classified as either dengue fever (DF) or dengue hemorrhagic fever (DHF). Hospital course and outcome were recorded; blood was obtained during the febrile illness (S1), after defervescence (S2), and 1 month after onset of disease (S4). Patients were classified as DF (n = 21) and DHF grades 1, 2, and 3; (DHF1, n = 8; DHF2, n = 30; and DHF3, n = 9). All had marked thrombocytopenia. Bleeding scores were assigned on the basis of bleeding site. Although there was no correlation between bleeding scores and pleural effusion index (a measure of vascular leakage) or bleeding scores and platelet counts, there was a correlation between pleural effusion index and platelet counts. Bleeding scores did not correlate with hemostatic data. Activated partial thromboplastin time was prolonged, with trends toward decreased fibrinogen and increased levels of prothrombin fragment F1.2 in the acute-phase samples. However, no factor level was dramatically decreased. We conclude that most patients with DF or DHF, even without overt hemorrhage, have consumptive coagulopathy. Nevertheless, hemorrhage in dengue without circulatory collapse is most likely due to activation of platelets rather than coagulopathy, which is well compensated. Our data suggest that vascular alteration may be the principal factor involved in the association of thrombocytopenia and hemorrhage with disease severity.

Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis.

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and 125I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.

The rabbit as a model for studies of fibrinolysis.

When compared to man, the rabbit shows marked prolongation of the dilute whole blood clot lysis time and an attenuated increase in plasminogen activator (PA) after the infusion of desmopressin (DDAVP). The levels of specific components of the plasma fibrinolytic system of the rabbit were compared to those in human plasma to ascertain their role in the differences between species. PA activity and plasminogen levels were similar in the two species. Anti-plasmin and plasminogen activator inhibitor (PAI) activity were lower in the rabbit than in man. The rabbit PAI, apparently similar to that described in man, was not increased by DDAVP infusion. The disparity between man and rabbit with respect to the lysis times of dilute blood clots and response to DDAVP cannot be explained by differences in functional plasma levels of inhibitors or activators of the fibrinolytic system.

Stimulated production of urokinase and plasminogen activator inhibitor-2 by the human promyelocytic leukemia cell line HL-60.

The effects of maturation inducing agents on the production of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) by the human promyelocytic leukemia cell line HL-60 were examined. PA activity, which was calibrated with a urokinase standard, was 3-6 mU/10(6) cells when measured in supernatants from control cells. This activity increased at least two-fold after dimethylformamide (DMF) or retinoic acid (RA) was added to cell cultures, and as much as ten to thirty-fold when cells were exposed to 12-O-tetradecanoylphorbol-13-acetate (PMA), an agent that induces monocytoid differentiation in HL-60 cells. The PA activity produced by control and induced cells had the same molecular weight as urokinase (UK), and was completely inhibited by antibodies to UK. Cells that were induced with PMA but not with RA or DMF also produced an inhibitor to UK that was identified as PAI-2, the plasminogen activator inhibitor that is produced by monocytes. Because of its dual capacity to produce both UK and PAI, the HL-60 cell line represents a useful model for studies of the fibrinolytic mediators that are generated and released by leukemia cells.

Role of chlorinated hydrocarbon pesticides in abortions and premature labour.

Premature labour and abortion possibly due to the high concentration of organochlorine pesticides have been investigated. Considerably higher amounts of organochlorine pesticide residues have been detected by gas liquid chromatography in the circulating blood and placental tissue of the women undergoing abortion or premature labour as compared with pregnant women in full term labour. The differences are highly significant for all pesticides estimated.

Inhibition of beta-interferon augmentation of murine natural killer cytotoxicity by gangliosides.

Interferons cause augmentation of natural killer (NK) cell activity, which might be a major reason for their antitumor effect. Antiviral and antigrowth effects of mouse beta interferon are inhibited by mono-, di-, and trisialogangliosides commonly found in brain extracts, but also in membranes of many other cells. Results presented in this report show that preincubation of mouse beta interferon with a brain ganglioside mixture or its isolated major components Gm1, Gd1a, Gd1b, and Gt1b (see Aberrations) prior to addition to effector spleen cells, inhibits NK-cell enhancement due to interferon in a dose-dependent manner. When spleen cells are treated with individual gangliosides alone, spontaneous NK cell activity is not affected. Pretreatment of effector cells with gangliosides prior to addition of interferon does not inhibit subsequent augmentation of NK cell activity by beta interferon. Also, target susceptibility remains unaltered in the presence of gangliosides. Thus the inhibitory effect of gangliosides appears to involve competition for interaction of beta interferon with the NK cells.

Effect of dengue virus on procoagulant and fibrinolytic activities of monocytes.

Some of the fibrinolytic and coagulation enzymes that monocytes produce are urokinase, a plasminogen activator (PA); a PA-specific inhibitor (PAI); and procoagulant activity (PCA), which has been characterized as tissue factor. Dengue infection in vivo is restricted to monocytes; however, it is unknown if dengue-infected monocytes undergo alterations in the production of PA, PAI, and PCA. This issue was addressed in studies in which monocytes were infected in vitro with dengue 2 virus in serum-free medium in the presence of enhancing antibody. No urokinase activity was detected in either control or infected cells or in their supernatants. Infection of monocytes with dengue 2 virus resulted in an almost threefold increase in PAI activity in cells and supernatants. No change in relation to the control was observed in PCA generated by the infected cells. These data indicate that dengue 2 infection enhances the production of PAI from monocytes without altering PA or PCA.

Recognition and management of heparin-induced thrombocytopenia (HIT) and thrombosis.

An immune response to heparin, which is clinically manifested by the development of thrombocytopenia with or without thrombosis, is stimulated by a complex of heparin with platelet factor 4 (PF4). The primary thrombotic events in patients with heparin-induced thrombocytopenia (HIT) are more frequently venous than arterial. The development of antibodies, however, does not always result in thrombocytopenia or in catastrophic events. The antibodies, which are of the IgG, IgM, and IgA isotypes, can be easily measured by an ELISA that contains a complex of heparin-platelet factor 4 (PF4). Initial antibody formation can be greatly reduced by limiting the exposure to unfractionated heparin or by the use of low-molecular-weight heparin. For those patients who require anticoagulation and who have antibodies to heparin-PF4, danaparoid (Orgaran), a low-molecular weight heparinoid that does not react with the antibodies, is now commercially available; argatroban, a thrombin-specific inhibitor, can also be obtained for compassionate use. The use of these agents during anticoagulation with warfarin is preferable to the simple discontinuation of heparin and intitiation of warfarin, because the latter treatment can result in ongoing thrombosis.

Kinetics of amino acid metabolism in the ovine fetus in utero.

The recycling of amino acids in chronically catheterized ovine fetuses was determined using a single intravenous injection of a mixture of fifteen (U-14C) amino acids. The specific activity--time data were fitted to a three exponential curve which best represented the disappearance of the tracer from the fetal blood plasma. Based on the rates of total entry and irreversible disposal of amino acid carbon, it was determined that 85% of the free amino acid pool was recycled. In addition, 23.6% of the administered label was recovered in fetal blood 14CO2. No radioactivity was detectable in the maternal plasma after the injection of tracer into the fetus. The mean total residence time of the label in the fetuses studied was 7.22 +/- 0.89 minutes and corresponded to 6.46 +/- 0.93 metabolic cycles. The high rate of recycling of amino acids in the plasma pool is a reflection of the rapid turnover of proteins in the actively growing fetus.