Quercetin 3,7,3',4'-tetrasulphated isolated from Flaveria bidentis inhibits tissue factor expression in human monocyte.

Sulphated esters of the flavonoids sulphated quercetin 3,7,3',4'-tetrasulphated (QTS) and quercetin 3-acetyl-7,3,4'-trisulphate (ATS), isolated from Flaveria bidentis, have demonstrated anticoagulant and antiplatelet properties. In this study, we examined if both compounds affected the expression of the procoagulant tissue factor (TF) induced by lipopolysaccharide (LPS) on human monocyte. Monocytes were pretreated with different concentrations of each flavonoid (0.1-500 µM), followed by a 4h incubation with LPS in order to induce TF expression. Results of the TF expression showed different behaviors for the two flavonoids studied. A slight inhibitory effect on the TF expression was detected at a QTS concentration of 0.1 µM, but from 1 µM onwards a significant inhibitory effect that remained up to 500 µM could be observed. In contrast, ATS induced a poor inhibitory effect on TF expression at all concentrations tested. These results suggest that QTS has another antithrombotic property, to be added to its already renowned ability as an anticoagulant and antiplatelet compound.

Cofactor dependence and isotype distribution of anticardiolipin antibodies in viral infections.

BACKGROUND: Antibodies to cardiolipin (aCLs) are often detected in patients with autoimmune disorders or infectious diseases. OBJECTIVE: To investigate the distribution of aCL isotypes and requirement of protein cofactor in viral infections in order to establish the importance, if any, of these antibodies in these infectious diseases. PATIENTS AND METHODS: The isotype distribution of aCLs in the sera from 160 patients with infection caused by HIV-1 (n=40), hepatitis A virus (n=40), hepatitis B virus (n=40), or hepatitis C virus (n=40) was studied by standardised enzyme linked immunosorbent assay (ELISA) in the presence and absence of protein cofactor (mainly beta2-glycoprotein I). Serum samples from healthy volunteers and patients with syphilis and antiphospholipid syndrome were also included and served as negative and positive control groups respectively. RESULTS: The prevalence of one or more aCL isotypes in serum of patients with HIV-1, hepatitis A virus, hepatitis B virus, or hepatitis C virus infection was 47%, 92%, 42%, and 17% respectively (principally IgM and/or IgA). Most of these antibodies were mainly cofactor independent. CONCLUSIONS: The presence of aCLs in viral infections is principally cofactor independent, suggesting that cofactor dependence of the aCLs should be assessed to distinguish subjects most likely to suffer from clinical symptoms observed in the presence of these antibodies.

Experimental retinopathy of prematurity: angiostatic inhibition by nimodipine, ginkgo-biloba, and dipyridamole, and response to different growth factors.

PURPOSE: To investigate whether commonly used vasodilating drugs ameliorate angiogenesis in experimental retinopathy of prematurity (ROP), and to study the response of these drugs to different growth factors. METHODS: We used a rat and mouse model of oxygen-induced ischemic retinopathy. Animals were treated with nimodipine, gingko-biloba and dipyridamole intraperitoneally starting the day before exposure to room air (day 1). Controls were injected with vehicle solution only. Eyes were processed histopathologically with serial sections and neovascularization was measured by counting the nuclei within the retinal internal limiting membrane, by a masked observer. Retinal and vitreous tissues were assayed by ELISA for VEGF, PDGF and TGFbeta2. RESULTS: Nimodipine significantly inhibited the growth of new vessels in rats. The number of nuclei was 310 +/- 69 in the control group (n:14) and 121 +/- 53 in the treated ones (n:14), (p<0.0005). Similar results were found with ginkgo-biloba extracts: 344 +/- 53 (n:15) in controls, and 136 +/- 29 (n:11) in treated ones (p<0.0005), and with dipyridamole: 303 +/- 69 (n:13) in controls, and 131 +/- 48.5 in treated rats (p<0.0005). Results were similar in mice. 186 +/- 45 (n:7) nuclei counted in controls against 90 +/- 25 (n:6) for dipyridamole treated (p<0.0005); and 81 +/- 21 for ginkgo-biloba treated animals (p<0.0005). A gradual, very significant increase in VEGF values in response to relative hypoxia (room air) contrasted with the significant inhibition noted both with ginkgo-biloba extracts and dipyridamole. TGFbeta2 and PDGF both showed a gradual increase in relative hypoxia at days 2 and 4 of room air (p<0.0005). Treated animals showed marked inhibition of the three growth factors. CONCLUSIONS: All three drugs markedly inhibited angiogenesis in experimental ROP. Growth factors were elevated in hypoxic conditions. Treated animals showed significant decreases of PDGF, VEGF, and TGFbeta2 in retinal and vitreous tissues.

Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

Distribution of lupus anticoagulant and anticardiolipin antibody isotypes in a population with antiphospholipid syndrome.

OBJECTIVE: To analyze the distribution of lupus anticoagulant (LAC) and anticardiolipin antibody (aCL) isotypes in a population with antiphospholipid syndrome and to explore whether there is an association with the site of thrombotic episodes and the number of recurrent spontaneous abortions. METHODS: Ninety-two patients (73 female, 19 male) with positive LAC and/or aCL were included as 2 groups: (1) 20 patients with secondary antiphospholipid syndrome (APS) (16 had thrombotic episodes and 4 thrombocytopenia); (2) 72 patients with primary APS (31 presented thrombotic episodes and 41 had recurrent spontaneous abortion). RESULTS: In Group 1 seven of 20 (35%) patients with secondary APS had IgG aCL, 9 (45%) had both IgG/IgM aCL, and 2 (10%) had IgM aCL; the remaining patients had combinations of aCL isotypes. In Group 2 patients with primary APS, IgG aCL was positive in 41%, IgG/IgM mixture in 21%, and 15% of patients had combinations of the 3 isotypes. Sixteen of 20 (80%) patients with secondary disease and 37 of 72 (51%) with primary disease tested positive for LAC. CONCLUSION: The presence of one or any mixture of isotype of aCL with or without LAC is not associated with the site of thrombosis (venous or arterial). On the contrary, in the patients with primary APS, the presence of the 3 aCL isotypes plus LAC was associated with a higher number of recurrent spontaneous abortions compared to other possible combinations of aCL isotypes.

Meningitis neonatal por estreptococcus pneumoniae / Neonatal meningitis caused by Streptococcus pneumoniae

Se comunica el caso de un recién nacido pretérmino que luego de un breve período libre de síntomas instaló una meningoencefalitis aguda supurada por Streptococcus pneumoniae, con estado de mal convulsivo refractario al tratamiento, falla multiorgánica y evolución fulminante. Se realizan consideraciones respecto a la frecuencia de esta entidad, los mecanismos patogénicos, la letalidad y el tratamiento, a la luz de la revisión bibliográfica

Enfermedad de Addison en un niño de raza negra / Addison's disease in a black boy

Se presenta la observación clínica de un niño de 4 años de raza negra, que fue admitido por una crisis addisoniana. La causa más frecuente de enfermedad de Addison es la adrenalitis autoinmune. Es una entidad rara en la infancia. Guiaron el diagnóstico la avidez por la sal, la severa deshidratación y los trastornos electrolíticos refractarios al tratamiento inicial. Lo confirmaron las dosificaciones hormonales y la excelente respuesta a la corticoterapia

Interaction of human tissue plasminogen activator (t-PA) with pregnancy zone protein: a comparative study with t-PA-alpha2-macroglobulin interaction.

Human pregnancy zone protein (PZP) is a major pregnancy-associated plasma protein strongly related to alpha2-macroglobulin (alpha2-M). Interactions of tissue plasminogen activator (t-PA) with PZP and alpha2-M were both investigated in vitro and the complexes were analyzed by polyacrylamide gel electrophoresis (PAGE). The results demonstrated that PZP-t-PA complex formation was evident within 1 h of incubation, whereas alpha2-M-t-PA complexes were formed after 18 h. Conclusions were supported by the following evidence: (i) PZP and alpha2-M complexes revealed changes of the mobility rate in non-denaturing PAGE, similar to those observed with alpha-Ms-chymotrypsin; (ii) both PZP and alpha2-M formed complexes of molecular size >360 kDa by SDS-PAGE, in accordance with the covalent binding of t-PA, which was previously reported for other proteinases; and (iii) PZP underwent a specific cleavage of the bait region with appearence of fragments of 85-90 kDa as judged by reducing SDS-PAGE. In contrast, the proteolytic attack on alpha2-M was found to occur more slowly, requiring several hours of incubation with t-PA for generation of an appreciable amount of fragments of 85-90 kDa. The appearance of free SH-groups of alpha-Ms was further investigated by titration with 5, 5'-dithiobis(2-nitrobenzoic acid). The maximal level of SH-groups raised was 3.9 mol/mol of PZP and 3.5 mol/mol of alpha2-M, indicating approximately one SH-group for each 180-kDa subunit. Finally, t-PA activity in PZP-t-PA complex was evaluated by measuring the hydrolysis of the chromogenic substrate Flavigen t-PA. Our results revealed that prolongation of the incubation period of this complex increased t-PA-mediated hydrolysis of Flavigen t-PA until a plateau was reached, approximately between 60 and 120 min. The present study suggests that PZP, by binding to t-PA, may contribute to the control of the activity of proteinases derived from fibrinolytic systems.

A novel functional assay of protein C in human plasma and its comparison with amidolytic and anticoagulant assays.

A simple and fast method for the quantitative determination of protein C activity in plasma is here described. The first step consists in the conversion of protein C in the test sample into activated protein C by means of an activator isolated from Southern Copperhead venom. Subsequently, the degradation of factor Va, in presence of protein C-deficient plasma, is measured by the prolongation of the prothrombin time which is proportional to the amount of protein C in the sample. The dose-response curve showed a linear relationship from 6 to 150% protein C activity and the inter- and intra-assay reproducibility was 3.5% and 5.6% respectively. In normal subjects, a mean of protein C level of 98 +/- 15% of normal pooled plasma was found. Comparison with the anticoagulant assay in samples of patients with oral anticoagulant, liver cirrhosis, disseminated intravascular coagulation and severe preeclampsia revealed an excellent correlation (r = 0.94, p less than 0.001). Also, a similar correlation (r = 0.93, p less than 0.001) existed between amidolytic assay and the method here proposed for all the samples studied without including the oral anticoagulant group. These results allowed us to infer that this method evaluates the ability of protein C to interact with protein S, phospholipids, calcium ions and factor Va.

Prothrombin time in the monitoring of oral anticoagulant: a comparison of results using different thromboplastins.

The objective of this report is to compare the prothrombin time performed with thromboplastin of different tissues and species in patients under oral anticoagulant therapy as well as the way of expressing the results. The results showed that the ISI of the thromboplastin of human and rabbit brain are very close to the IRP BCT/253 (1.2 vs 1.1) and RBT/79 (1.3 vs 1.4), respectively. In contrast, the rabbit lung thromboplastin showed the greatest differences in the ISI values (1.6 vs 1.4) and in the CV (6.1%). The authors found significant statistical differences with the results of the prothrombin time as expressed in activity percentage (p less than 0.001) in three plasma pools of patients under different oral anticoagulant level for all thromboplastins studied. However, if the results are expressed in terms of INR, the values obtained are almost the same. The results here reported would demonstrate that the prothrombin time as INR allows the use of only one scheme for oral anticoagulant control when the thromboplastin reagent is calibrated according to the recommendations of the WHO.

Serologic study by competitive enzyme-linked immunosorbent assay of pregnancy-associated alpha 2-glycoprotein in hepatitis B virus infection.

Serum levels of pregnancy-associated alpha 2-glycoprotein (alpha 2-PAG) in patients with hepatitis B virus (HBV) infection were compared with those of healthy male subjects used as controls by a competitive enzyme-linked immunosorbent assay (ELISA). Assay parameters were optimized, and minimal detectable concentration was 100 ng/ml. The alpha 2-PAG levels in 22/26 acute HBV patients showed a very significant statistical difference when compared with controls (x2 = 19.93, p less than 0.0005). On the other hand, 8/8 chronic persistent HBV patients showed high levels with a range between 51 to 200 ug/ml (x2 = 18.16, p less than 0.0005). There was no significant difference between asymptomatic HBsAg carriers and controls. Although alpha 2-PAG apparently exhibits immunosuppressive properties similar to other factors present in HBV infection, follow-up studies are needed to elucidate its role in the natural evolution of this disease.