Microparticle-mediated thrombin generation assay: increased activity in patients with recurrent thrombosis.

BACKGROUND: Circulating cell-derived microparticles (MP) are important players in thrombogenesis, attributed in part to tissue factor (TF) carried on them. We developed MP-mediated thrombin generation assay (TGA) and measured a series of patients with thrombosis (TBS) and normal controls (NC). METHODS: MP were isolated from plasma of 66 patients with TBS and 34 NC. The MP were resuspended in normal pooled particle-free plasma (PFP) containing corn trypsin inhibitor (to inhibit contact pathway). MP mediated TGA yields three parameters: lag time, peak and rate. This method is not influenced by anticoagulant therapy. Of the TBS patients, 41 had only a single thrombosis (S-TBS) and 25 had recurrences (R-TBS) within a 5-year period. In parallel, MP were quantitated by flow cytometry, and cell origin was determined: endothelial cells (EMP), leukocytes (LMP), red cells (RMP) and platelets (PMP). RESULTS: MP from all TBS patients exhibited higher thrombin generation than NC by all three TGA parameters. R-TBS had significantly greater TGA values than S-TBS, reflected in higher peak and rate, and shorter lag time. MP numbers were also higher in TBS vs. NC, for all MP subtypes, and were significantly higher in R-TBS than S-TBS (except LMP). All MP levels correlated with thrombin generation (P < 0.0001), most closely between PMP and peak (R = 0.47) and rate (R = 0.43). CONCLUSIONS: MP-mediated TGA is a novel way to assess functional procoagulant activity of MP. Enhanced MP-mediated TGA was demonstrated in TBS patients, and significantly higher activity in R-TBS. These findings support a major role of MP in thrombogenesis.

Mathematical modeling of T-cell activation kinetic.

T-cell activation is a crucial step in mounting of the immune response. The dynamics of T-cell receptor (TCR) specific recognition of peptide presented by major histocompatibility complex (MHC) molecule decides the fate of the T cell. Several biochemical interactions interfere resulting in a highly complex mechanism that would be difficult to understand without computer help. The aim of the present study was to define a mathematical model in order to approach the kinetics of monoclonal T-cell-specific activation. The reaction scheme was first described and the model was tested using experimental parameters from the published data. Simulations were concordant with experimental data showing proportional decrease of membrane TCR and production of interleukin-2 (IL-2). Agonist and antagonist peptides induce different levels of intracellular signal that could make the yes or no decision for entry to cell cycle. Different conditions (peptide concentrations, initial TCR density and exogenous IL-2 levels) can be tested. Several parameters are missing for parameters estimation and adjustment before it could be adapted for a polyclonal T-cell reaction model. However, the model should be of interest in setting experiments, simulation of clinical responses and optimization of preventive or therapeutic immunotherapy.

Endothelial microparticles induce formation of platelet aggregates via a von Willebrand factor/ristocetin dependent pathway, rendering them resistant to dissociation.

Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL(-1)), EMP induced up to 95% of platelets to aggregate. EMP-platelet interaction was 80% blocked by anti-CD42b, or by 0.1 microm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate-P) dissociated 50% within 15-25 min following 1:20 dilution. In contrast, aggregates formed with EMP persisted two- to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin-induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

RGS14 is a novel Rap effector that preferentially regulates the GTPase activity of galphao.

In an attempt to elucidate the physiological function(s) of the Ras-related Rap proteins, we used the yeast two-hybrid system and isolated a cDNA encoding a protein that interacts with both Rap1 and Rap2, but not with Ras; the use of Rap2 mutants showed that this interaction is characteristic of a potential Rap effector. This protein was identified as RGS14, a member of the recently discovered family of RGS ('regulators of G-protein signalling') proteins that stimulate the GTPase activity of the GTP-binding alpha subunit of heterotrimeric G-proteins (Galpha). Deletion analysis, as well as in vitro binding experiments, revealed that RGS14 binds Rap proteins through a domain distinct from that carrying the RGS identity, and that this domain shares sequence identity with the Ras/Rap binding domain of B-Raf and Raf-1 kinases. RGS14 is distinguished from other RGS proteins by its marked preference for Galpha(o) over other Galpha subunits: RGS14 binds preferentially to Galpha(o) in isolated brain membranes, and also interacts preferentially with Galpha(o) (as compared with Galpha(i1)) to stimulate its GTPase activity. In adult mice, RGS14 expression is restricted to spleen and brain. In situ hybridization studies showed that it is highly expressed only in certain areas of mouse brain (such as the CA1 and CA2 regions of the hippocampus), and that this pattern closely resembles that of Rap2, but not Rap1, expression. Double in situ hybridization experiments revealed that certain cells in the hippocampus express both RGS14 and Galpha(o), as well as both RGS14 and Rap2, showing that the interaction of RGS14 with Galpha(o) and Rap2 is physiologically possible. Taken together, these results suggest that RGS14 could constitute a bridging molecule that allows cross-regulation of signalling pathways downstream from G-protein-coupled receptors involving heterotrimeric proteins of the G(i/o) family and those involving the Ras-related GTPase Rap2.

Establecimiento en Cuba de la técnica de fertilización in vitro (FIV) y transferencia embrionaria (TE): informe preliminar / Establishent in Cuba of fertilization in vitro technique (FIV) and embryo transference (ET): preliminary report

La infertilidad constituye un serio problema de salud al nivel mundial con repercusiones psicosociales de extrema importancia. La fertilización in vitro y transferrencia embrionaria es considerada actualmente el tratamiento de elección en gran parte de las causas de infertilidad masculina y femenina. Se estableció en nuestro país el primer Programa de este tipo y se logró el primer éxito a los 10 meses de comenzado el trabajo. Hasta el presente se han obtenido 8 embarazos, de los cuales hay 3 nacimientos, 1 embarazo en curso, 1 ectópico y 3 abortos espontáneos tempranos. La tasa de embarazo es de 12,3

Establecimiento en Cuba de la técnica de fertilización in vitro (FIV) y transferencia embrionaria (TE): informe preliminar / Establishment in Cuba of fertilization in vitro technique (FIV) and embryo transference (ET): preliminary report

La infertilidad constituye un serio problema de salud al nivel mundial con repercusiones psicosociales de extrema importancia. La fertilización in vitro y transferrencia embrionaria es considerada actualmente el tratamiento de elección en gran parte de las causas de infertilidad masculina y femenina. Se estableció en nuestro país el primer Programa de este tipo y se logró el primer éxito a los 10 meses de comenzado el trabajo. Hasta el presente se han obtenido 8 embarazos, de los cuales hay 3 nacimientos, 1 embarazo en curso, 1 ectópico y 3 abortos espontáneos tempranos. La tasa de embarazo es de 12,3

Chromatographic pattern of circulating prolactin in ovulatory hyperprolactinemia.

Hyperprolactinemia is a major cause of anovulation and female infertility. However, we found 22 hyperprolactinemic patients with preserved ovarian function as assessed by midluteal plasma progesterone levels greater than 7 ng/ml. We performed a gel chromatography on the plasma of six of those patients to study the circulating pattern of prolactin (PRL). In all six patients we found that the vast majority of circulating PRL corresponds to big,big and big PRL, with only a small proportion of monomeric biologically active PRL. This finding might explain the preserved luteal function in those women and might have therapeutic implications.