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Biomedical subjects

Y Gruel

Publications and source records attributed to Y Gruel.

At least 37 records · Page 2Linked to original sources

Thromboembolic prophylaxis with danaparoïd (Orgaran) in a high-thrombosis-risk pregnant woman with a history of heparin-induced thrombocytopenia (HIT) and Widal's disease.

There is no consensus concerning thromboembolic prophylaxis in high-risk pregnant women with a previous history of heparin-induced thrombocytopenia. An alternative anticoagulant therapy is danaparoïd, whereas unfractioned and low-molecular-weight heparin therapy is contraindicated. We report a case of successful thrombosis prophylaxis using danaparoïd in a high-thrombosis-risk pregnant woman with a history of heparin-induced thrombocytopenia during a previous pregnancy and Widal's disease.

Adult↗

Antibodies to platelet factor 4-heparin after cardiopulmonary bypass in patients anticoagulated with unfractionated heparin or a low-molecular-weight heparin : clinical implications for heparin-induced thrombocytopenia.

BACKGROUND: Cardiopulmonary bypass (CPB) induces platelet activation with release of platelet factor 4 (PF4), and patients are exposed to high doses of heparin (H). We investigated whether this contributes to the development of antibodies to H-PF4 and heparin-induced thrombocytopenia (HIT). METHODS AND RESULTS: CPB was performed with unfractionated heparin (UFH) in 328 patients. After surgery, patients received UFH (calcium heparin, 200 IU. kg-1. d-1) (group 1, n=157) or low-molecular-weight heparin (LMWH, Dalteparin, 5000 IU once daily) (group 2, n=171). Eight days after surgery, antibodies to H-PF4 were present in 83 patients (25.3%), 46 in group 1 and 37 in group 2 (P=0.12). Most patients (61%) had IgG1 to H-PF4, but only 8 samples with antibodies induced platelet activation with positive results on serotonin release assay. HIT occurred in 6 patients in group 1, but no thrombocytopenia was observed in subjects receiving LMWH, although 2 had high levels of antibodies with positive serotonin release assay results. When antibodies to H-PF4 were present, mean platelet counts were lower only in patients with FcgammaRIIA R/R131 platelets. CONCLUSIONS: These results provide evidence that the development of antibodies to H-PF4 after CPB performed with UFH is not influenced by the postoperative heparin treatment. The antibodies associated with high risk of HIT are mainly IgG1, which is present at high titers in the plasma of patients continuously treated with UFH.

Adolescent↗

Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription-polymerase chain reaction.

We developed fast and sensitive reverse transcription-polymerase chain reaction (RT-PCR) procedures to study the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI-1) mRNA in human endothelial cells and monocytes. The sensitivity of the technique was checked by performing RT-PCR with limited numbers of cells. Cells were stimulated either with tumor necrosis factor (TNF-alpha) or endotoxin to induce TF mRNA expression or with phorbol ester to increase TFPI-1 mRNA expression. Thus, RT-PCR specific for TF mRNA provided detection from as few as 10(3) TNF-alpha stimulated endothelial cells and 5 x 10(2) monocytes stimulated by endotoxin. TF mRNA expression was increased by TNF-alpha in endothelial cells and in monocytes stimulated by endotoxin. Elevated expression of TF mRNA in monocytes without stimulation by endotoxin was mainly related to cell adhesion. TFPI-1 mRNA was constitutively expressed in endothelial cells and was detected in only 5 x 10(2) unstimulated cells and 10(2) phorbol ester-stimulated cells. Expression was increased upon stimulation with phorbol ester. With this technique, TFPI-1 mRNA in monocytes was rather low even when cells were stimulated with phorbol ester or after adhesion.

Blood Cells↗

Decision analysis for use of platelet aggregation test, carbon 14-serotonin release assay, and heparin-platelet factor 4 enzyme-linked immunosorbent assay for diagnosis of heparin-induced thrombocytopenia.

The value of the platelet aggregation test, carbon 14-labeled serotonin release assay (SRA), and heparin-platelet factor 4 enzyme-linked immunosorbent assay (H-PF4 ELISA) for the diagnosis of heparin-induced thrombocytopenia was evaluated by studying blood samples from 100 patients with suspected heparin-induced thrombocytopenia, and categorized into 4 clinical groups: unlikely (n = 22), possible (34), probable (36), and definite (8) thrombocytopenia. Results of the platelet aggregation test were positive in 40 of 44 patients with probable or definite heparin-induced thrombocytopenia (sensitivity 91%) and in 5 of 22 unlikely to have heparin-induced thrombocytopenia (specificity 77%). The SRA exhibited sensitivity of 88% and negative predictive value of 81%, close to those values for the platelet aggregation test; specificity and positive predictive value were 100%. The sensitivity of the heparin-PF4 ELISA was 97%, with specificity 86%, and a positive correlation was recorded between the level of antibodies to H-PF4 and clinical score (P = 0.66). When ELISA was used with the platelet aggregation test or SRA, positive predictive value and specificity were 100% when both tests yielded positive results, and negative predictive value was 100% when both tests yielded negative results. A biologic flow chart was designed that presented a choice based on the results of the platelet aggregation test or SRA in association with ELISA, and enabled more accurate and specific identification of heparin-induced thrombocytopenia.

Carbon Radioisotopes↗

Human T lymphocyte proliferative response to resting porcine endothelial cells results from an HLA-restricted, IL-10-sensitive, indirect presentation pathway but also depends on endothelial-specific costimulatory factors.

To investigate the mechanisms of cellular rejection in pig-to-human xenotransplantation, the proliferation of different human purified lymphocyte subpopulations in response to swine leukocyte Ag class II-negative porcine aortic endothelial cells (PAEC) was measured in the presence or absence of human autologous adherent cells (huAPC). CD8+ lymphocytes proliferated moderately in the absence of huAPC, and the immune response was slightly increased when huAPC were added. CD56+ lymphocytes failed to proliferate in response to PAEC whether huAPC were present or not. CD4+ lymphocytes alone did not proliferate in response to PAEC, but a strong proliferative response was observed in the presence of metabolically active huAPC. This response was totally abolished by mAbs directed against HLA class II molecules or by pretreatment of huAPC by human IL-10. Even in the presence of huAPC, CD4+ lymphocytes failed to respond to fixed PAEC or to PAEC-lysates, suggesting that PAEC must be viable to support lymphocyte proliferation. Finally, none of the nonendothelial porcine adherent cells tested was able to induce human lymphocyte proliferation, despite the fact that they also provided a large set of xenogeneic peptides. Our results show that the indirect presentation pathway of xenoantigens by huAPC to CD4+ lymphocytes is crucial in the response to porcine endothelial cells, and that IL-10 could be of therapeutic interest to prevent human lymphocyte activation by this pathway. Furthermore, we demonstrated that stimulatory signals specifically provided by endothelial cells are also necessary for this huAPC-restricted proliferative response.

Animals↗

Human allogeneic lymphocytes trigger endothelial cell tissue factor expression by a tumor necrosis factor-dependent pathway.

Lymphocyte adhesion to endothelial cells and the extravascular deposition of fibrin are 2 important processes during pathologic situations such as allograft rejection. Tissue factor (TF) expression was therefore measured on human umbilical vein endothelial cells (HUVECs) after coculture with allogeneic lymphocytes (PBLs) by a factor Xa generation assay. When cocultured with PBLs, HUVECs expressed strong procoagulant activity related to the TF/factor VII-dependent pathway, which was enhanced when endothelial cells were treated with interferon-gamma (IFN-gamma). The highest TF activity was measured when 10(5) lymphocytes were incubated with 10(4) HUVECs (ratio 10: 1) for 4 hours, a time-dependent course similar to that obtained with tumor necrosis factor-alpha (TNF-alpha), and direct contact between the 2 cell types was necessary. PBL-induced TF activity was inhibited by cycloheximide or actinomycin D, indicating active protein synthesis that was confirmed by the increase in TF mRNA detected by reverse transcription-polymerase chain reaction. It was then demonstrated that 1 of the primary signaling pathways leading to endothelial cell TF expression was a rapid initial interaction between membrane TNF expressed on PBLs and the 75-kd TNF receptor, with subsequent involvement of platelet-activating factor and P-selectin. Finally, we showed that the transduction of external signals involving the activation of protein kinase C and protein tyrosine kinases also contributed to the regulation of TF expression.

Cells, Cultured↗

The kinetics of antibodies against hepatitis C virus may predict viral clearance in exposed hemophiliacs.

Several studies have reported the spontaneous loss of hepatitis C virus (HCV) antibodies in HCV-exposed persons. However, the relationship between seroreversion and spontaneous virus clearance has yet to be precisely determined in a single homogeneous population of untreated immunocompetent patients. In this study, 32 human immunodeficiency virus-seronegative hemophiliacs who had been exposed to HCV were followed for a mean duration of 141 months; 22 remained chronic carriers (68.8%). All but 1 of the nonviremic patients (90.0%) showed partial (8 cases) or complete (2 cases) seroreversion. In contrast, all but 1 of the viremic patients (95.1%) had a stable serologic profile when analyzed by a recombinant immunoblot assay. The results indicate that any HCV antibody-positive immunocompetent patient with no detectable serum HCV RNA and normal alanine aminotransferase values and whose serial samples show a progressive decrease in the level of HCV antibodies present may be considered as having a resolved infection.

Adolescent↗

[Heparin-induced thrombopenia. Clinical manifestations and physiopathology].

CLINICAL SIGNS: Heparin-induced thrombocytopenia is a clinically interesting model of immunological thrombocytopenia because thrombus formation occurs much more frequently than hemorrhage, in some patients, major thrombocytopenia is observed with signs of consumption coagulopathy. HIGH INCIDENCE OF THROMBUS FORMATION: Venous thrombi predominate with severe consequences such as pulmonary emboli or more rarely distal gangrene. Artenal thrombi are less frequent but are highly suggestive and often lead to acute ischemia in the lower limbs. IMMUNOLOGICAL MECHANISM: Severe forms of heparin-induced thrombocytopenia result from an immunological mechanism and are associated with IgG antibody production. The Fc fragment of these antibodies activate platelets. Heparin-dependent antibodies preferentially recognize antigenic complexes formed by the heparin-platelet facto 4 association although certain other antigens (IL-8 or NAP-2 are also recognized. Thrombus formation results from platelet activation and enhanced coagulability which can be favored by antibody binding to endothelial cells and synthesis of tissue factor. PRACTICAL ASPECTS: Severe heparin-induced thrombocytopenia is particularly frequent in medical and surgical situations involving platelet activation and treatments by infractionated heparins. No particular phenotype of the IgG Fc receptor has been identified. Likewise no specific risk factors have been found. As effective antithrombin therapy is required in most cases, Xa inhibitors or thrombin inhibitors are useful.

Anticoagulants↗

Differences in specificity of heparin-dependent antibodies developed in heparin-induced thrombocytopenia and consequences on cross-reactivity with danaparoid sodium.

Heparin-induced thrombocytopenia (HIT) is frequently associated with antibodies (Abs) to heparin-PF4 complexes (H-PF4). In order to investigate whether there are variations in specificity of Abs, we studied 63 samples from patients with suspected HIT. Two groups of samples were separated after comparing their reactivity against H-PF4 or recombinant PF4 (r-PF4) using ELISA. In group Ab1 (n = 46), Abs only or mainly bound to H-PF4 complexes and thus most of the epitopes recognized probably involved both heparin and PF4. In group Ab2 (n = 17), Abs exhibited similar reactivity to r-PF4 and H-PF4, and the antigens recognized were possibly neoepitopes mainly expressed by modified PF4 and by H-PF4 complexes. Platelet activation tests were positive with 56 samples containing high titres of Abs to H-PF4. Most samples (n = 59) contained IgG antibodies, often associated with IgA antibodies which were more frequently found in group Ab2, and/or IgM. With unfractionated heparin treatment, HIT was associated with Ab1 or Ab2 antibodies, whereas only Ab1 antibodies were detected after low-molecular-weight heparin (LMWH). Furthermore, cross-reactivity with danaparoid sodium was present only in group Ab1 and mainly involved LMWH-treated patients.

Adult↗

Pseudo-homozygous activated protein C resistance due to coinheritance of heterozygous factor V Leiden mutation and type I factor V deficiency. Variable expression when analyzed by different activated protein C resistance functional assays.

The laboratory diagnosis of activated protein C (APC) resistance is based on a weak anticoagulant response to APC using a chronometric procedure confirmed in almost all cases by molecular diagnosis of the FV Leiden mutation. A recently-developed Xa-based assay (Accelerimat, Biomerieux) was compared with two different activated partial thromboplastin time (APTT)-based procedures (Coatest APC resistance and Modified Coatest, Chromogenix) in 115 patients with a personal or familial history of thrombotic disease, or both, being studied for the FV Leiden mutation. Our results confirmed the improvement in specificity for the FV Leiden mutation when the APTT-based assay was performed after dilution of samples in FV-deficient plasma (Modified Coatest). However, five patients who were heterozygous for the FV Leiden mutation appeared to be homozygous when tested by both APTT-based assays. These patients, belonging to three different families, had a FV type I deficiency with FV plasma levels between 43 and 64%. In contrast, the Xa-based method was not influenced by the decrease in plasma FV levels. Thus, this procedure is more specific than APTT-based assays to predict the genotype status of the FV Leiden mutation.

Adolescent↗

[Thrombopenia induced by heparin. From physiopathology to treatment].

Heparin-induced thrombocytopenia is an uncommon but potentially dangerous adverse effect of heparin therapy. Late onset thrombocytopenia is usually observed several days after initiating treatment and can be distinguished from early-onset benign thrombocytopenia which is more moderate and transitory and which results from a direct interaction between heparin and platelet membrane proteins which potentialize ADP-induced aggregation. Severe late-onset thrombocytopenia clearly results from an immunological mechanism due to the development of heparin-independent antiplatelet antibodies, often IgGs. These antibodies do not cause cell lysis but have a platelet-activating effect with release of the contents of the dense alpha granulations. This cell activation requires the formation of a heparin-dependent antibody-platelet complex. In most cases, platelet factor 4, an alpha granule protein, would be implicated. The antibody-platelet interaction has an activating effect following binding of the IgG Fc fragment to the Fc gamma RII receptor. The antibodies could also bind, favoring the development of thrombosis. The diagnosis of heparin-induced thrombocytopenia is evidenced by a platelet count under 100 Giga/l, usually from the 5th to 20th week of heparin therapy. Occasionally, the only sign is the low platelet count (drop of over 40% from pretreatment levels). Coagulation activation can lead to diffuse consumption coagulopathy in about 25% of the cases. Clinically, thrombosis is observed in about one half of the cases. Arterial thrombosis is the most characteristic and concerns the aorta and its branches as well as cerebral, coronary, mesenteric, renal and upper limb arteries. Venous thrombosis may be underestimated as they often occur as paradoxical recurrence after heparin therapy. Hemorrhage is much less frequent (less than 20% of cases) and often benign. To diagnose heparin-induced thrombocytopenia, one must eliminate other potential causes (infection, drug...), observe complete normalization of platelet count after heparin withdrawal, and demonstrate heparin-dependent antibodies in the plasma or serum. Different laboratory tests are quite helpful but have variable sensitivity. The incriminated heparin must be discontinued immediately. Use of low-molecular-weight heparins, even when cross-reactivity is not demonstrated in vitro, is not recommended. Other compound however, such as Orgaran 10,172 (or Lomoparan, appear to be the best choice. The action of antivitamin K agents is delayed and, due to the early dissociated drop in protein C at the beginning of treatment further raise the major risk of thrombosis. Classic antiplatelet agents such as aspirin are ineffective if used alone. More powerful compounds such as Ilomedine, are not available for this indication and are difficult to titrate. Part of the therapeutic problem with heparin-induced thrombocytopenia may be resolved with the advent of molecules with a direct antithrombin effect such as hirudine or its analogues. As suggested by a recent study, widespread use of low-molecular-weight heparin will undoubtedly have a highly significant effect on reducing the number of cases of severe thrombocytopenia.

Anticoagulants↗

Evolution of blood coagulation activators and inhibitors in the healthy human fetus.

Blood coagulation proteins were determined in 285 healthy fetuses from 19 to 38 weeks' gestation and compared with those of 60 normal full-term newborns and 40 adult controls. Prolongation of the coagulation screening tests, prothrombin time, activated partial prothrombin time, and thrombin clotting time, in fetuses throughout intrauterine life was explained by low levels of vitamin K-dependent factors (II, VII, IX, and X), contact factors (XI, XII, prekallikrein, and high-molecular-weight kininogen), factor V, factor VIII, and fibrinogen. Low levels of antithrombin III, heparin cofactor II, protein C and protein S, and tissue factor pathway inhibitor were also found, and these probably contributed to a satisfactory hemostatic balance. Some of these parameters were evaluated by both immunologic and functional assays to detect possible "fetal" proteins. An increase in factor levels was observed after the thirty-fourth week of intrauterine life for most of the coagulation activators and inhibitors, but only factors V and VIII reached adult values at birth. This study therefore showed that fetal hemostasis is a dynamic system that evolves gradually toward the neonatal state and then toward the adult state.

Adult↗

Human lymphocyte adhesion to xenogeneic porcine endothelial cells: modulation by human TNF-alpha and involvement of VLA-4 and LFA-1.

Considering that in the allogeneic situation the adhesion of recipient lymphocytes to donor endothelial cells initiates the cellular rejection, we questioned the possible occurrence of a similar process in the xenogeneic situation. The adhesion of human peripheral blood lymphocytes (PBL) to porcine aortic endothelial cells (PAEC) was thus studied in an in vitro porcine-to-human xenogeneic model. It was found that 25.9% of human PBL adhered to resting PAEC. Furthermore, this adhesion increased significantly when the PAEC were stimulated by the human cytokine TNF-alpha (tumor necrosis factor-alpha). The effect of human TNF-alpha was concentration- and time-dependent and was maximal (from 25.9% to 35.6%) with 100 U/ml during 6 h. Moreover, blocking experiments with monoclonal antibody (mAb) demonstrated the role of the PBL adhesion molecules LFA-1 and especially VLA-4. Indeed, an anti-CD11a mAb decreased PBL adhesion to resting PAEC by 17.1% and to TNF-alpha stimulated PAEC by 16.9%, whereas an anti-CD49d mAb decreased dramatically PBL adhesion to resting PAEC by 53.1% and to TNF-alpha stimulated PAEC by 41.0%. Finally, phenotypic analysis of the adherent PBL showed that 50.5% of adherent cells to resting PAEC were NK (natural killer) cells, whereas 50.7% of adherent cells to TNF-alpha stimulated PAEC were T lymphocytes, showing the preferential adhesion of NK cells to resting PAEC, and that the stimulation of the PAEC with human TNF-alpha affects predominantly T lymphocyte adhesion. These results indicate that human PBL could bind to xenogeneic PAEC and that this interaction could be a first step of a xenogeneic cellular rejection.

Animals↗