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G Potron

Publications and source records attributed to G Potron.

At least 19 recordsLinked to original sources
PubMed1999

Coexpression of tissue factor and tissue factor pathway inhibitor by human monocytes purified by leukapheresis and elutriation. Response of nonadherent cells to lipopolysaccharide.

BACKGROUND: Counterflow centrifugal elutriation is the method of choice for obtaining a large quantity of highly purified monocytes. In spite of the fact that this technique has been used for many years to isolate monocytes for cellular immunotherapy, it is not known whether the process of elutriation can stimulate tissue factor (TF) expression and therefore trigger coagulation in patients receiving these cell preparations. The aim of the present study is thus to identify TF and TF pathway inhibitor (TFPI) in elutriated monocytes and to evaluate their ability to trigger thrombin generation. STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis and elutriation in sterile, endotoxin-free conditions. TF and TFPI mRNA is detected by reverse transcription-polymerase chain reaction. TF and TFPI are measured by enzyme-linked immunosorbent assay in cell lysates. TF antigen expression on cell surface is evidenced by direct-flow cytometry. Two functional tests (a chronometric test and an amidolytic assay) assess the capacity of monocytes to trigger thrombin generation. The response to lipopolysaccharide (LPS) is evaluated with each technique. Monocytic cell line THP-1 is used as a positive control. RESULTS: Elutriated monocytes coexpress TF mRNA and TFPI mRNA. The expression of TF mRNA is dramatically increased by LPS activation. This is correlated with a 100-fold increase in the amount of TF antigen in monocyte lysates. Flow immunocytometry confirms the expression of TF antigen on cell membrane in response to LPS stimulation, whereas TFPI mRNA is slightly increased after LPS stimulation. The amount of TFPI antigen in cell lysates is small when compared to that in plasma. Elutriated monocytes have a strong potential to trigger thrombin generation in response to LPS. CONCLUSION: In spite of the coexpression of TF mRNA and TFPI mRNA, elutriated monocytes are capable of supporting prothrombinase activity. This should be taken into account in the evaluation of the safety of adoptive cellular immunotherapy.

Antigens↗
PubMed1999

[Vascular disease and cellular activation: exploration of cell adhesion phenotype by quantitative flow cytometry].

The pathogenesis of atheromatous and/or thrombotic vascular diseases involves rheological parameters, soluble mediators and cellular agents. The many studies that have tried to establish correlations between plasma factors, shear stress and the risk of ischemia have left some questions unanswered. Current exploration methods are now focusing on the determining role of cells. Activated cells express adhesion molecules on their membranes, which allow to communicate in a homo- or heterotypical manner. Quantifying adherence molecules on the surface of platelets, leukocytes and endothelial cells provides an assessment of the "adhesive phenotypical profile". Quantitative cytometry, using beads coated with a known amount of immunoglobulins as calibrators, is perfectly suited, through its multiple parameter analyses and the specificity provided by monoclonal antibodies, for the quantification of membrane antigens. Measuring the adhesive profile on the surface of cells that are implicated in vascular disease makes it possible to correlate that phenotype to the ischemic risk in such diversified pathologies or circumstances as intermittent angor, myocardial infarction, angioplasty, insulin-dependent diabetes or pre-eclampsia. In addition, that quantification permits monitoring the action of new therapeutical agents targeting adherence molecules.

Angioplasty, Balloon, Coronary↗
PubMed1999

Free and total platelet glycoprotein IIb/IIIa measurement in whole blood by quantitative flow cytometry during and after infusion of c7E3 Fab in patients undergoing PTCA.

A quantitative flow cytometry assay was used to evaluate the ex vivo kinetics of c7E3 Fab platelet effect in 16 patients undergoing PTCA treated with abciximab and compared with aggregometry assay. Immunolabeling of platelets was directly assessed on whole blood, using in parallel two monoclonal antibodies (Mabs) raised against GPIIIa, Mab1, the binding of which is inhibited by c7E3 Fab, and Mab2, the binding of which is not affected by c7E3 Fab. We found a severe and sustained inhibition of both GPIIb/IIIa receptors and platelet functions. The inter-individual variation in response to abciximab was low. A significant transient increase at H24 and H48 in the binding of Mab2 was found as an unexpected result, and confirmed in vitro. Results demonstrate that flow cytometry is a reliable method in agreement with aggregation. In addition, our results show that it is a standardized tool and a time-saving technique.

Abciximab↗
PubMed1999

Inhibition of fMLP-triggered respiratory burst of human monocytes by adenosine: involvement of A3 adenosine receptor.

Adenosine (Ado) is a potent anti-inflammatory agent acting on a variety of cell functions. However, its effects on human monocytes have been less well characterized. We investigated the effect of Ado and its receptor-specific analogs on NADPH oxidase activity with the use of luminol-enhanced chemiluminescence (CL). Adenosine inhibited fMLP-triggered NADPH oxidase activity with a maximal inhibition of 55+/-5%. IB-MECA, a selective A3 Ado receptor agonist reduced fMLP triggered NADPH oxidase activity more potently than the A2 receptor agonist CGS 2180 HCl (CGS) and the A1 Ado receptor agonist N-2-phenylethyl-adenosine (R-PIA). The inhibitory effect of Ado was reversed by neither the A1 Ado receptor antagonist 1,3-dipropyl-8(2-amino-4chlorophenyl)-xanthine (PACPX) nor the A2 Ado receptor antagonist 3,7-dimethyl-1-(2-propynyl)xanthine (DMPX). It was significantly reversed by the A1/A3 Ado receptor antagonist xanthine amine congener (XAC). Pretreatment of monocytes by cytochalasin B reversed the effect of Ado but not of dibutyryl cAMP (dBcAMP) on fMLP-CL response. KT 5720, a specific cAMP-dependent protein kinase inhibitor completely counteracted the inhibition of NADPH oxidase activity by dBcAMP but not by Ado. Using flow cytometry, we observed that Ado did not inhibit intracellular oxidative metabolism, whereas dBcAMP did. Furthermore, the inhibition of NADPH oxidase activity by Ado was not mediated by changes in cytosolic calcium. These results demonstrated that Ado inhibited NADPH oxidase activity via A3 Ado receptor independently of cAMP elevation or changes in calcium mobilization.

Adenosine↗
PubMed1998

Vitamin D differentially regulates B7.1 and B7.2 expression on human peripheral blood monocytes.

The hormonal active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1, 25(OH)2D3), inhibits (through an unknown mechanism) the ability of monocytes/macrophages to induce T-cell activation. For T cells to be optimally activated, recognition of antigen/major histocompatibility complexes (MHC) by the T-cell receptor (TCR) must be accompanied by a second costimulatory signal. Considerable experimental data now suggest that this costimulatory signal is predominantly generated by B7.1 and/or B7.2 molecules, expressed on antigen-presenting cells (APC), when engaged to their counter-receptor, CD28, present on T cells. To determine whether the inhibitory effect of 1,25(OH)2D3 on monocytes/macrophages might involve modulation of the expression of B7.1 and B7.2 molecules, we analysed (by flow cytometry) the influence of 1,25(OH)2D3 and an analogue, KH 1060, on the expression of these two molecules at the surface of resting human peripheral blood monocytes. In parallel, we tested the effect of these two agents on human monocyte expression of cell-surface markers (CD14 and CD4) and antigen-presenting molecules (MHC class I and MHC class II). Our results showed that both 1,25(OH)2D3 and KH 1060 inhibited the basal expression of B7.2 in a dose- and time-dependent manner, without affecting B7.1. Moreover, these two compounds increased CD14 and reduced MHC class II and CD4 expression. Furthermore, the effect of 1,25(OH)2D3 on B7 molecule expression in combination with lipopolysaccharide (LPS) or cytokines, including interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha), was studied. The 1,25(OH)2D3-induced B7.2 down-regulation was still detectable when monocytes were activated by IL-10, IFN-gamma and TNF-alpha but not with LPS. Moreover, the induction of B7.1 by TNF-alpha was inhibited by addition of 1, 25(OH)2D3. We conclude that the ability of 1,25(OH)2D3 to decrease B7.2 expression on human monocytes might contribute to its inhibitory effect on APC-dependent T-cell activation and to its immunosuppressive properties observed in autoimmune diseases and organ transplantation.

Antigen Presentation↗
PubMed1998

Adherent-free generation of functional dendritic cells from purified blood monocytes in view of potential clinical use.

There is increasing interest in dendritic cells (DC) that are capable of initiating antitumor immune responses. An in vitro cell differentiation method has recently been developed that uses GM-CSF and IL-4 to generate human DC from adherent blood mononuclear cells cultured on tissue culture plastic. These cells are competent for antigen uptake but express relatively low levels of co-stimulatory molecules and thus correspond to immature resident tissue DC. We have adapted this method to consider some variables that are pertinent to clinical use, including a large scale differentiation of functional DC in a culture system suitable for clinical use. We report here that sizable numbers of monocytes purified by elutriation from blood leukocytes and cultured in Teflon bags develop with high efficiency into typical DC, as defined by morphology and membrane phenotype. When compared with usual adherent DC, cells generated under our adherent-free conditions exhibited lower CD1a expression and antigen capture capacity, but maintained the ability to present soluble antigens to T cells. They neoexpressed a high level of the co-stimulator molecule B7-2 (CD86) and was potent accessory cells for T cell proliferation, but they lacked the CD83 marker of DC full maturation. This study may constitute a prerequisite step for clinical investigations in tumor immunotherapy.

Antigens, CD↗
PubMed1998

[Modulators of leukocytic functions].

Polymorphonuclear neutrophils (PMN) and monocytes play a role in vascular diseases. Animal experimental models, using deleukocytation or injection of anti-CD11b-anti-CD18 monoclonal antibodies (inhibition of leukocytic adhesion and of interaction with the endothelial cell) have confirmed this role in the ischemia-reperfusion syndrome and in myocardial infarction. In man, increased production of oxygen radicals, PMN release of elastase, increased monocyte formation of tissue factor (TF) and integrins have been noted in coronary artery disease, in chronic arteriopathy of the lower limbs and in association with vascular risk factors such as diabetes. Pharmacological alteration of leukocyte hyperactivity therefore seems to be justified. Pentoxifylline, used with good effect in arteriopathy of the lower limbs, affects numerous leukocytic functions: diminution in adherence and in PMN production of free radicals, diminution in the formation of TF and cytokines (TNF). Gingkolides reduce leukocytic interactions and platelet activation through an anti-PAF (Platelet Activation Factor) action. Aspirin may interfere with free radicals and inhibit TF formation. alpha-tocopherol blocks the activation, by free radicals, of the transcription factor NF k B. By altering the TNF and IL-1 cytokines, leukocytic activation can be controlled. Other cytokines (IL-4, IL-10) have an immunosuppressive action and reduce the formation of TF. The pharmacological targets are therefore multiple. Their use in vascular diseases is only at a very preliminary stage.

Animals↗
PubMed1997

Factor V Leiden: detection in whole blood by ASA PCR using an additional mismatch in antepenultimate position.

Factor V Leiden mutation was initially detected in thrombophilic patients and relatives by PCR RFLP (Restriction Fragment Length Polymorphism) according to Bertina (1). This technique presents some drawbacks and the current trend is to simplify the diagnosis. We describe a technique of Allele Specific Amplification (ASA) which is optimized in terms of reliability: an additional mismatch in antepenultimate position enables to obtain the same specificity as PCR RFLP. Furthermore, coamplification of internal control warrants an optimal sensitivity. All the PCR have been simplified: the DNA extraction improvement allows to analyse the genotype with only a few microliters of whole blood whatever the anticoagulant and the procedure of preservation (freezing, dried blood spots, storage at +4 degrees C for several days). This technique saves time. Moreover, full automation of the ASA technique may be shortened thanks to the lack of extraction and the positive/negative reading of the PCR signal.

Alleles↗
PubMed1996

Tissue origin and extracellular matrix control neutral proteinase activity in human fibroblast three-dimensional cultures.

Remodeling of the extracellular matrix by fibroblasts is an important step in the process of wound healing and tissue repair. We compared the behavior of fibroblasts from two different tissues, dermis and gingiva, in three-dimensional lattices made of two different extracellular matrix macromolecules, collagen and fibrin. Cells were grown in monolayer cultures from normal skin or gingiva and seeded in three-dimensional lattices made of either collagen of fibrin. Photonic and scanning electron microscopy did not reveal any morphological differences between the two types of fibroblasts in both sets of lattices. Both types of fibroblasts retracted collagen lattices similarly and caused only a slight degradation of the collagen substratum. By contrast, when seeded in fibrin lattices, gingival fibroblasts completely digested their substratum in less than 8 days, whereas only a slight fibrin degradation was observed with dermal fibroblasts. The ability of gingival but not dermal fibroblasts to express high levels of tissue plasminogen activators (tPA) when cultured in fibrin lattices was assessed on an immunological basis. Also, deprivation of plasminogen-contaminating fibrinogen preparations or use of tPA inhibitors markedly inhibited both fibrinolysis and retraction rates of fibrin lattices by gingival fibroblasts. Casein-zymography confirmed the intense proteolytic activity induced by fibrin in gingival fibroblasts. It was inhibited by aprotinin and phenyl methylsulfonyl fluoride (PMSF), two non-specific inhibitors of serine proteinases, and by epsilon-amino-caproic acid (epsilon ACA), an inhibitor of plasminogen activators. Monolayer cultures exhibited only trace amounts of caseinolytic activity. Our results demonstrate that the expression of proteinases by fibroblasts is dependent not only on their tissue origin but also on the surrounding extracellular matrix. The intense fibrinolytic activity of gingival fibroblasts in fibrin lattices may explain partially the high rate of healing clinically observed in gingiva.

Adult↗
PubMed1996

Differential spontaneous expression of mRNA for IL-4, IL-10, IL-13, IL-2 and interferon-gamma (IFN-gamma) in peripheral blood mononuclear cells (PBMC) from atopic patients.

Distinct cytokine-producing T cell subsets are well known to play a major role in IgE production and to be differentially regulated in allergic patients, although the characterization of the type 1/type 2 cytokine pattern in PBMC during allergic responses remains to be clearly defined. The aim of this study was to determine whether different cytokine profiles are observed directly in PBMC of atopic donors. We attempted to study several cytokines (IL-2, IFN-gamma, IL-4, IL-10 and IL-13) using not only ELISA but also polymerase chain reaction (PCR) techniques, because the frequency of cytokine-producing cells in peripheral blood is very low. All the patients were selected during their acute symptomatologic phase. Data showed a significantly higher production of IL-4 (P = 0.05) and IL-10 (P < 0.005) as determined by ELISA in phytohaemagglutinin (PHA)/phorbol myristate acetate (PMA)-stimulated mononuclear cells of atopic donors compared with controls, although spontaneous IL-4 production without stimulation was never detected within either atopic or control groups. The reverse-transcriptase (RT)-PCR technique appeared to be advantageous in that it allowed the detection of the spontaneous expression of cytokine mRNA in cells without stimulation. We found a clear expression of IL-4 mRNA spontaneously in all atopic patients, whereas normal donors in most cases did not show specific signals (P < 0.0001). Less differences between atopic subjects and controls were found in IL-10 mRNA expression. Although the technique of RT-PCR amplification used in this study is semiquantitative, a reproducible and significant (P < 0.001) enhancement of IL-10 mRNA expression was observed in atopic donors. A heterogeneous expression of IL-13 mRNA was observed in individuals from the two groups studied, although mean levels in atopic donors were slightly enhanced compared with controls (P = 0.02). Furthermore, we did not observe any alteration in the expression of the type 1-derived cytokines such as IFN-gamma and IL-2. In addition, we showed a lack of correlation between the expression of serum IgE (total or specific) and spontaneous IL-4 mRNA expression. This study showed a tendency of PBMC from atopic donors to express a type 2-like cytokine pattern, with IL-4 as the most discriminatory cytokine. Additionally, as the level of serum IgE has a low predictive value in allergic disease, and as the elevated expression of IL-4 that we found was not correlated with serum IgE, we could strongly suggest that the measurement of IL-4 in blood mononuclear cells may be of great value in the analysis of allergic responses in atopic donors.

Adolescent↗
PubMed1996

Early neonatal diagnosis of congenital toxoplasmosis: value of comparative enzyme-linked immunofiltration assay immunological profiles and anti-Toxoplasma gondii immunoglobulin M (IgM) or IgA immunocapture and implications for postnatal therapeutic strategies.

Diagnostic strategies for congenital toxoplasmosis have changed profoundly in recent years. Immunological diagnostic methods, long considered disappointing, can now be used at a very early stage. Over a 3-year period, 1,050 infants at risk of congenital toxoplasmosis (born to 1,048 mothers infected during pregnancy) were monitored for a minimum of 12 months and a maximum of 7 years. More than 6,000 serum specimens were analyzed by comparative mother-infant immunological profiles (CIPs) based on an enzyme-linked immunofiltration assay (ELIFA) and an immunocapture method for the detection of specific immunoglobulin M (IgM) and IgA. IgG antibodies were also titrated. One hundred three cases of congenital toxoplasmosis were demonstrated. The CIP-ELIFA method had a better diagnostic yield (sensitivity, 90%) than specific IgM and/or IgA detection by immunocapture assay (sensitivity, 77%). By using a combination of these tests, congenital infection was diagnosed in the first month and the first 3 months of life in 90 and 94% of infants with toxoplasmosis, respectively, with a specificity of 99.8% and a positive predictive value of 99% at 8 months of age. This dual diagnostic approach (ELIFA and IgM-IgA immunocapture) is highly efficient and has important implications for therapy. Indeed, early postnatal diagnosis based on objective evidence enables therapy with pyrimethamine-sulfadoxine to be started immediately for 24 months, while spiramycin (which used to be given preventively for 9 to 12 months to all infants at risk) can be stopped after the first 3 months of life.

Animals↗
PubMed1996

Validation protocol of analytical hemostasis systems: measurement of anti-Xa activity of low-molecular-weight heparins.

A standard validation protocol adapted to the chromogenic assay of anti-Xa activity of low-molecular-weight heparins was used in a multicenter study to assess its suitability for comparing and evaluating analytical hemostasis systems. The protocol included: familiarization with the system (repeatability); assessment of limits of linearity, detection limits, and cross-contamination; and validation (reproducibility and accuracy of measurements of treated patients' plasmas). We calibrated the systems with the same range of lyophilized plasmas daily and evaluated repeatability and reproducibility by using a single batch of lyophilized plasmas at three anti-Xa activities. The two automated systems tested [SB 300 (Gilford) and ACL (IL)] and the two semiautomated systems [ST 888 (D. Stago) and Chromotimer (Behring)] gave similar mean values. Dispersion of results was lower with the automated systems than with the semiautomated ones, especially at low anti-Xa activities, a tendency that also was observed for reproducibility. Because each analytical system gave linear results for activities as great as 1000 IU/L, suitable sample dilution is advisable for higher anti-Xa activities. Accuracy was greater in the automated systems. We conclude that this protocol is feasible and is applicable to validation of other analytical hemostasis instruments, in particular the latest generation of fully automated instruments.

Autoanalysis↗
PubMed1996

[Evaluation of multidrug resistance phenotype on medullary specimens from patients with acute leukemia by determination of nuclear efflux of tetrahydropyranyl-doxorubicin. Approach by confocal laser microspectrofluorometry].

Confocal microspectrofluorometry allows the analysis of fluorescent molecules such as anthracylines in isolated living cells. An optical microscope fitted with a phase-contrast 100 X water-immersion objective enables simultaneous observation of the sample, focusing of the laser beam on the selected cell fraction (nucleus) and collection of the fluorescence emitted from the sample. The resulting intranuclear spectra are interpreted according to a quantitative model of the fluorescence spectra of both free and DNA-bound anthracycline. The intranuclear drug concentration can thus be determined. This technique has been applied to blast cells collected in patients with acute leukemia. Leukemic cells are aspirated from bone marrow, separated by Ficoll sedimentation and resuspended in RPMI-1640 containing 10% fetal calf serum and 200 nM tetrahydropyranyl-doxorubicin (THP-DOX). After one hour, 20 cells are analyzed and the mean nuclear drug content is determined (C1). Cells are then resuspended in the same medium but without anthracycline for 3 hours and the mean intranuclear drug concentration is then also determined (C3). From C1 and C3 the retention rate (RR) is calculated. Firstly, the accuracy of the method was checked. In 4 AML patients, two different samples aspirated on the same day were divided into two portions. Thus, two measurements were made on each one (4 values per patient). Coefficients of variation were satisfactory (4, 6, 12, and 12%). Secondly, blast cells collected in patients with AML and ALL at diagnosis or in relapse were studied. P-glycoprotein (P-gp) and CD34 expression was also studied using respectively immunohistochemistry land flow cytometry. Results obtained from the first 21 patients showed that there was no correlation between RR and either P-gp or CD34 expression. This could result from the efflux of THP-DOX by other mechanisms and/or low sensitivity of the staining technique.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

[Fibrinogen: a risk factor].

More than 10 epidemiologic studies have established that a high fibrinogen level is a thrombotic risk factor. The role of fibrinogen in arterial occlusion is multiple : the atheroma plaque involvement in formation thrombus, erythrocyte aggregation, whole blood and plasma viscosity. Fibrinogen level is high during inflammation and increases with ageing and in tobacco addicts. In coronary disease, it is an independent risk factor of prognosis value. In arterial peripheral disease, it is a risk factor of postsurgical reocclusion. After a stroke, a high level of fibrinogen is a sign of severe disease. The dosage of fibrinogen is quite easy but requires a precise calibration. The determination of genetic polymorphism associated with high fibrinogen level is promising. Many circumstances can modify fibrinogen level and are targets for prophylaxis treatments. The influence of genetic factors is still discussed.

Arteries↗