Biomedical subjects

M Broussas

Publications and source records attributed to M Broussas.

5 recordsLinked to original sources

PubMed2000

Separation of dendritic cells from highly purified human monocytes by counterflow centrifugation induces tissue factor expression.

BACKGROUND: In vitro generation of dendritic cells (DCs) from human monocytes represents a promising tool in immunotherapy. However, it is not known whether the separation of DCs from monocytes induces tissue factor expression and therefore may trigger coagulation in patients receiving these DC preparations. The aim of this study is thus to analyze tissue factor expression on monocyte-derived DCs and to compare their ability to trigger thrombin generation to that of macrophages obtained from the same monocytes. STUDY DESIGN AND METHODS: Human monocytes are separated by leukapheresis and washed by using counterflow centrifugation in sterile, endotoxin-free conditions. Macrophages are grown from human monocytes in the presence of GM-CSF alone and immature DCs are grown in the presence of GM-CSF plus IL-4 for 5 days with fetal calf serum (IDC-FCS). Immature DCs are also grown from human monocytes for 7 days in the presence of GM-CSF plus IL-4 with human group AB serum (IDC-HS). The addition of prostaglandin E(2) and TNFalpha in this culture medium at Day 5 leads to mature DCs (MDC-HS). Tissue factor mRNA expression is studied by RT-PCR analysis. Tissue factor antigen is measured by ELISA in cell lysates and by direct flow cytometry. The procoagulant activity of intact cells is assessed by using an amidolytic assay or a chronometric assay. RESULTS: IDC-FCS express tissue factor mRNA and antigen and trigger thrombin generation. Procoagulant activity of IDC-FCS is dependent on both tissue factor expression and exposure to anionic phospholipid. Monocyte-derived macrophages cultured for 5 days with GM-CSF alone express lower levels of tissue factor mRNA, tissue factor antigen, and procoagulant activity than IDC-FCS. IDC-HS and MDC-HS also express high levels of tissue factor mRNA and antigen and support procoagulant activity. CONCLUSION: Monocyte-derived DCs express a high level of functional tissue factor and support procoagulant activity. This finding should be taken into account in clinical trials.

Animals↗

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

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↗

PubMed1999

[Measurement of monocyte activation: perspectives in clinical application in the investigation of the diabetic patient].

Monocytes play a pivotal role in the complex processes of inflammation, immunologic responses and atherothrombosis. Clinical studies essentially reported an increased procoagulant activity in diabetes and coronary disease, suggesting an overexpression of tissue factor. This was further confirmed by the direct measurement of tissue factor on monocyte membrane by flow cytometry. Many receptors can be measured on monocytes by flow cytometry: beta 2 integrins (CD 11 a-b-c/CD 18) involved in adhesion, EPR-1 receptor, receptors for advanced glycation products, urokinase receptor U-PAR. Flow cytometry allows a cell analysis in whole blood. Modern methods allow a standardization of the procedures and a quantification of the number of sites expressed by the cell. However, the respect of preanalytical and analytical conditions is mandatory to obtain reliable data. Besides, clinical studies in diabetes should carefully define the subgroups of patients: type of diabetes, metabolic abnormalities, risk factors, infective complications.

Diabetes Mellitus↗

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↗