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Biomedical subjects

D Dumas

Publications and source records attributed to D Dumas.

At least 19 recordsLinked to original sources
PubMed2003

The strength of integrin binding between neutrophils and endothelial cells.

The firm adhesion of activated polymorphonuclear neutrophils to endothelial cells in blood vessels is achieved through binding of the integrin intercellular adhesion molecule. To contribute to the better understanding of this adhesion step, our investigation is aimed at the relationship between integrin expression and the strength of neutrophil binding to endothelial cells. Flow cytometry and 3D scanning microscopy are used to study integrin expression and distribution, respectively. It is found that CD11b/CD18 integrin expression is localized in clusters distributed irregularly over the neutrophil surface. After cell activation, the cluster distribution polarizes, increasing the local CD11b/CD18 density concurrently with nearly doubled integrin expression. The neutrophil adhesion efficiency is measured in a flow chamber coated successively by various substrates, including endothelial cells in an activated state. Analysis of the flow dependence of the number of attached cells reveals the prevailing number of neutrophils with stronger binding to the endothelium when both cells are in the activated state in comparison with non-activated cells.

CD11b Antigen↗
PubMed2003

The cooperative effect of L-selectin clusters and velocity-dependent bond formation that stabilizes leukocyte rolling.

The role of clustered of L-selectin receptors on the leukocyte surface is discussed in connection with the postulated velocity-dependent formation of selectin-ligand tether bonds to interpret the mechanism of leukocyte tethering to, and rolling along, the vascular endothelium. The distinct feature of this step-wise process is a weak dependence of leukocyte rolling velocity on the hydrodynamic forces of ambient flow due to the increased number of selectin bonds with increasing flow shear rate and also their clustering. The contact zones on the leukocyte surface are separated by distances with distribution which corresponds to the distribution of distances of the observed L-selectin clusters. It suggests that the localization of L-selectin receptors to clusters and the way of their approach to the ligand molecules creates such conditions for binding of L-selectin and ligand molecules that resulting number of bonds stabilizes rolling velocity.

Blood Flow Velocity↗
PubMed2003

Comparison of wide-field/deconvolution and confocal microscopy in bioengineering. Interest of multi-photon microscopy in the study of articular cartilage.

The increase in lateral and spatial resolutions is one of the major targets of research and development in the field of optical microscopies applied to living tissue. The optical geometry of Confocal Laser Scanning Microscopy (CLSM) demonstrates its undeniable advantage on conventional fluorescence microscopy by segregating the planes outside the focussing plane. The methodological and technological advances of the last five years have been fast evolving, especially with regard to the optimisation of CLSM and deconvolution process. The limited analysis in thick tissue have given rise to the development of other techniques, multi-photon excitation microscopy in particular. In this paper, we have applied these techniques on major biological applications in bioengineering (endothelial cell, chondrocyte in 3D-culture, human cartilage) and discussed the technical limitations and perspectives.

Actins↗
PubMed2002

In vitro study of intracellular IL-1beta production and beta1 integrins expression in stimulated chondrocytes--effect of rhein.

The purpose of the present study was to investigate the intracellular IL-1beta production and beta1 integrins (alpha4/beta1 and alpha5/beta1) expression on chondrocytes. Chondroytes monolayer (human chondrosarcoma cell line HEM-C55) were incubated for 12, 24 and 48 hours in the presence of Tumor Necrosis Factor-alpha (TNF-alpha, Sigma, France) or recombinant human IL-1alpha (rh-IL1alpha, Becton Dickinson, France). After direct immunolabelling, cells were either analyzed on FACScan flow cytometer (Becton Dickinson, France), or observed under an epi-fluorescence inverted microscope equipped with the CellScan EPR optical scanning acquisition system (IPLab-Scanalytics, USA). We found that the IL-1beta mean fluorescence intensity in flow cytometry and in 3D microscopy was increased in the presence of TNF-alpha or rh-IL-1alpha, and alpha4/beta1 or alpha5/beta1 expression was higher on stimulated cells than on control cells. On the other hand, we have evaluated the in vitro effects of rhein (10(-5) M, Negma, France), an active metabolite of diacerein, on the intracellular IL-1beta and beta1 integrins expressed by stimulated or no-stimulated chondrocytes. The results indicated that rhein leads to a reduction of IL-1beta synthesis whereas a weak decrease of beta1 integrins receptors expression is observed. From this study, it seems that rhein partially reduce cytokine-induced intracellular IL-1beta production, and it has a weak action on alpha4/beta1 or alpha5/beta1 receptors.

Anthraquinones↗
PubMed2001

Sodium alginate sponges with or without sodium hyaluronate: in vitro engineering of cartilage.

Studies are underway to design biosystems containing embedded chondrocytes to fill osteochondral defects and to produce a tissue close to native cartilage. In the present report, a new alginate three-dimensional support for chondrocyte culture is described. A sodium alginate solution, with or without hyaluronic acid (HA), was freeze-dried to obtain large-porosity sponges. This formulation was compared with a hydrogel of the same composition. In the sponge formulation, macroscopic and microscopic studies demonstrated the formation of a macroporous network (average pore size, 174 microm) associated with a microporous one (average pore size, 250 nm). Histological and biochemical studies showed that, when loaded with HA, the sponge provides an adapted environment for proteoglycan and collagen synthesis by chondrocytes. Cytoskeleton organization was studied by three-dimensional fluorescence microscopy (CellScan EPR). Chondrocytes exhibit a marked spherical shape with a nonoriented and sparse actin microfilament network. Type II collagen was detected in both types of sponges (with or without HA) using immunohistochemistry. In conclusion, the sponge formulation affords new perspectives with respect to the in vitro production of "artificial" cartilage. Furthermore, the presence of hyaluronate within the alginate sponge mimics a functional environment, suitable for the production by embedded chondrocytes of an extracellular matrix.

Alginates↗
PubMed2000

Bovine beta-lactoglobulin receptors on transformed mammalian cells (hybridomas MARK-3): characterization by flow cytometry.

Flow cytometry was used to demonstrate the presence of beta-lactoglobulin (betaLG) receptors on living murine hybridoma MARK-3 cells using a fluorescein isothiocyanate-betaLG conjugate (FITC-betaLG: molar ratio of 5:1). A site occupation curve was produced using a shift in the mean channel fluorescence at various concentrations of FITC-betaLG. The binding of labelled ligand was concentration dependent and was inhibited by unlabelled betaLG. The on-rate constant was 3.2x10(2) M(-1) min(-1) and the off-rate constant was 0.002 min(-1). Scatchard plot analysis gave a dissociation constant (K(d)) of 44+/-21x10(-7) and 39+/-24x10(-5) M (n=3). Flow cytometry indicated that at least 15% of the FITC-betaLG were internalized for 5 min and that internalization was temperature- and time-dependent. The internalization was confirmed by 3-D fluorescence microscopy (CELLScan system).

Animals↗
PubMed2000

[Determination of polymorphonuclear neutrophil adhesion receptors. Effect of pre-analytic factors].

UNLABELLED: Polymorphonuclear neutrophil (PMN) adherence receptors expression varies with leukocyte activation state. Their quantification need accurate and inter-laboratories reproducible methods, without artefactual activation. OBJECTIVE: The aim of this study was to study the influence of cell preparation on PMN adherence receptors expression. MATERIALS AND METHODS: It was proposed to quantify, using immunolabeling standard (QIFIKIT(R), Dako), surface expression of the main adherence receptors (L-selectin and B(2) integrins), from different preparations of PMN: total blood collected with EDTA, isolated PMN by density gradient Polymorphprep(TM) 1,113 (Nycomed Pharma) and formaldehyde fixed PMN. RESULTS: A decrease of all receptors was noted after isolation and fixation of PMN, in comparison with whole blood PMN analysis. These results differed from data previously reported since, in these studies, activated phenotypes (increased of B(2) integrins) were observed after isolation and fixation methods. CONCLUSION: The present study provides strong evidence that pre-analytical conditions are sources of biological variations and thus extreme care must be taken in the interpretation of results. It underlines the interest of consensual practices for these pre-analytic and analytic parameters in order to compare results in multicenter and longitudinal studies.

CD18 Antigens↗
PubMed2000

The role of 3D-microscopy in the study of chondrocyte-matrix interaction (alginate bead or sponge, rat femoral head cap, human osteoarthritic cartilage) and pharmacological application.

The potentialities of a new non-invasive optical scanning microscopy technique were evaluated through 3D analysis of chondrocyte-matrix interactions. Five different 2D or 3D culture systems were used: (1) MonoLayer (ML) of human chondrosarcoma cell line; (2) rat or human chondrocytes encapsulated in Alginate Bead (AB); (3) human chondrocytes encapsulated in Alginate Sponge (AS); (4) Rat Femoral Head Cap (RFHC); (5) slices of knee human Osteoarthritic Cartilage (HOAC). Chondrocytes ML, AB, RFHC were incubated for 24 h in vitro in the presence of recombinant human interleukin1-beta (rhIL1-beta) and the effects on cytoskeleton organisation (F-actin filament), Focal Adhesion Kinase (FAK) expression (tyrosine kinase), collagenase B expression (metalloprotease) were studied. Furthermore, the production of intracellular IL1-beta by LPS- or rhIL1-beta-stimulated chondrocytes was shown to be partly suppressed by rhein (active metabolite of diacerhein) in all culture systems. This high resolution light microscopy gave complementary information that could be important for a better understanding of the interaction of chondrocytes with the extracellular matrix in a variety of culture devices.

Actins↗
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

Hemorheology and vascular endothelial cells.

The vascular endothelium is a biologically active monolayer of cells provided an interface between blood and tissues. Vascular endothelial cells (ECs) have two functional states, which are allowed by their different properties: (i) vaso regulating properties: ECs releases vasomotor components, as endothelin (vaso constriction), prostacyclin and nitrite oxide (vaso dilatation); (ii) antithrombotic and hemostatic properties; and (iii) anti-adhesion properties. The endothelium is normally antithrombotic and anti-adhesive to ensure blood fluidity. During many cardio-vascular diseases, these properties may be reversed. Thus, the ECs have a determinant role in hemodynamic control through these various metabolic activities. Otherwise, many studies have demonstrated that local blood flow conditions have a crucial role on the EC properties (mechanotransduction concept). The knowledge of the properties of ECs and the control of the phenomena which define their functions is a key element in the cardiovascular diseases understanding.

Animals↗
PubMed1999

Development and evaluation of an automatic method for the study of platelet osmotic response.

Study of the osmotic resistance to hypotonic medium of platelets has often been suggested as a global test to assess the viability of these cells in transfusion or to study modification during haematological pathologies. A number of authors have analysed the behaviour of platelets in hypotonic media by a variety of methods (cell count, determinations of substances released, morphology, etc.), but most studies are currently based on the so-called "Hypotonic Shock Response" test (HSR). In this study, the authors describe a new automated and reproducible apparatus, called fragilimeter, using slow dialysis to assess platelet osmotic resistance. The variations in light transmission through a platelet suspension according to ionic strength are linked to the change in cellular volume and lysis and characterise the osmotic behaviour of the cells. The results revealed the good reproducibility and sensibility of the technique. This apparatus allows also the realisation of the "HSR" test.

Adult↗
PubMed1999

Membrane fluidity and oxygen diffusion in cholesterol-enriched endothelial cells.

In this study, we measured the influence of cholesterol rigidification on oxygen permeability in human endothelial cell monolayer membranes (ECs). Cholesterol-induced membrane rigidification was assessed at different membrane depths by a fluorescence polarization method with diphenyl-hexatriene (DPH) and 1-(4-trimethylamino)-6-phenylhexatriene (TMA-DPH). Fluorescence quenching by oxygen was probed in preferentially labelled membrane with pyrene butyric acid (PyC4) and pyrene dodecanoic acid (PyC12), as shown with a 3D fluorescence microscope (CellScan System). With both probes the experiments revealed a decrease in oxygen diffusion as the cholesterol concentration increased in the medium culture (from 3.42 microM to 17.11 microM). We showed that very low concentrations of cholesterol (about 1000 times below normal value, 6.2 mM) particularly decrease oxygen levels or diffusion rate in the middle region of the membrane. In conclusion, these findings prove in a direct manner that cholesterol significantly affect the endothelial barrier function and molecular oxygen transfer to underlying tissues. Risk factors (cholesterol) directly would contribute to tissue ischemia.

Biological Transport↗
PubMed1997

Membrane fluidity and oxygen diffusion in cholesterol-enriched erythrocyte membrane.

This work studied the effect of cholesteryl hemisuccinate incorporation on membrane fluidity and on the kinetics of oxygen diffusion at different depths in the erythrocyte membrane. Cholesterol concentration in the membrane was expressed as the cholesterol-protein ratio (C/Pt). The membrane fluidity, as assessed by a fluorescence polarization method with diphenyl-hexatriene and 1-(4-trimethylamino)-6-phenylhexa-1,3,5-triene, decreased as the C/Pt ratio increased. Time-resolved fluorescence spectroscopy of pyrene dodecanoic acid (PDA) under an increasing C/Pt ratio in the erythrocyte membrane revealed enhanced oxygen diffusion in the middle of the membrane bilayer (in which PDA was incorporated), which was not the case with pyrene butyric acid (PBA) incorporated in the internal part of the membrane surface. It has generally been accepted that increased membrane fluidity reduces the physical barrier to oxygen permeation. Such conflicting observations on oxygen permeation in the rigidified erythrocyte membrane could be due to variations in oxygen solubility (preferential partitioning) in different polarity microdomains (cholesterol and phospholipid partitions).

Cholesterol↗
PubMed1997

Clobenzorex: evidence for amphetamine-like behavioral actions.

Clobenzorex, an optically active N-substituted derivative of (+)amphetamine, has been identified on the illicit market. Because so little is known regarding the pharmacology or abuse potential of this agent, it was examined in tests of stimulus generalization in rats trained to discriminate 1 mg/kg of (+)amphetamine from vehicle to determine if it would produce amphetamine-appropriate responding. Clobenzorex (ED50 = 6.6 mg/kg) substituted for (+)amphetamine (ED50 = 0.3 mg/kg) but was approximately twenty times less potent than the training drug. Clobenzorex was also compared with (+)amphetamine and cocaine for its ability to induce locomotor stimulation and rearing frequency in mice. Clobenzorex was active in both assays but was less potent than either (+)amphetamine or cocaine. It is concluded that, although weaker than (+)amphetamine, clobenzorex constitutes an agent with amphetamine-like central stimulant behavioral properties.

Amphetamine↗