Biomedical subjects

C Foa

Publications and source records attributed to C Foa.

At least 19 recordsLinked to original sources

PubMed1998

Adhesion-related glycocalyx study: quantitative approach with imaging-spectrum in the energy filtering transmission electron microscope (EFTEM).

Large polysaccharide molecules composing the glycocalyx have been shown to prevent cell adhesion. However, this process was not observed microscopically. Terbium labeling, combined with a new quantitative imaging method based on electron energy loss spectroscopy, allowed specific glycocalyx staining with excellent contrast. Image analysis enabled us to compare glycocalyx structure in free membrane areas and contacts between monocytic cells and bound erythrocytes. Apparent glycocalyx thickness, in contact areas, was half of the sum of glycocalyx thicknesses in free areas without label density increase. Ultrastructural immunogold localization of CD43 molecules, a major component of glycocalyx, was also demonstrated to be excluded from contact areas during adhesion. Thus, both approaches strongly suggest that some glycocalyx elements must exit from contact to allow binding of adhesion molecules.

Antigens, CD↗

PubMed1997

Thymocytes in Thy-1-/- mice show augmented TCR signaling and impaired differentiation.

Thy-1, a single variable-like immunoglobulin superfamily domain anchored in the plasma membrane by a glycosyl phosphaditylinositol tail [1], is a major surface glycoprotein in adult mammalian neurons and rodent thymocytes [2]; the function of Thy-1 has remained enigmatic since its discovery [3]. Studies in vitro have implicated Thy-1 in homotypic and heterotypic cell-cell interactions [2,4]. Ligation of Thy-1 initiates transmembrane signaling pathways that lead to diverse physiological outcomes in different cells [2,5-7]. In rodents, Thy-1 is highly expressed on the surface of CD4+CD8+ double-positive immature thymocytes and downregulated in mature T cells. Here, we report that thymocytes from Thy-1-/- mice [8] had altered cell-cell contacts, and hyperresponsiveness to T-cell receptor (TCR) triggering as demonstrated by the heightened activation of p56lck, phosphorylation of TCR subunits, Ca2+ fluxes and cell proliferation. Thy-1-/- thymocytes exhibited impaired maturation from the double positive to single positive stage of thymocyte development, possibly due to inappropriate negative selection, and were prone to T lymphomas in aged mice. These observations indicate that Thy-1 negatively regulates TCR-mediated signaling and controls activation thresholds during thymocyte differentiation.

Animals↗

PubMed1997

Leukosialin (CD43) behavior during adhesion of human monocytic THP-1 cells to red blood cells.

To understand the modulation and the behavior of glycocalyx elements during adhesion, we explored one of its components, the CD43 molecule, on human monocytic THP-1 cells exposed to cytokine stimulation and its redistribution during heterotypic adhesion to opsonized erythrocytes. First we demonstrated by immunofluorescence and immunoprecipitation that CD43 is dys-sialylated in monocytic THP-1 cells stimulated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and stimulation increased correlated to heterotypic adhesion. CD43 anti-adhesive effect seemed to be related to sialic acid moeties because an increase in adhesion was also induced by sialidase treatment and by monoclonal antibodies recognizing sialic acid-dependent epitopes on CD43. Second, a redistribution of CD43 molecules was observed after adhesion, resulting in the exclusion of CD43 molecules from contact areas as demonstrated by immunofluorescence and by ultrastructural immunogold localization. We therefore demonstrated in monocytic THP-1 cells that some glycocalyx molecules can be modulated by cytokines and redistributed during adhesion. These results support the concept that CD43 can regulate cell interactions.

Antibodies, Monoclonal↗

PubMed1994

Relationship between phagosome acidification, phagosome-lysosome fusion, and mechanism of particle ingestion.

The fate of pathogens ingested by macrophages is dependent on phagosome acidification and fusion with different intracellular vesicles. Whereas the mode of particle recognition by the phagocyte seems the main determinant of phagosome-lysosome fusion, the influence of membrane reorganization, fusion events, and cell activation in phagosome acidification is not well known. We looked for a relationship between the nature of receptors involved in phagocytosis, phagosome acidification, and phagosome-lysosome fusion. Murine macrophage-like P388D1 cells were made to ingest sheep erythrocytes coated with immunoglobulin G (EIgG) or IgM and complement (EIgMC) or treated with glutaraldehyde and periodate (EGP). The following results were obtained: (1) As expected, the adhesion of the three particle types was differentially inhibited by monoclonal antibodies specific for Fc gamma RII and CD11b/CD18. (2) The phagosomes containing all three particle types displayed similar acidification kinetics with a pH decrease to 6 within the first 10 min after ingestion. (3) Only phagosomes containing EIgG or EIgMC were fused with peroxidase-loaded secondary lysosomes. (4) Coating EGP with IgG only partially restored fusion, even when the surface density of IgG was markedly higher than found on EIgG. It is concluded that phagosome acidification and fusion are regulated by different mechanisms. Also, the lack of fusion observed with EGP is not entirely accounted for by the absence of stimulation of suitable receptors on the phagocyte membrane, because it cannot be restored by providing such a stimulus.

Animals↗

PubMed1994

Programmed cell death in Dictyostelium.

Programmed cell death (PCD) of Dictyostelium discoideum cells was triggered precisely and studied quantitatively in an in vitro system involving differentiation without morphogenesis. In temporal succession after the triggering of differentiation, PCD included first an irreversible step leading to the inability to regrow at 8 hours. At 12 hours, massive vacuolisation was best evidenced by confocal microscopy, and prominent cytoplasmic condensation and focal chromatin condensation could be observed by electron microscopy. Membrane permeabilization occurred only very late (at 40-60 hours) as judged by propidium iodide staining. No early DNA fragmentation could be detected by standard or pulsed field gel electrophoresis. These traits exhibit some similarity to those of previously described non-apoptotic and apoptotic PCD, suggesting the hypothesis of a single core molecular mechanism of PCD emerging in evolution before the postulated multiple emergences of multicellularity. A single core mechanism would underly phenotypic variations of PCD resulting in various cells from differences in enzymatic equipment and mechanical constraints. A prediction is that some of the molecules involved in the core PCD mechanism of even phylogenetically very distant organisms, e.g. Dictyostelium and vertebrates, should be related.

Animals↗

PubMed1994

A novel role for E- and P-selectins: shape control of endothelial cell monolayers.

The migration of neutrophils from blood vessels to peripheral tissues is a key step of inflammation. This requires the formation of transient gaps between endothelial cells with concomitant leucocyte squeezing through these narrow apertures and immediate restoration of endothelium continuity. It is currently considered that the main role of selectins is to mediate the initial contact between flowing leucocytes and endothelial cells. We show here that the binding of E- or P-selectins by specific antibodies induces a marked 'rounding up' of interleukin-1- or thrombin-activated human endothelial cells, respectively. Also, anti-E-selectin antibodies trigger a transient increase in cytosolic calcium involving intracellular calcium stores. No such effect is observed when von Willebrand factor or intercellular adhesion molecule 1 are similarly bound. Thus, in addition to promoting the initial interaction between activated endothelium and moving leucocytes, selectins might play a role in the induction of subsequent endothelial deformation, which would facilitate leucocyte arrest and transmigration towards peripheral tissues, and enhance the diffusion of soluble molecules between intravascular and peripheral compartments. Our results are consistent with this hypothesis and demonstrate a new property of endothelial selectins.

Antibodies, Monoclonal↗

PubMed1992

Motion of cells sedimenting on a solid surface in a laminar shear flow.

Cell adhesion often occurs under dynamic conditions, as in flowing blood. A quantitative understanding of this process requires accurate knowledge of the topographical relationships between the cell membrane and potentially adhesive surfaces. This report describes an experimental study made on both the translational and rotational velocities of leukocytes sedimenting of a flat surface under laminar shear flow. The main conclusions are as follows: (a) Cells move close to the wall with constant velocity for several tens of seconds. (b) The numerical values of translational and rotational velocities are inconsistent with Goldman's model of a neutrally buoyant sphere in a laminar shear flow, unless a drag force corresponding to contact friction between cells and the chamber floor is added. The phenomenological friction coefficient was 7.4 millinewton.s/m. (c) Using a modified Goldman's theory, the width of the gap separating cells (6 microns radius) from the chamber floor was estimated at 1.4 micron. (d) It is shown that a high value of the cell-to-substrate gap may be accounted for by the presence of cell surface protrusions of a few micrometer length, in accordance with electron microscope observations performed on the same cell population. (e) In association with previously reported data (Tissot, O., C. Foa, C. Capo, H. Brailly, M. Delaage, and P. Bongrand. 1991. Biocolloids and Biosurfaces. In press), these results are consistent with the possibility that cell-substrate attachment be initiated by the formation of a single molecular bond, which might be considered as the rate limiting step.

CD4 Antigens↗

PubMed1992

Localization of calcium changes in stimulated rat mast cells.

We studied intracellular free, bound, and sequestered calcium in rat mast cells after various stimulations. The use of a fluorescent probe combined with digitized imaging on individual living cells demonstrated transient increases of free Ca2+ in the micromolar range. The use of histochemical techniques (K pyroantimonate and anhydrous fixation), together with X-ray microanalysis, energy electron-loss spectroscopy, and electron spectroscopic imaging, revealed large amounts of stored calcium within the cells (in the millimolar range). Chelation experiments and stimulations enabled us to identify at least two pools of bound calcium which exhibited different dynamic behaviors. Stimulation in the presence of EGTA did not modify calcium from granules, granule membranes, and heterochromatin, whereas it decreased calcium from other cell compartments. Stimulation triggered variations in the amount of bound calcium but they did not parallel free calcium movements. Hence, whereas free calcium is implicated in exocytosis, bound calcium may be involved in altogether different cell functions.

Animals↗

PubMed1990

Use of conjugates made between a cytolytic T cell clone and target cells to study the redistribution of membrane molecules in cell contact areas.

In many models of cell-cell adhesion, it was reported that some cell membrane molecules might be redistributed into contact areas. However, this phenomenon was not subjected to precise quantification. In the present work, fluorescence microscopy, immunolabelling and digital image processing were combined to analyse quantitatively the spatial organization of specific or nonspecific conjugates made with a cytolytic T (CTL) lymphocyte clone (BM3.3) and target cells (EL4 or RDM4). Binding was achieved under calcium-free conditions to study the earliest steps of cell interaction, preceding CTL activation. Fluorescent antibodies were used to label class I histocompatibility molecules on both killer and target cells, and T cell receptor, CD3, CD8 and LFA-1 (CD18/CD11a) on the killer cells. Membrane bilayers were stained with a fluorescent phospholipid, glycoconjugates were labelled with periodic oxidation and Lucifer Yellow uptake, and polymerized actin was revealed with a fluorescent phallacidin derivative. Also, the fine geometry of killer-target interaction area was studied with electron microscopy and computer-assisted contour analysis. It is concluded that: (1) qualitative examination of fluorescence photomicrographs cannot permit accurate comparison between different fluorescence densities. (2) The cell-cell contact area was about fourfold higher in specific conjugates than in non-specific ones. (3) The surface density of adhesion molecules exhibited similar increases (between 30 and 80%) in the contact areas of both specific and nonspecific conjugates. (4) However, the amount of redistributed surface molecules was higher when cell-cell interaction was enhanced either by specific immunological recognition (in specific conjugates) or periodate oxidation. (5) Since redistribution did not require extracellular calcium and it was detected on nonspecific conjugates, this did not require full lymphocyte activation. Spatial reorganization of cell surface molecules may thus be a general consequence of adhesion, cell surface mobility and intermolecular forces.

Animals↗

PubMed1988

T-cell-mediated cytolysis: analysis of killer and target cell deformability and deformation during conjugate formation.

T-cell-mediated cytolysis is initiated by the formation of strong adhesions between killer and target cells. The present work was aimed at determining whether T lymphocytes might exert some mechanical stress on targets during the binding process. Target S194 myeloma cells were thus conjugated to cytotoxic T lymphocytes (CTLs) raised by mixed lymphocyte culture or a cloned lymphoid line that was no longer cytolytic (TG2OUA2). After incubation periods of various lengths, conjugates were processed for electron microscopy and micrographs were digitized for computerized analysis: the cell surface curvature (at the micrometre level) and rugosity (at the submicrometre level) were quantified in free and adhesion-involved regions. Also, the size of cell interaction areas and the distribution of intermembrane distances were measured. Finally, TG2OUA2 and target cells were aspirated into glass micropipettes with calibrated pressure in order to assay their resistance to deformation by mechanical forces. The following conclusions were suggested. (1) Formation of extensive contact zones (with a linear size of several micrometres) with tight intermembrane adhesion (more than 30% of the membrane contours in adhesive zones were separated by an apparent distance lower than 500 A) was essentially completed within less than one minute. (2) CTLs or TG2OUA2 cells were more villous than their targets, and they seemed to deform in adhesive zones in order to adapt to the target contour, rather than imposing some deformation on the target. This may be a general feature of actively adherent cells. (3) Some CTLs, but no TG2OUA2 cells, exhibited conspicuous protrusions extending towards the bound target. In this case, the target cell but not the CTL displayed markedly increased roughness in the adhesion area. (4) TG2OUA2 cells were more resistant to mechanical deformation than S194 target cells, in accordance with previous reports suggesting that the membrane of CTLs was more resistant than that of target tumour cells. Hence, CTLs might transiently impose mechanical stress on the target membrane during the course of lethal-hit delivery.

Animals↗

PubMed1987

Study of CTL and LAK contacts to target cells after treatment with mitomycin C and adriamycin.

We tried to understand the role of Mitomycin C and Adriamycin in the increased killing of target cells by Cytotoxic T lymphocytes (CTL) and lymphokine activated killers (LAK). For this purpose, we used an objective method allowing quantitative evaluation of the roughness of cell contours on electron micrographs. We compared the folding of the membranes of LAK and CTL as well as conjugated targets exposed to different treatments. We demonstrated first that CTL and LAK displayed similar morphological patterns: the killer cells were more villous than targets in the free areas, and second that the former cells exhibited significant smoothing on the areas of contact with these targets. These results suggest that the binding process (as distinct from the recognition step) is dependent on killer properties which are the same in CTL, LAK and probably NK cells.

Animals↗

PubMed1987

Phenotypic evolution of CTL B-lines in vitro.

This study demonstrates that cytolytic T-cell lines exhibit progressive in-vitro modifications of their phenotype and of their growth behaviour and may use different pathways for their multiplication. Comparing three established cell lines, we firstly demonstrated that the expression of LFA-I is stable but the Lyt 2, 3 is rapidly lost. In this case, a high lectin-dependent cytotoxicity appears. Secondly, we demonstrated that two of the cell lines used the interleukin 2-interleukin 2 receptors (IL-2-IL-2R) binding pathway. Two different monoclonal antibodies showed that the IL-2 receptors distribution does not correlate with the number of functional sites which determines the IL-2 requirement. In contrast, the third cell line, although bearing high levels of IL-2 receptors, grows without the addition of IL-2; this cell growth is not inhibited by anti-IL-2 receptors monoclonal antibodies. Thirdly, it appears that the new property of IL-2 independence is associated with acquisition of the simultaneous capacity to induce tumour grafts in nude mice. As it has been recently reported that cytolytic T-lymphocytes against tumour cells could be promising immunotherapeutic agents, the spontaneous malignant transformation of such CTL lines should be taken into account before using them for adoptive immunotherapeutic purposes.

Animals↗

PubMed1986

Quantification of cell surface roughness; a method for studying cell mechanical and adhesive properties.

The presence on the surface of nucleated cells of a variety of asperities of different size and shape plays a prominent role in cell-cell and cell-substrate interaction. Also, the organization of these asperities is directly related to cellular cytoskeletal elements. In the present report, we describe a simple and objective method of studying electron micrographs to quantify the roughness of cell contours. Constant-length segments of cell boundaries are compared to reference circular segments with common extremities and enclosing the same area. This procedure was performed with a digitizer connected to a microcomputer, and it was used to analyse model contours or electron micrographs of (i) target tumour cells bound by cytotoxic T lymphocytes and (ii) thymocytes sticking to concanavalin A-coated surfaces. It is shown that this method allows precise quantification of cell deformation in adhesive zones, which may allow absolute evaluation of adhesive stimuli.

Animals↗

PubMed1985

Study of cell deformability by a simple method.

Cell deformability plays an important role in many immunological processes, such as phagocyte chemotaxis and endocytosis. The most widely used method of assay consists in aspirating cells into glass micropipettes and measuring the length of the protrusion induced by a given pressure, or the minimum pressure required to drive cells into the micropipette. This procedure requires specialized equipment and delicate manipulation. The present report describes a simpler procedure: cells are centrifuged in petri dishes floating on a water cushion, then fixed and coated with 0.8 micron diameter latex beads, which allows rapid and accurate determination of their height. This method is compared with the micropipette technique by studying lymphocyte and macrophage-like cell lines in physiological medium and in the presence of a divalent cation chelator or a microfilament inhibitor. In addition to simplicity, the main advantages of this technique are that (i) many cells may be examined within a reasonable period of time, which allows testing of heterogeneous cell populations, and (ii) unexpectedly, centrifugation was quite harmless under our experimental conditions, since it did not impair cell proliferative ability nor phagocytic ability. It is concluded that the method may be used in clinical laboratories to explore phagocyte dysfunctions, as well as in experimental studies.

Animals↗

PubMed1985

Unexpected cell surface labeling in conjugates between cytotoxic T lymphocytes and target cells.

Specific binding of target cells by cytotoxic T lymphocytes (CTL) is an example of tight interaction between two different cell types. The molecular events that occur at the cell membranes during these interactions are largely unknown. In the present report, we describe an electron microscopic immunostaining study made on CTL-target cell conjugates. Various membrane structures were labeled with monoclonal antibodies specific for structures possibly relevant to cytolysis (Lyt-2, LFA-1, and target cell class I major histocompatibility antigens) or probably unrelated to the cytolytic process (effector cell class I major histocompatibility antigens). Antibodies against Thy-1 were also used. Staining was achieved with immunoperoxidase or immunoferritin. With both techniques nonconjugated cells were either stained or not, depending on whether they bore the antigen corresponding to the antibody used. However, when conjugated to an antigen-bearing cell, a "non-antigen bearing" cell was labeled near the cell interaction area. No increased Fc receptor activity could be detected on bound cells near the interaction area. These data are consistent with the occurrence of limited exchange of membrane macromolecules between bound CTL and target cell.

Animals↗

PubMed1984

Functional characteristics of cytotoxic T lymphocyte polyoma virus-transformed fibroblast hybrids.

By two different fusions between a cytotoxic T-lymphocyte (CTL) clone and a polyoma virus (Py)-transformed fibroblast line, 40 hybrid clones have been generated. It has been demonstrated that they were all TCGF independent for multiplication. Moreover, some of these hybrids were functional for cytolytic expression, whether or not TCGF was present either at the time of fusion or in the selective media. Two clones generated from the same fusion were markedly cytolytic and were able to remove TCGF from their culture medium, suggesting that they possessed TCGF receptors. These clones also secreted discrete amounts of a TCGF-like factor. The effect of TCGF on hybrid cell proliferation is discussed.

Animals↗

PubMed1984

Nonspecific cell surface properties: contact angle of water on dried cell monolayers.

Measuring contact angle of water on dried cell or bacterium monolayers allowed van Oss (1) and others (2) to find a correlation between particle hydrophobicity and ingestion by phagocytic cells. The present study was undertaken to understand what was actually assayed with this method. Monolayers were prepared with different cell types at different densities, and they were dried under atmospheres with varying humidity before being studied with scanning electron microscopy and contact angle techniques. It is concluded that a) contact angles are independent of the cell density and substrate structure when more than 30% of the substrate area is covered with cells. b) Initial cell shape should not influence contact angle. c) Contact angles are markedly dependent on the nature of tested cells. d) Contact angles are substantially influenced by the cell drying procedure. e) A very small fraction of the energies we measured would be sufficient to account for cell-cell interactions. Hence these might play a role in some situations of biological interest.

Animals↗

PubMed1983

Functional and ultrastructural alterations of autologous platelets labeled with 111In-oxine.

High activity labeled platelets could be useful for the detection and observation of small foci of thrombosis by gamma-camera imaging. Therefore platelets were labeled with 111In-oxine containing increasing activities of 111In to determine the elimiting amount of this tracer that did not cause cell damage. A labeling procedure was employed so that all the chemical parameters remained constant except the amount of 111In. Platelet damage was studied by ADP-induced aggregability according to the Born procedure and by scanning and ultrastructural electron microscopy. Platelets labeled with the lowest activity 2.2 MBq/ml of platelet suspension (10(9) cells/ml) showed no alterations. With the highest activity studied, (22 MBq/ml) aggregability decreased by two-thirds and great changes in the shape of the platelets were seen by electron microscopy. These modifications were attributed to the decay of 111In predominantly located in the platelet cytosol. Labeling of platelets with an activity higher than 7.4 MBq/ml is unsuitable for detection of thrombosis since normal platelet functions are not retained.

Blood Platelets↗