Biomedical subjects

A M Benoliel

Publications and source records attributed to A M Benoliel.

At least 19 recordsLinked to original sources

PubMed1998

Thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106).

Thrombin, a central molecule in coagulation, is also involved in inflammation. Notably, thrombin induces endothelial neutrophil adhesion, P- and E-selectin expression, and chemokine production. We show here that thrombin induces expression of intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) on human umbilical vein endothelial cells (HUVECs) associated with increased adhesion of monocytes. Thrombin increased mRNA steady-state levels and expression of ICAM-1 over 24 hours. Thrombin-induced VCAM-1 expression exhibited unusual kinetics, reaching maximum levels after 6 to 12 hours, but decreasing to near baseline after 24 hours. Thrombin activity on HUVECs was mediated through interaction with its specific receptor, because ICAM-1 and VCAM-1 expression were similarly induced by the 14-amino acid thrombin receptor-activating peptide. Thrombin-induced ICAM-1 and VCAM-1 expression was significantly inhibited by hirudin, but not by interleukin-1 receptor antagonist or anti-tumor necrosis factor alpha monoclonal antibody (MoAb). Thrombin-activated HUVECs significantly increased greater numbers of adhering THP-1 macrophagic cells, peripheral blood mononuclear cells, or purified monocytes than unstimulated HUVECs. This adhesion was inhibited by anti-CD18 and anti-CD49d MoAb, demonstrating that thrombin-induced ICAM-1 and VCAM-1 were functional. These results show that, in addition to selectins, thrombin directly induces a cytokine-independent expression of adhesion molecules of the Ig superfamily on HUVECs that may support firm leukocyte attachment during inflammation.

Antibodies, Monoclonal↗

PubMed1998

Experimental study of the interaction range and association rate of surface-attached cadherin 11.

We describe a method allowing quantitative determination of the interaction range and association rate of individual surface-attached molecules. Spherical beads (1.4 micro(m) radius) were coated with recombinant outer domains of the newly described classical type II cadherin 11, a cell adhesion molecule. Beads were driven along cadherin-coated surfaces with a hydrodynamic force of approximately 1 pN, i.e., much less than the mechanical strength of many ligand-receptor bonds. Spheres displayed periods of slow motion interspersed with arrests of various duration. Particle position was monitored with 50 Hz frequency and 0.025 micro(m) accuracy. Nearly 1 million positions were recorded and processed. Comparison between experimental and computer-simulated trajectories suggested that velocity fluctuations might be related quantitatively to Brownian motion perpendicular to the surface. The expected amplitude of this motion was of order of 100 nm. Theoretical analysis of the relationship between sphere acceleration and velocity allowed simultaneous determination of the wall shear rate and van der Waals attraction between spheres and surface. The Hamaker constant was estimated at 2.9 x 10(-23) J. The frequency of bond formation was then determined as a function of sphere velocity. Experimental data were consistent with the view that the rate of association between a pair of adhesion molecules was approximately 1.2 x 10(-3) s-1 and the interaction range was approximately 10 nm. It is concluded that the presented methodology allows sensitive measurement of sphere-to-surface interactions (with approximately 10 fN sensitivity) as well as the effective range and rate of bond formation between individual adhesion molecules.

Cadherins↗

PubMed1998

Thyrotropin chronically regulates the pool of thyroperoxidase and its intracellular distribution: a quantitative confocal microscopic study.

The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1-50 microM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 microM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation 1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and 2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells.

Animals↗

PubMed1997

Interest of image processing in cell biology and immunology.

Microscopy is a basic tool for cell biologists. Recent progress of electronics and computer science made powerful methodologies for digital processing of microscopic images easily available. These methods allowed impressive increase of the power of conventional microscopy. Dramatic image enhancement may be achieved by combination of filtering techniques, computer-based deblurring and contrast enhancement. Quantitative treatment of digitized images allows absolute determination of the density of different components of the observed sample, including antigens, intracellular calcium and pH. Morphometric studies are also greatly facilitated by image processing techniques. The capture of fast phenomena may be performed by transfer of small portion of microscopic images into computer memory as well as particular use of confocal microscopy. Finally, improved display of experimental data through coded colors or other procedures may enhance the amount of information that can be conveyed by visual examination of microscopical images. The purpose of the present review is to describe the basic principles of image processing and exemplify the power of this approach with a variety of illustrated applications to conventional, fluorescence or electron microscopy as well as confocal microscopy.

Image Processing, Computer-Assisted↗

PubMed1997

The dependence of the association rate of surface-attached adhesion molecules CD2 and CD48 on separation distance.

The kinetics of bond formation between spherical beads coated with CD48 and CD2-derivatized surfaces was studied with a flow chamber. For a given shear rate, the binding frequency was exquisitively sensitive to the particle velocity. Flow equations were used to derive the particle-to-surface distance from the velocity, thus yielding a relationship between this distance and the binding rate. Numerical values of the binding site densities allowed absolute determination of the rate of association between two individual molecules as a function of the distance between attachment points. In our model, this rate was about 0.03 s-1 at 10 nm separation, and it was inversely proportional to the cube of the distance.

Animals↗

PubMed1997

Insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor.

Insulin-mediated cell motility as well as the role of transcription factors in insulin-activated intracellular signal events have not been extensively studied. In this report we have examined whether insulin could mediate haptotactic migration of cultured human epidermal keratinocytes through activation of transcription factor NF-kappa B. Insulin caused a dose-dependent stimulation of keratinocyte migration that maximally reached 2-fold at 2 x 10(-7) M hormone. This phenomenon was independent of the nature of the extracellular matrix component (collagen I or laminin5/nicein) on which the cells migrated, indicating that a specific integrin-ligand complex is not required. A 10(-7) M insulin treatment of keratinocytes resulted in activation of a major kappa B DNA binding complex within 15 to 30 minutes, which was identified as the p65/p50 NF-kappa B heterodimer by electrophoretic mobility shift assays. The activation induced nuclear translocation of cytosolic pools of NF-kappa B factor. Pyrrolidine dithiocarbamate and N-acetyl-leucinyl-leucinyl-norleucinal H (two compounds that differentially inhibit I kappa B alpha degradation and, thus, NF-kappa B activation) reversed the insulin-stimulated keratinocyte haptotactic migration without affecting insulin receptor activation. These compounds inhibited the insulin-induced nuclear translocation of NF-kappa B as detected by confocal laser scanning microscopy. Taken together our experiments demonstrate that insulin stimulates haptotactic migration of human epidermal keratinocytes through activation of NF-kappa B transcription factor. They emphasize the ability of insulin to stimulate keratinocyte movement and provide a first clue to the mechanism of insulin-induced haptotactic signaling.

Antioxidants↗

PubMed1997

Analysis by confocal laser scanning microscopy imaging of undilated bile canaliculi F-actin staining in the hepatocytes of human extrahepatic cholestatic liver.

Many studies have demonstrated the role of bile canalicular microfilaments in bile secretion and bile flow. It is now admitted that modification of bile canalicular network of microfilaments play a role in dysfunction of bile secretion observed in many cases of cholestasis. This work intends to study F-actin, a major component of microfilaments, in human hepatocytes in extrahepatic cholestasis. Normal and extrahepatic cholestatic liver were studied. F-actin was stained with fluorescent phallotoxin and quantified by using confocal laser scanning microscopy and an image analysis method. Mean specific fluorescence (MSF) of bile canaliculi was measured. Since dilated and bile plugged canaliculi were rarely observed in cholestatic liver sections, only undilated bile canaliculi were analysed. Bile canalicular MSF was significantly increased (p < 0.05) in cholestatic hepatocytes (1.3 to 1.7 fold higher than in controls). These data demonstrate a pericanalicular thickening of F-actin microfilaments in human extrahepatic cholestatis, similar to that described in literature in many cases of human intrahepatic and extrahepatic cholestasis cases as well as in experimentally induced cholestasis. However, further studies are needed to understand this increase in F-actin pericanalicular microfilaments in human extrahepatic cholestasis.

Actin Cytoskeleton↗

PubMed1996

Determination of the lifetime and force dependence of interactions of single bonds between surface-attached CD2 and CD48 adhesion molecules.

We studied single molecular interactions between surface-attached rat CD2, a T-lymphocyte adhesion receptor, and CD48, a CD2 ligand found on antigen-presenting cells. Spherical particles were coated with decreasing densities of CD48-CD4 chimeric molecules then driven along CD2-derivatized glass surfaces under a low hydrodynamic shear rate. Particles exhibited multiple arrests of varying duration. By analyzing the dependence of arrest frequency and duration on the surface density of CD48 sites, it was concluded that (i) arrests were generated by single molecular bonds and (ii) the initial bond dissociation rate was about 7.8 s-1. The force exerted on bonds was increased from about 11 to 22 pN; the detachment rate exhibited a twofold increase. These results agree with and extend studies on the CD2-CD48 interaction by surface plasmon resonance technology, which yielded an affinity constant of approximately 10(4) M-1 and a dissociation rate of > or = 6 s-1. It is concluded that the flow chamber technology can be an useful complement to atomic force microscopy for studying interactions between isolated biomolecules, with a resolution of about 20 ms and sensitivity of a few piconewtons. Further, this technology might be extended to actual cells.

Animals↗

PubMed1996

Measuring bonds between surface-associated molecules.

Adhesive interactions play an essential role in immune function. Much information on these phenomena was recently obtained by applying sophisticated methods such as the surface forces apparatus, atomic force microscopy, lipid vesicle-based technology or flow chambers. In the present review it is shown that the use of hydrodynamic flow allows quantitative study of the formation and dissociation of individual molecular bonds between receptor-bearing cells or particles and ligand-derivatized surfaces. In addition, it should be possible to determine particle-surface interaction forces with subpiconewton sensitivity and nanometer resolution. Data analysis shows that the classical concepts of bond strength, or association and dissociation rates must be reexamined in order to achieve a correct understanding of the behavior of individual molecules.

Cell Adhesion Molecules↗

PubMed1995

Measuring the lifetime of bonds made between surface-linked molecules.

It is not well known how the kinetic constants of association between soluble receptors and ligands may be used to predict the behavior of these molecules when they are bound to cell surfaces. Spherical beads were coated with varying densities of anti-rabbit immunoglobulin monoclonal antibodies and driven along glass surfaces derivatized with rabbit anti-dinitrophenol. Particle motion was analyzed. The velocity, attachment frequency, and duration of binding events were determined on individual particles. It was found that i) beads exhibited frequent arrests lasting between a few tenths of a second and more than one minute; ii) when antibodies were diluted, the median arrest duration remained fairly constant (approximately 1 s) whereas binding frequency varied as the first power of the antibody concentration, suggesting that most particle arrests were due to the formation of a single bond; iii) when the shear rate was increased 7-fold, the duration of transient binding events remained constant. The disruptive force exerted on attachment points was estimated to range between about 6 and 37 piconewtons; and iv) the distribution of arrest durations suggested that binding was not a monophasic reaction but involved at least one intermediate step. Therefore, transient binding events reflected the formation of unstable associations that are not detected with standard techniques.

Animals↗

PubMed1995

Immunocytochemical study of NA+ K(+)-ATPase alpha 1 and beta 1 subunits in human and rat normal hepatocytes using confocal microscopy.

Hepatic Na+ K(+)-ATPase was recently shown to be composed of two alpha 1 and beta 1 subunits similar to that of kidney. Its localization on hepatocyte plasma membranes was not clearly established. We have studied the localization of alpha 1 and beta 1 subunits using immunocytochemical method and confocal microscopy. In accordance with previous cytochemical findings, the catalytic alpha 1 subunit was distributed in basolateral and bile canalicular membranes of hepatocytes. The beta 1 subunit was not demonstrated, due to its very low amount in the liver. The controversy about the bile canalicular localization was discussed.

Animals↗

PubMed1995

Use of thermal fluctuations to study the length and flexibility of ligand-receptor bonds.

We describe an experimental approach yielding new information on the behavior of ligand-receptor bonds. Spherical particles of 1.4 microns radius were coated with anti-rabbit immunoglobulin monoclonal antibodies and deposited on surfaces derivatized with rabbit immunoglobulins. Brownian motion was studied. When particles where bound through multiple bonds, the mean square displacement during a 1 s interval was 0.0038 micron2 as compared to 0.245 micron2 for unbound particles. Under the same conditions, the mean square displacement of particules coated with limiting dilutions of binding sites and bound by single molecular bonds was 0.0774 micron2. Results are compatible with the concept that the latter particles behaved as spheres transiently bound to the substratum by links of 2.7 nm length, allowing brownian oscillations with an angular amplitude of 0.062 radian.

Animals↗

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

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↗

PubMed1993

Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow.

The adhesion of moving cells to receptor-bearing surfaces is a key step to many important biological processes. Attachment was subjected to extensive modeling. However, the numerical values of kinetic bonding parameters relevant to realistic models of cell adhesion remain poorly known. In this report, we describe the motion of human granulocytes to interleukin-1-activated endothelial cells in presence of a low hydrodynamic drag (a few piconewtons) estimated to be much weaker than a standard ligand-receptor bond. It was thus expected to visualize the formation and rupture of individual bonds. We observed multiple short-time cell arrests with a median duration of 2.43 s. Stop frequency, not duration, was significantly inhibited by anti-E-selectin antibodies. Binding efficiency exhibited an almost linear relationship with the inverse of cell velocity. The distribution of arrest duration was determined: results were consistent with the view that these arrests reflected the formation/dissociation of single ligand-receptor bonds with a spontaneous dissociation rate of 0.5 s-1. The rate of bond formation was on the order of 0.04 s-1 when cells were freely rolling (mean velocity: 19 microns/s) and it exhibited an approximately 10-fold increase after the formation of a first adhesion.

Cell Adhesion↗

PubMed1993

Localization of myosin in normal human liver hepatocytes using immunohistochemical amplification methods.

Different immunohistochemical amplification systems were used to visualise myosin in normal human hepatocytes. With biotin-streptavidin-peroxidase and immunogold silver staining, myosin was distributed along the plasma membranes and at the bile canaliculi. With biotin-streptavidin-rhodamine, myosin was mainly found at the bile canaliculi level, however with confocal microscopy, the plasma membrane fluorescent staining was more apparent. The staining pattern of myosin appeared to be similar to that of actin in normal human hepatocytes.

Actins↗

PubMed1993

Role of calcium in the shape control of human granulocytes.

The possible role of cytoplasmic calcium in the shape control of human blood granulocytes was explored with two complementary sets of experiments. First, cells were stimulated with N-formyl methionyl leucyl phenylalanine (FMLP) with or without depletion of free intracellular calcium (by incubation with BAPTA/AM in calcium-deprived medium). Control cells displayed a rapid and transient rise of free intracellular calcium (peaked after a few seconds, returned to the basal level after 2-3 minutes), extensive morphological polarization (more than 90% polarized cells after 15 minutes), and reorganization of actin filaments (as assessed by image analysis on cells labeled with a fluorescent phallacidin derivative). Calcium-depleted cells displayed no calcium rise, but both morphological polarization and actin reorganization were indistinguishable from those of controls. In a second set of experiments, individual granulocytes were labeled with fluorescent calcium probes and aspirated in a micropipette on the stage of a confocal laser microscope. About half of the cells displayed a transient calcium rise, which was concomitant with the formation of a protrusion in most cases. However, extensive protrusions were shown by the cells that did not exhibit calcium flashes. Furthermore, if cells were treated with ionomycin, in calcium-containing or calcium-deprived medium, then aspirated, they showed protrusions comparable to those of controls. Finally, when cells were treated with ionomycin during micropipette aspiration, a calcium rise was followed by a moderate increase of the rate of protrusion growth. It is concluded that free calcium alone cannot play a major role in the control of cell shape.

Actin Cytoskeleton↗

PubMed1992

Phagolysosomal alkalinization and the bactericidal effect of antibiotics: the Coxiella burnetii paradigm.

Most infections due to intracellular bacteria respond poorly to antibiotic treatment. The chemical conditions within the subcellular site of bacteria may change antibiotic activity. Coxiella burnetii multiplies within phagolysosomes. The antimicrobial activity of antibiotics combined with the lysosomotropic agents amantadine (1 microgram/mL), chloroquine (1 microgram/mL), and ammonium chloride (1 mg/mL), which alkalinized Coxiella burnetii-containing phagolysosomes from pH 4.8 to 5.3, 5.7, and 6.8, respectively, was evaluated. Percentages of residual viable bacteria (RVB) in cell cultures were significantly reduced after exposure to combinations of doxycycline (4 micrograms/mL) with amantadine (RVB = 18.2% +/- 8.7%, P < .05), chloroquine (RVB = 0.64% +/- 0.38%, P < .01), or ammonium chloride (RVB = 0.29% +/- 0.17%, P < .01); the same was seen with pefloxacin (1 microgram/mL) with chloroquine (RVB = 27.6% +/- 10.8%, P < .05) or ammonium chloride (RVB = 3.72% +/- 1.1%, P < .05). Such bactericidal activity correlated with increased phagolysosomal pH, as determined by Pearson's correlation coefficient, suggesting that phagolysosomal alkalinization is critical for the bactericidal effect of antibiotics.

Amantadine↗