Search PubMed⌕ Search

Biomedical subjects

P Bongrand

Publications and source records attributed to P Bongrand.

At least 19 recordsLinked to original sources

Is there a predictable relationship between surface physical-chemical properties and cell behaviour at the interface?

There is much interest in predicting and controlling the outcome of interaction between artificial surfaces and living cells. However, although there is an impressive amount of information on the behaviour of many cell populations deposited on a variety of surfaces, there is presently no available theory to explain or even summarize these data. Indeed, it is not even obvious that such a theory may exist. The aim of the present review is to emphasize the problems encountered when one attempts to build such a theory. Three sequential steps of cell surface interactions are considered: 1) protein adsorption is a preliminary step liable to involve irreversible interaction between the surface and several hundreds of molecular species occurring in blood or plasma. 2) the second step is the formation of adhesive bonds. Several theoretical frameworks were suggested to account for this step, including DLVO theory, physical chemistry of surfaces, and formation of specific ligand-receptor bonds. It is concluded that present evidence supports the latter approach, although this involves serious difficulties. 3) The last step is the triggering of a specific cell program such as apoptosis, proliferation, migration, differentiation or activation. Recent evidence suggests that in addition to the nature and amount of stimulated surface receptors, additional cues such as substratum mechanical or topographical properties may significantly affect cell behaviour.

Adsorption↗

Assessment of T lymphocyte cytokine production in induced sputum from asthmatics: a flow cytometry study.

BACKGROUND: Asthma results from a bronchial inflammation in which Th2 lymphocytes play a pivotal role, as shown in invasive bronchial biopsies and broncho-alveolar lavages. Induced sputum (IS) is a non-invasive method of recovery of bronchial cells, which can be repeated in the same patients. However, lymphocyte activation has not been studied in IS to date, because of the low number of T cells recovered. Herein we took advantage of flow cytometry, a method suitable for the study of small cell populations, to assess T cell cytokine production in IS. OBJECTIVES: (1) To assess induced sputum T cell cytokine production by flow cytometry in asthmatic subjects and controls. (2) To compare the T cell cytokine production between symptomatic and non-symptomatic asthmatics. METHODS: Thirteen asthmatics and 19 controls were included. Sputum was induced by a hypertonic saline. Sputum cells were stimulated and intracellular IL-13 and IFN-gamma were detected in T cells by flow cytometry. RESULTS: Stimulation induced an increase of IL-13 and IFN-gamma production by T cells. This increase was higher in asthmatics. IL-13-producing T cells were increased in asthmatics after stimulation. In symptomatic asthma, IFN-gamma-producing T cells were in higher proportion than in controlled asthma. CONCLUSION: IS T cell cytokine production indicates a basic Th2 bias in asthma, accompanied during symptoms by a Th1-like activation. These results open the field for longitudinal studies of the variation of T cell activation in asthma.

Adult↗

Cell fitting to adhesive surfaces: A prerequisite to firm attachment and subsequent events.

Cell adhesion usually involves extensive shape reorganization. This process is important because i) it is required for efficient cross-linking of interacting surfaces by adhesion receptors the length of which does not exceed several tens of nanometers and ii) it influences subsequent cell differentiation and activation. This review focuses on the initial phase of cell deformation, preceding the extensive reorganization process known as spreading. This first phase includes local flattening at the micrometer scale and membrane alignment at the nanometer level, resulting in fitting of the cell to an adhesive surface. Three main points are considered. First, experimental methods available to study cell apposition to a surface are described, with an emphasis on interference reflection microscopy. Second, selected experimental evidence is presented to show that there is a quantitative relationship between "adhesiveness" and "contact extension", and some theoretical models aimed at relating these parameters are briefly sketched. Third, experimental data on the kinetics of initial contact extension are described and possible mechanisms for driving this extension are discussed, including nonspecific forces, receptor-mediated interactions, active cell movements or passive membrane fluctuations. It is concluded that both passive physical phenomena and random active cell movements are possible candidates for the initial triggering of contact extension.

Journal Article↗

Increased soluble p55 and p75 tumour necrosis factor-alpha receptors in patients with hepatitis C-associated mixed cryoglobulinaemia.

To investigate whether tumour necrosis factor alpha (TNFalpha) plays a role in the pathogenesis of hepatitis C virus-associated mixed cryoglobulinaemia (HCV-MC), we measured soluble TNFalpha and its soluble p55 (sTNFR1) and p75 (sTNFR2) receptors in the serum of patients with HCV-MC. TNFalpha, sTNFR1 and sTNFR2 were measured in the serum of 32 patients with HCV-MC, 18 patients with hepatitis C without MC (HCV) and 18 healthy volunteers, using specific immunoassays. Correlations between clinical and biological parameters and the concentrations of TNFalpha and sTNFRs were established by studying detailed clinical records of the 32 HCV-MC patients. Although higher, TNFalpha levels were not significantly different in HCV-MC patients compared with healthy or HCV controls. sTNFR1 and sTNFR2, however, were significantly higher in HCV-MC compared with controls or with HCV patients, and higher concentrations of sTNFR1 and sTNFR2 were observed in patients with severe visceral vasculitis, compared with patients with limited purpura. sTNFR1 concentrations positively correlated with fibrinogen levels but TNFalpha, sTNFR1 and sTNFR2 did not correlate with other biological parameters such as rheumatoid factor concentrations, CH50 or C4 values. These data suggest a role for TNFalpha in the pathogenesis of the immune complex-mediated vasculitis associated with HCV-MC.

Aged↗

The IL-6-soluble IL-6Ralpha autocrine loop of endothelial activation as an intermediate between acute and chronic inflammation: an experimental model involving thrombin.

Thrombin is a procoagulant and proinflammatory molecule in vivo. In vitro, thrombin has been shown to induce endothelial activation, notably IL-8 secretion and adhesion molecule expression. In this study, we showed that thrombin may induce a new cascade leading from acute to chronic inflammation. Thrombin was able to induce the production of both IL-6 and monocyte chemotactic protein-1 (MCP-1) by HUVEC independently of IL-1alphabeta and TNF-alpha. Addition of physiological concentrations of exogenous soluble IL-6Ralpha (sIL-6Ralpha) to thrombin-activated HUVEC was sufficient to increase the amounts of MCP-1 produced, but not those of IL-8. These effects could be blocked by anti-IL-6 or anti-sIL-6Ralpha blocking mAb, demonstrating the existence of an autocrine loop of MCP-1 secretion, involving the IL-6/IL-6Ralpha/gp130 complex on HUVEC. In addition, we identified IL-8-activated neutrophils as a potential source of sIL-6Ralpha because IL-8 induced IL-6Ralpha shedding from the neutrophil membranes and increased in parallel sIL-6Ralpha concentrations in neutrophil supernatants. Furthermore, addition of neutrophils to thrombin-activated HUVEC significantly increased MCP-1 secretion, which could be decreased by blocking IL-6. Thus, thrombin-activated endothelium may induce a cascade of events characterized by IL-8 secretion, neutrophil local infiltration, and the release of IL-6Ralpha from neutrophil membranes. sIL-6Ralpha may then complex with IL-6 and increase the amount of MCP-1 produced by thrombin-activated endothelium, favoring monocyte infiltration, and the transformation of acute into chronic inflammation.

Acute Disease↗

Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells.

Endothelial cells play a key role in prominent immunological and pathological processes such as leukocyte trafficking, inflammation, atheroma or cancer cell metastasis. Umbilical veins are probably the most widely used source for human endothelial cells, since they are more easily available than many other vessels, they are free from any pathological process and they are physiologically more relevant than many established cell lines. Here, we describe a standard protocol for preparation, maintenance and quality control of these cells.

Cell Culture Techniques↗

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzman's law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force of approximately 5 pN and to assess the distance dependence within the tens of nanometer range.

Algorithms↗

Venom immunotherapy induces monocyte activation.

BACKGROUND: Venom immunotherapy (VIT) is an efficient treatment of hymenoptera venom allergy. The mechanism of VIT is based on the induction of tolerance of allergen-specific Th2 cells. The mechanisms of this T cell modulation are unknown, and could depend on cytokines produced by other cell types such as interleukin (IL)-12, tumour necrosis factor (TNF)-alpha and IL-10 by monocytes. OBJECTIVE: To assess if VIT modifies the monocyte production of IL-12, TNF-alpha and IL-10 during the 45 first days of treatment. METHODS: Fourteen patients and seven controls were included. Blood samples were taken once in controls and at day (D)1, D30 and D45 of VIT in patients. Monocytes were isolated, cultured with and without lipopolysaccharide (LPS), and the culture supernatant was harvested. IL-10, IL-12 and TNF-alpha were assayed in supernatants by ELISA. RESULTS: Baseline cytokine levels were not statistically different between patients and controls. During treatment, an increase of spontaneous monocyte production of IL-12 and TNF-alpha was observed at D15 and D45. The production of IL-10 increased at D15 and D45 but not significantly. After LPS-stimulation, IL-12, TNF-alpha and IL-10 monocyte production was not modified by VIT. CONCLUSION: VIT induces a monocyte activation characterized by a delayed overproduction of IL-12 and TNF-alpha. These cytokines could be relevant to the inhibition of Th2 cells during VIT. Therefore, VIT-induced tolerance could depend not only on the specific action of venom antigens on T cells, but also on a secondary non-specific action of monocytes.

Adult↗

Radiation-induced expression of functional Fas ligand in EBV-positive human nasopharyngeal carcinoma cells.

Ionizing radiation remains a major therapeutic tool against human cancers, especially epithelial tumors, which account for the majority of human malignancies. Although Fas and Fas-L are essential determinants of apoptosis, few data support their role in the cytotoxic effect of ionizing radiation. Epstein-Barr-virus (EBV)-positive nasopharyngeal carcinoma (NPC) were chosen to address this question owing to their known sensitivity to ionizing radiation and their constitutive expression of the Fas-receptor. We here report that, in xenografted NPC cells, Fas-L expression, which was very low in basal conditions, was dramatically increased by tumor irradiation. Both the Fas receptor and the Fas ligand were found to be functional in this model, and a high proportion of irradiated NPC cells underwent apoptosis following tumor irradiation. Induction of Fas-L expression and apoptosis were observed for doses as low as 2 Gy. These data show an increase in Fas-L expression upon irradiation exposure, and strongly suggest that, in some epithelial malignancies, Fas-mediated apoptosis can play a major role in the anti-tumor effect of ionizing radiation, in the range of doses used for therapeutic applications.

Animals↗

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level.

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins.

Actin Cytoskeleton↗

Increased soluble vascular cell adhesion molecule 1 concentrations in patients with primary or systemic lupus erythematosus-related antiphospholipid syndrome: correlations with the severity of thrombosis.

OBJECTIVE: Recent studies have shown that in vitro endothelial cells are activated by antiphospholipid antibodies and may support leukocyte adhesion. We studied levels of soluble intercellular adhesion molecule 1 (sICAM-1, sCD54), soluble vascular cell adhesion molecule 1 (sVCAM-1, sCD106), and soluble E-selectin (soluble endothelial leukocyte adhesion molecule 1 [sELAM-1, sCD62E]) in sera from patients with primary antiphospholipid syndrome (primary APS), and compared them with those from patients with systemic lupus erythematosus-associated APS (SLE-APS) or pure SLE, as well as with those from 2 control groups composed of healthy volunteers and patients with thrombosis unrelated to autoimmune diseases. METHODS: Serum samples from 24 patients with primary APS, 15 patients with SLE-APS, 22 patients with pure SLE, 48 control patients with thrombosis, and 18 healthy volunteers were examined using enzyme-linked immunosorbent assays specific for sICAM-1, sVCAM-1, and sELAM-1. RESULTS: Serum levels of sVCAM-1, but not sICAM-1 or sELAM-1, were significantly increased in all patient study groups compared with thrombosis control patients and healthy volunteers, but did not differ between the groups of patients with primary APS, SLE-APS, or pure SLE. Concentrations of sVCAM-1 were significantly higher in primary APS or SLE-APS patients with severe, recurrent thrombosis and were negatively correlated with platelet counts in primary APS patients. In patients with primary APS, sVCAM-1 levels were higher if there was thrombotic kidney involvement and correlated with creatinemia. CONCLUSION: Serum sVCAM-1 concentrations are increased in patients with primary APS, especially those with repeated thrombotic events or kidney involvement. These findings suggest that endothelial/ monocyte interaction may be important in the pathogenesis of primary APS.

Adult↗

Effect of dietary lipid (soybean lecithin and triacylglycerol) on hepatic F-actin microfilaments in cyclosporine A-treated rats: image analysis by confocal laser scanning microscopy.

We studied and quantified the effect of cyclosporine A on hepatic F-actin on bile canalicular and basolateral membranes in rats fed either soybean lecithin, triacylglycerol-enriched diet, or low-fat diet by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was quite normal in the lecithin-cyclosporine A group but decreased significantly in the other cyclosporine A-treated groups (by 40% and 25% of control in triacylglycerol-cyclosporine A and cyclosporine A groups, respectively). The alteration of F-actin by cyclosporine A, related to cholestasis evidenced by a decrease in bile salt secretion, was prevented by dietary soybean lecithin and amplified by dietary soybean triacylglycerol.

Actins↗

Relationships between natural T cells, atopy, IgE levels, and IL-4 production.

BACKGROUND: Th2 cells govern allergic disorders. Mechanisms leading to the Th2 commitment are dominated by the requirement of IL-4. A potential source of this triggering IL-4 could be the CD4 + subset of a small population of T cells, natural T (NT) cells. Indeed, this subset is involved in IgE responses in mice and produces promptly high amounts of IL-4 in both mice and man. METHODS: NT cells were identified in peripheral blood by flow cytometry with antibodies against Valpha24 and Vbeta11, recognizing the T-cell receptor specific for NT cells. Simultaneous staining with anti-CD3, anti-CD4, or anti-CD8 antibodies was performed. The frequency of NT cells in man was studied according to the presence of atopy defined by the positivity of skin tests, according to total IgE levels in serum, and according to IL-4 concentration of whole-blood culture supernatants determined by a flow cytometer microsphere-based assay. RESULTS: Seventy subjects were included, of whom 30 were atopic. The number of CD4+ NT cells was higher in atopics than in nonatopics (P=0.009). This number was correlated to the total IgE levels (r = 0.34, P = 0.03). In addition, the number of CD4 + NT cells, but also of CD8 + NT cells, was correlated to the levels of IL-4 (r=0.71, P=0.01, and r=0.6, P=0.03, respectively). CONCLUSIONS: These results show that the number of NT cells, particularly the CD4+ subset, is related to atopy, IL-4 production, and IgE levels. Therefore, this population of T cells is likely to play a role in the Th2 commitment initiating atopic diseases.

Adolescent↗

Comparative CD43 behavior on monocytes and lymphocytes in kidney transplants.

In human cultured monocytic cells stimulated by cytokines, CD43 was demonstrated to exhibit a modification of sialylated epitopes (dys-sialylation) [Soler et al: Leukoc Biol 1997;61:609-618]. Therefore, we chose to investigate CD43 behavior on patients who present pathological status implicating monocytes after renal graft (KTR). We performed flow cytometry after immune staining using monoclonal antibodies to CD43 sialic acid-dependent (L60) and -independent (L10) epitopes. Compared to normal controls, mean fluorescence intensity was never altered on lymphocytes. Conversely, on monocytes, we found different profiles with L60: 26% of patients having normal CD43 expression, 54% displayed decreased values and 20% had a double population of monocytes, the major one being normal and the minor one with a very low staining. Decreased values were more frequent among KTR during the first 3 months following transplantation. L10 immunostaining was not altered on monocytes in patients with low values of CD43 staining by L60, confirming that the mechanism involved was a CD43 dys-sialylation. We investigated a possible role of cyclosporin (CsA) on human monocytic (THP-1) and lymphoid (Jurkat) cell lines. CsA decreases CD43 expression in monocytic and not in lymphoid cell lines and could be responsible for the specific dys-sialylation of KTR monocytes. Whatever, CD43 dys-sialylation might lead to functional abnormalities of monocytes in KTR, possibly involving the adhesion process.

Adolescent↗

Assessment of the Th1/Th2 paradigm in whole blood in atopy and asthma. Increased IFN-gamma-producing CD8(+) T cells in asthma.

Atopy is characterized by an immune system that is biased to T helper cell, type 2 (Th2) activation. This condition predisposes to asthma, a disease in which a Th2 activation was found in blood and lungs. However, most blood studies have considered purified cells, which might give an incomplete view of immune reactions. In this study, we assessed in whole blood cultures the Th1/Th2 paradigm in atopy and asthma. Sixty-nine subjects (31 atopic asthmatics, six nonatopic asthmatics, 13 atopic nonasthmatics, and 19 control subjects) were included in this study. Interleukin-4 (IL-4), interferon gamma (IFN-gamma), and IL-12 were assayed in stimulated whole blood culture supernatants by using a flow cytometer microsphere-based assay. Intracellular IL-4 and IFN-gamma were detected in T cells and CD8(+) T cells by flow cytometry. Atopy was characterized by a higher production of IL-4, which was correlated to total IgE levels, and by an impairment of the T-cell capacity to produce IFN-gamma. This impairment was correlated to the number of positive skin tests. In asthma, the overproduction of IL-4 was still found if atopy was present. Unexpectedly, an overproduction of IFN-gamma was found, which was related to an increased capacity of CD8(+) T cells to produce IFN-gamma. The number of IFN-gamma-producing CD8(+) T cells was related to asthma severity, to bronchial hyperresponsiveness, and to blood eosinophilia. In addition, this number was correlated to IL-12 production. These results show that in addition to the well-known Th2 inflammation in asthma, there are IFN-gamma-producing CD8(+) T cells in the blood, possibly controlled by IL-12.

Adult↗

Glycocalyx modulation is a physiological means of regulating cell adhesion.

Here we present experimental evidence that phagocytic cells use modulation of specific components of their glycocalyx to regulate their binding capacity. Particles coated with antibodies specific for the CD32 medium affinity IgG receptor were driven along human monocytic THP-1 cells (expressing CD32) in a flow chamber operated at low shear rate. Surprisingly, only minimal adhesion was observed. However, when cells were activated by exposure to fibronectin-coated surfaces and/or soluble &ggr; interferon, adhesion efficiency was dramatically increased, whereas the apparent glycocalyx thickness displayed 20% decrease, and the surface density of CD43/leukosialin carbohydrate epitopes displayed 30-40% decrease on activated cells. The existence of a causal link between adhesion increase and glycocalyx alteration was strongly supported by the finding that (i) both phenomena displayed similar kinetics, (ii) an inverse relationship between THP-1 cell binding capacity and glycocalyx density was demonstrated at the individual cell level, and (iii) adhesion enhancement could not be ascribed to an increased binding site density or improved functional capacity of activated cells. Additional experiments revealed that cell-to-particle adhesion resulted in delayed (i.e. more than a few minutes) egress of CD43/leukosialin from contact areas. Since the time scale of particle attachment was less than a second, surface mobility should not affect the potential of CD43 to impair the initial step of adhesion. Finally, studies performed with fluorescent lectins suggested that THP-1 cell activation and increased adhesive potential were related to a decrease of O-glysosylation rather than N-glycosylation of surface glycoproteins.

Cell Adhesion↗

[Adhesion molecules and cancer].

INTRODUCTION: This review was aimed at summarizing recent advances in the understanding of cell adhesion in order to discuss the possible relevance of new knowledge to the exploration of cancer patients and elaboration of therapeutic strategies. CURRENT KNOWLEDGE AND KEY POINTS: During the last 10 years, many adhesion molecules were identified, thus allowing to determine their tissue distribution and functional regulation. The concept of adhesiveness was refined. It is now well known that adhesive rate (i.e., the minimal contact time required for bond formation) and binding strength (i.e., the minimal force required to detach bound cells) are distinct parameters. They may be regulated independently, and influence the cell behavior in different ways. It is now possible to achieve accurate control of tumor cell adhesiveness, either by inhibiting an adhesive mechanism (through monoclonal antibodies, competitive ligands, or inhibition of receptor expression with antisense strategy or gene knock-out) or by promoting a binding mechanism (with receptor transfection or pro-inflammatory stimulation). FUTURE PROSPECTS AND PROJECTS: Recent progress opens new possibilities for diagnosis and treatment. First, the interpretation of experimental data may be improved. Cell adhesive behavior is not entirely accounted for by the density of membrane adhesion receptors. Indeed, adhesion is influenced by receptor connection to the cytoskeleton and structure of the cell coat. An adhesion receptor may be anti-metastatic through an increase in tumor cohesion and cell differentiation, or pro-metastatic, through facilitation of cell migration towards a target tissue. New therapeutic strategies may include anti-adhesive procedure aimed at preventing metastasis formation. The potential importance of a better control of inflammatory processes is also emphasized in view of the influence of these processes on the expression of adhesion molecules.

Cell Adhesion↗