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F Dignat-George

Publications and source records attributed to F Dignat-George.

At least 19 recordsLinked to original sources
PubMed2006

Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol.

BACKGROUND: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. AIM: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. METHODS: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. RESULTS: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4 degrees C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. CONCLUSION: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

CD146 Antigen↗
PubMed2006

The imbalance between circulating endothelial cells and progenitors in cardiovascular diseases: a mirror of disrupted endothelial integrity.

The disruption of endothelial integrity is at the crossroads of the initiation and progression of multiple cardiovascular diseases pathophysiology. During these last years, it has been evidenced that endothelium is a highly dynamic tissue in equilibrium with a circulating compartment composed of various sub-populations offering important opportunities for a non-invasive exploration. Among these, circulating endothelial cells (CEC) are mature cells detached from injured vessels. Rarely found in blood in the health, CEC number is raised in a wide variety of pathological conditions associated with profound vascular insult. An additional population are the endothelial progenitor cells derived from bone marrow and characterised by their ability to participate in endothelial repair. Reduced number and impaired function of EPC have been related to situations with increased cardiovascular risk or to clinical atherosclerotic diseases. This review will focus on present data concerning CEC and endothelial cells and progenitors (PEC) in the setting of cardiovascular diseases. Particular emphasis will be given on the clinical value of these endothelial biomarkers to monitor endothelial homeostasis depending on the balance between endothelial injury and repair.

Biomarkers↗
PubMed2005

Elevation of circulating endothelial microparticles in patients with chronic renal failure.

BACKGROUND: Chronic renal failure patients are at high risk of cardiovascular events and display endothelial dysfunction, a critical element in the pathogenesis of atherosclerosis. Upon activation, the endothelium sheds microparticles, considered as markers of endothelial dysfunction that also behave as vectors of bioactive molecules. AIM: To measure plasma levels of endothelial microparticles (EMPs) in chronic renal failure patients (CRF), either undialyzed or hemodialyzed (HD), and to investigate the ability of uremic toxins to induce EMP release in vitro. METHODS: Circulating EMPs were numerated by flow cytometry, after staining of platelet-free plasma with phycoerythrin (PE)-conjugated anti-CD144 (CD144+ EMP) or anti-CD146 (CD146+ EMP) monoclonal antibodies. Platelet MP (CD41+ PMP), leukocyte MP (CD45+ leukocyte microparticles (LMP)), and annexin-V+ MPs were also counted. In parallel, MPs were counted in supernatant of human umbilical vein endothelial cells incubated with uremic toxins [oxalate, indoxyl sulfate, p-cresol, and homocysteine (Hcy)], at concentrations found in patients. RESULTS AND CONCLUSIONS: CD144+ EMP and CD146+ EMP levels were significantly higher in CRF and HD patients than in healthy subjects. Furthermore, annexin-V+ MPs were elevated in both groups of uremic patients, and CD41+ PMP and CD45+ LMP were increased in CRF and HD patients, respectively. In vitro, p-cresol and indoxyl sulfate significantly increased both CD146+ and annexin-V+ EMP release. Increased levels of circulating EMP in CRF and HD patients represent a new marker of endothelial dysfunction in uremia. The ability of p-cresol and indoxyl sulfate to increase EMP release in vitro suggests that specific uremic factors may be involved in EMP elevation in patients.

Adult↗
PubMed2005

[NK cells: new insights on physiology and clinical implication in diseases].

INTRODUCTION: Natural killer cells are cytotoxic lymphocytes of innate immunity. These last ten years our knowledge about the mechanisms that regulates NK cell function has greatly improved. Our purpose is to present a review of these new acquisitions and their potential implications in human disease. CURRENT KNOWLEDGE AND KEY POINTS: NK cell function is regulated by a repertoire of NK cell receptors and is diversified by recognition of MHC class I by a multigenic and multi-allelic family of NK receptors. Analysis of NK cell repertoire has been used to investigate features that characterize NK cells in pathological situations. Apart from their direct cytotoxic potential to eliminate target cells, recently identification of mechanisms that control NK cell mediated cytokine production and cross talk with dendritic cells emphasize the role of NK cells in the regulation of acquired immune response. FUTURE PROSPECTS AND PROJECTS: These findings have lead to a better knowledge of the importance of the NK cells in several human diseases. It has been shown that NK cells are actors of the immunosurveillance of tumoral and infectious challenges. Allo or auto reactivity of the NK cell compartment have also been suggested in autoimmune diseases, infertility or foetal loss and transplantation. Ongoing research on NK cells in the fields of human diseases is increasing and will clarify the utility of the evaluation of the NK cell compartment and their receptors in clinical practice.

Animals↗
PubMed2004

[New insight in physiopathology of preeclampsia and intra-uterine growth retardation: role of inflammation].

Inflammatory response is a major component in physiopathology of preeclampsia and intra-uterine growth retardation. Endothelium is a main connection between placental ischemia and clinical manifestations during vascular pregnancy complications. In this review recent findings concerning inflammatory response and its links with endothelium are reported. Studies concerning isolated intra-uterine growth retardation confirm the hypothesis of a similar pathophysiology with an activation confined to utero-placental bed or at a lower level. Current information on oxidative stress, atherosclerosis, and apoptosis in vascular pregnancy complications are available in this review. These concepts offer innovative possibilities of treatment.

Apoptosis↗
PubMed2002

Analysis of donor NK and T cells infused in patients undergoing MHC-matched allogeneic hematopoietic transplantation.

We retrospectively analyzed the percentages and absolute numbers of T cells, natural killer (NK) cells and NK cell subsets in cryopreserved samples of either bone marrow or blood non-T cell-depleted allogeneic MHC-matched hematopoietic grafts. Using flow cytometry, we found higher numbers of NK cells in aphereses than in bone marrow collections. We further investigated the distribution of NK cell subsets, defined by the cell surface expression of MHC class I-specific receptors, in these allogeneic grafts. The distribution of NK cell subsets from the two different origins were similar, with the exception of the CD158a/h(+) NK cell subset, whose size appeared to be smaller in bone marrow. The search for relations between the numbers of infused cells and post-transplantation events demonstrated that increasing numbers of infused T cells but not NK cells are related with decreased overall survival. Our study highlights the toxicity of infused T cells but not NK cells in allogeneic MHC-matched hematopoietic grafts. These data pave the way for further trials to investigate the effect of NK cell infusion in MHC-matched allogeneic transplantation, and in particular whether ex vivo NK cell expansion and activation may enhance the anti-tumoral effect of the procedure and decrease its morbidity.

Adolescent↗
PubMed2002

Platelet associated u-PA up-regulates u-PA synthesis by endothelial cells.

Adhesion of platelets to endothelium has been shown to induce important changes in endothelial properties. In this study, we examined the effect of platelet-endothelial cell interactions on the expression of urokinase-type plasminogen activator (u-PA) by human microvascular endothelial cells. After incubation of endothelial cells with platelets, a dose-dependent increase in the expression of u-PA Ag was observed and reached a plateau for a ratio of 300 platelets per endothelial cells. The u-PA Ag upregulation resulted from an increase in u-PA mRNA that originated from a synthesis by endothelial cells since no u-PA mRNA was detected in platelets. The platelet-induced u-PA synthesis was inhibited when the endothelial cells were pre-treated with phospholipase C to remove the u-PA receptor, or when the platelets were incubated with an antibody that blocks the binding of u-PA to u-PAR. Taken together, these data indicate that u-PA present on the platelet surface interacts with u-PAR on the endothelial cells and induces the u-PA synthesis. This mechanism may represent a physiological control of platelet-mediated intravascular fibrin deposition.

Adenosine Diphosphate↗
PubMed2001

Identification of CD146 as a component of the endothelial junction involved in the control of cell-cell cohesion.

CD146 is a cell-surface molecule belonging to the immunoglobulin superfamily and expressed in all types of human endothelial cells. Confocal and electron microscopic analysis of confluent human umbilical vein endothelial cells (HUVECs) were used to demonstrate that CD146 is a component of the endothelial junction. Double immunolabeling with vascular endothelial cadherin showed that CD146 is localized outside the adherens junction. Moreover, CD146 expression is not restricted to the junction, since part of the labeling was detectable at the apical side of the HUVECs. Interestingly, cell-surface expression of CD146 increased when HUVECs reached confluence. In addition, the paracellular permeability of CD146-transfected fibroblast cells was decreased compared with that of control cells. Finally, CD146 colocalized with actin, was partly resistant to Triton X-100 extraction, and had its expression altered by actin-disrupting agents, indicating that CD146 is associated with the actin cytoskeleton. These results show the regulated expression of CD146 at areas of cell-cell junction and strongly suggest involvement of CD146 as a mediator of cell-cell interaction.

Actins↗
PubMed2001

Outside-in signaling pathway linked to CD146 engagement in human endothelial cells.

CD146 (S-Endo 1 Ag or MUC18) is a transmembrane glycoprotein expressed on endothelial cells on the whole vascular tree. CD146 is located at the intercellular junction where it plays a role in the cohesion of the endothelial monolayer. CD146 engagement initiates an outside-in signaling pathway involving the protein tyrosine kinases FYN and FAK as well as paxillin. Here we report that CD146 engagement by its specific monoclonal antibody in human umbilical vein endothelial cells induces a Ca(2+) influx that is sensitive to thapsigargin and EGTA treatment, indicating that CD146 engagement initiates a store-operated calcium mobilization. In addition, biochemical and pharmacological analysis revealed that CD146 engagement initiates the tyrosine phosphorylation of phospholipase C-gamma, Pyk2, and p130(Cas). Pharmacological inhibition of Ca(2+) flux with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acetoxymethyl ester and EGTA indicated that an increase in Ca(2+) is required for Pyk2 and p130(Cas) tyrosine phosphorylation. Moreover, a complex association was observed between Pyk2, p130(Cas), and paxillin. These results indicate that CD146 is coupled to a FYN-dependent pathway that triggers Ca(2+) flux via phospholipase C-gamma activation leading subsequently to the tyrosine phosphorylation of downstream targets such as Pyk2, p130(Cas), FAK, and paxillin. In addition to its role in cell-cell adhesion, CD146 is a signaling molecule involved in the dynamics of actin cytoskeleton rearrangement.

Antigens, CD↗
PubMed2001

Maternal endothelial soluble cell adhesion molecules with isolated small for gestational age fetuses: comparison with pre-eclampsia.

OBJECTIVE: 1. To evaluate the activation profile of the endothelium in pregnancies complicated by small for gestational age fetuses compared with pre-eclampsia and normal pregnancy, by measuring the plasma levels of soluble adhesion molecules soluble E-selectin, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. 2. To determine whether soluble adhesion molecules were related to the severity of small for gestational age fetuses and pre-eclampsia. DESIGN: Observational study. PARTICIPANTS: Sixteen women with small for gestational age fetuses; 15 women with pre-eclampsia and 15 healthy primigravidae were recruited as controls. METHODS: Plasma levels of soluble E-selectin, intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 were measured by ELISA. RESULTS: Compared with the healthy controls, soluble E-selectin was significantly increased in both small for gestational age fetuses and pre-eclampsia, whereas intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 were increased only in pre-eclampsia. In the small for gestational age fetuses group, soluble E-selectin correlated inversely with the ratio between birthweight and the expected normal birthweight (r = -0.4, P = 0.007). In the pre-eclampsia group, a significant correlation was observed between vascular cell adhesion molecule-1 and blood pressure (r = 0.54, P = 0.039). CONCLUSIONS: Endothelial activation, reflected by raised levels of soluble E-selectin, is a feature of small for gestational age fetuses and is correlated with the severity of the disease. Differences in the profile of soluble cell adhesion molecules suggest variations in the degrees of endothelial activation between pre-eclampsia and small for gestational age fetuses.

Adult↗
PubMed2000

Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin.

Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-gamma and tumor necrosis factor-alpha) and chemokines (e.g., macrophage inflammatory protein-1alpha) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR(+) NK cells to specific tissues.

Amino Sugars↗
PubMed2000

Circulating endothelial cells in vascular disorders: new insights into an old concept.

The endothelial contribution to vascular disorders has been widely documented in experimental models. However, its implication in human pathology is difficult to investigate, owing to the paucity of noninvasive methods and of specific endothelial markers. The enumeration of circulating endothelial cells (CEC) released in peripheral blood after vascular injury represents a direct exploration of the endothelium. For this purpose, we have produced a monoclonal antibody (S-Endo 1), which recognizes CD 146, a molecule expressed on all types of human endothelial cells but absent from haemopoietic cells. Using this antibody, we have designed a specific and sensitive immunocapture test, which allowed us to detect high numbers of CEC in thrombotic, infectious or immunological disorders, while CEC were found to be very rare ( < 3/ml) in normal subjects. This quantitative approach using CEC might prove useful as a marker of vascular wall injury. Their enumeration is of interest in the clinical follow-up of vascular disorders, in the evaluation of therapeutic effectiveness or in the direct diagnosis of infectious diseases involving intra-endothelial microbial agents. Furthermore, an immunological and/or functional study of CEC could allow one to assess their procoagulant and proadhesive properties, as well as their viability, opening new perspectives for CEC investigation in vascular pathology.

Antigens, CD↗
PubMed2000

Impact of highly active anti-retroviral therapy (HAART) on cytokine production and monocyte subsets in HIV-infected patients.

HIV infection is associated with cytokine production by monocytes and expansion of a monocyte subset that expresses high levels of CD16. Our study was designed to investigate the effects of anti-retroviral therapies on these immune parameters. Four groups of HIV+ patients were included in the study. The first group comprised drug-naive patients (n = 20); the second included patients who received two inhibitors of HIV reverse transcriptase (n = 45); the third group received a therapy combining these two inhibitors and one inhibitor of HIV protease (HAART) (n = 35); the fourth consisted of patients who had stopped their treatment (n = 20). The release of inflammatory cytokines (tumour necrosis factor, IL-1beta, IL-6) and immunoregulatory cytokines such as IL-10 by monocytes was determined by ELISA. The monocyte subsets expressing low or high levels of CD16 were studied by flow cytometry. Monocytes from patients naive of treatment released higher amounts of inflammatory cytokines and IL-10 than HIV- individuals. Each anti-retroviral therapy restored a normal pattern of cytokine secretion. Nevertheless, the release of cytokines increased again after the arrest of the treatment. The expansion of the monocyte subset that expresses high levels of CD16 was significantly decreased by HAART but not by the treatment including two inhibitors of reverse transcriptase. These results suggest that only HAART controls monocyte activation in the treatment of HIV infection.

Adult↗