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H Lecoeur

Publications and source records attributed to H Lecoeur.

At least 37 records · Page 2Linked to original sources
PubMed1998

All-trans retinoic acid inhibition of anti-CD3-induced T cell apoptosis in human immunodeficiency virus infection mostly concerns CD4 T lymphocytes and is mediated via regulation of CD95 ligand expression.

This study analyzes the influence of all-trans retinoid acid (tRA) on apoptosis of peripheral lymphocytes from human immunodeficiency virus (HIV)-positive patients. tRA inhibits the ex vivo apoptosis in T cells; a more potent effect was observed on activation-induced apoptosis. Phenotypic characterization of T cell subsets prevented from anti-CD3-induced apoptosis by tRA revealed a more potent effect on CD4 T cells. A central regulatory system for apoptosis is the CD95 system, and inappropriate induction of this pathway is thought to contribute to AIDS pathogenesis. In investigation of CD95-based apoptosis, tRA had no effect on activation-dependent induction of CD95 on T lymphocytes, but it inhibited the induction of CD95 ligand expression on anti-CD3-activated T cells. The previously reported in vivo effect of tRA inhibiting HIV-associated apoptosis and the present observations suggest that tRA could be considered to down-regulate apoptosis associated with AIDS pathogenesis.

Animals↗
PubMed1997

Strategies for phenotyping apoptotic peripheral human lymphocytes comparing ISNT, annexin-V and 7-AAD cytofluorometric staining methods.

The present article compares the reliability of four previously described cytofluorometric methods of apoptosis quantification for phenotyping apoptotic human lymphocytes. Each of these assays detects distinct cellular alterations of the apoptotic process. Alteration in plasma membrane integrity can be evaluated following 7-AAD incorporation and the translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane can be detected through the FITC annexin V staining. DNA strand breaks in apoptotic nuclei can be evidenced by the ISNT assay and finally morphological modifications can be followed with FSC/SSC criteria. Comparative analysis of apoptosis in cultured PBMC from HIV-infected patients considering the FSC/SSC parameters, 7-AAD stainability and annexin V fixation revealed that the latter identifies early apoptotic cells, also characterized as 7-AAD(low) with a reduced FSC. Moreover these three methods proved to be reliable and gave statistically similar results when combined with cell surface detection of antigens such as CD4, CD8 and CD19 by specific mAbs. Importantly, the 7-AAD assay easily allowed the identification of debris/apoptotic bodies, which were still stained by anti-cell surface mAbs and might therefore significantly distort the apoptosis percentage in a given lymphocyte subset. In the present report we also point out that the ISNT assay is not appropriate for phenotyping apoptotic lymphocytes in PBMC. Indeed it can particularly underestimate the rate of apoptosis in the B-cell subset. This was found to be related to the apoptosis-associated decrease in cell surface antigen expression, which is dramatically exacerbated in the ISNT assay because of the stripper effect of ethanol used for cell permeabilization. Finally, we propose a three step analytical strategy to accurately phenotype apoptotic peripheral human lymphocytes. It includes two gating steps performed on FSC/SSC criteria and 7-AAD/FSC parameters to eliminate monocytes, granulocytes and debris-apoptotic bodies, the third step being the phenotyping step itself, performed in dual or triple staining experiments. Altogether these observations emphasize that it is essential to assess critically the ability of a cytofluorometric method to phenotype apoptotic cells in complex lymphoid populations and that inaccurate identification of cell subsets undergoing apoptosis can be readily overcome by gating properly the lymphoid population, and using assays which preserve cell surface structure.

Annexin A5↗
PubMed1997

Potential deleterious effect of anti-viral cytotoxic lymphocyte through the CD95 (FAS/APO-1)-mediated pathway during chronic HIV infection.

The potential deleterious effect through a CD95-based pathway of anti-viral cytotoxic lymphocyte (CTL) during HIV-infection was studied. The present paper reports that a Nef specific CTL line derived from an HIV-infected person is able to kill not only Nef-expressing target cells but also CD95+ compliant Jurkat cells. The two mechanisms of cytotoxicity, i.e. perforin-vs-CD95-dependent were differentiated according to their respective Ca(2+)-dependence. The existence of the dual killing machinery in the anti-HIV CTL line was correlated with the coexpression in these cells of perforin and CD95-L molecules. A model of AIDS pathogenesis involving the deleterious effect through the CD95 pathway of the viral specific CTL response is discussed.

Antigen-Presenting Cells↗
PubMed1997

Lack of chronic immune activation in HIV-infected chimpanzees correlates with the resistance of T cells to Fas/Apo-1 (CD95)-induced apoptosis and preservation of a T helper 1 phenotype.

Chimpanzees are one of the few species, along with humans, susceptible to persistent HIV-1 infection. However, HIV-infected chimpanzees do not exhibit the marked immune system alterations seen in humans and remain relatively resistant to AIDS. In humans, HIV infection leads to unresponsiveness of T cells in response to TCR stimulation, associated with increased T cell death by apoptosis. In an effort to understand some of the mechanisms used to limit lentivirus infection in African nonhuman primates, we compared apoptosis in infected humans vs chimpanzees in CD4 and CD8 T cells in relation with the expression of Bcl-2 and Fas molecules. The intensity of apoptosis in CD4 and CD8 T cells from infected chimpanzees was very low, was not inducible by several TCR-dependent activators, and was comparable to that detected in noninfected chimpanzees. Moreover, CD45RO+ and HLA-DR+ subsets, which were shown to exhibit ex vivo a high propensity to undergo apoptosis in infected humans, were not modified in infected chimpanzees. Interestingly, in contrast to the situation found in infected humans, Fas ligation by agonistic Abs or recombinant human Fas ligand on CD4 and CD8 T cells from infected chimpanzees did not induce apoptosis in these subsets even when Bcl-2 was down-regulated. Finally, this resistance to apoptosis was associated with the predominance of CD3 T cells with a Th1 phenotype. Together these observations argue for a strong relationship among the absence of chronic immune stimulation in HIV-1-infected chimpanzees, the normal control of lymphocyte survival, and the resistance to disease progression.

Animals↗
PubMed1996

Comparative analysis of flow cytometric methods for apoptosis quantitation in murine thymocytes and human peripheral lymphocytes from controls and HIV-infected persons. Evidence for interference by granulocytes and erythrocytes.

The present article reports a multiparametric cytofluorimetric analysis of apoptosis in murine thymocytes and human PBMC from healthy donors or HIV-infected patients. We have evaluated four previously described cytofluorimetric methods of apoptosis quantification, each of them detecting distinct cellular alterations of the apoptosis process. Reduced DNA stainability was detected with the PI assay on nuclei and the AO/EB dual staining method was evaluated on entire and non-fixed cells. DNA strand breaks were detected following in situ nick translation, and alterations in membrane integrity were evaluated following 7-AAD incorporation. When apoptosis was quantified in murine thymocytes under various conditions of induction, the combined analysis of FSC/SSC criteria and 7-AAD or AO/EB staining on the same samples permitted the identification of distinct steps in the apoptosis process. Moreover these four methods proved to be reliable and gave statistically similar results both on murine thymocytes and PBMC from healthy donors. However, in HIV-infected persons, some discordant apoptosis determinations were observed with PI and 7-AAD staining assays. We found that after Ficoll isolation, PBMC from AIDS patients were enriched in erythrocytes and granulocytes. On the one hand, granulocytes were found to be responsible for a poor apoptosis estimation with the PI assay whereas erythrocytes were responsible for an underestimation rate of apoptosis in the 7-AAD assay. To prevent such interference, we propose some modifications which render these methods more suitable for application to PBMC from HIV-infected patients. Taken together these observations indicate that it is essential to assess critically the apoptosis quantification methods with respect to their applicability to complex lymphoid populations such as those from AIDS patients.

Animals↗
PubMed1996

Peripheral V gamma 9/V delta 2 T cell deletion and anergy to nonpeptidic mycobacterial antigens in asymptomatic HIV-1-infected persons.

Gamma delta T cells represent a minor population of human peripheral lymphocytes, the majority of them expressing the V delta 2/V gamma 9 TCR. Their accumulation in infectious disease lesions and their reactivity toward mycobacterial Ags suggest that V gamma 9/V delta 2 T cells play a role during infectious diseases. We have shown previously a significant expansion of the V delta 1 subset parallel to a dramatic decrease of the V delta 2 subset in PBMC from HIV-infected persons. To understand the mechanisms involved in the deletion of V delta 2 T cells, we analyzed their ability to respond in vitro to several V gamma 9/V delta 2 t cell-specific ligands. We observed that in 60% of asymptomatic HIV-infected persons, V delta 2 T cells exhibited a functional anergy to Daudi and to Mycobacterium tuberculosis stimulations. These observations were supported by the defective expansion of this subset to the recently described nonpeptidic phosphorylated Ag, TUBAg-1. Since V delta 2 responsiveness to mycobacterial Ags was shown to be normally dependent on IL-2 secretion by Th1-type CD4 T cells, the ability of IL-2 to restore V delta 2 T cells' responsiveness to TUBAg-1 was tested. V delta 2 T cell anergy persisted in spite of the presence of IL-2, and was frequently correlated with a defect in CD25 expression on stimulated V delta 2 T cells. Since V delta 2 anergy was associated with an in vivo depletion of this subset, we studied whether programmed cell death could be involved in this process, particularly because of their activated phenotype. Although peripheral V delta 2 T cells from some HIV-infected persons showed an increased susceptibility to spontaneous and activation-induced apoptosis, statistical comparison between HIV+ and HIV- donors indicated that there was no difference between both groups in the rate of V delta 2 apoptosis. Finally, V delta 2 complementarity-determining region 3 TCR analysis indicated that, in vivo, the remaining V delta 2 T cells were still polyclonal. All together these results suggest that the qualitative and quantitative alterations of the V delta 2 subset in the course of HIV infection are the consequence of a chronic antigenic stimulation, and raise the question of the contribution of a cellular ligand induced or modified by chronic HIV infection.

Antigens, Bacterial↗
PubMed1996

Programmed cell death in peripheral lymphocytes from HIV-infected persons: increased susceptibility to apoptosis of CD4 and CD8 T cells correlates with lymphocyte activation and with disease progression.

We analyzed the potential causes of the increased susceptibility to apoptosis of peripheral lymphocytes from a large cohort of HIV-infected persons that we followed during a 3-yr period. By using quantitative cytofluorometric methods, we demonstrate that all lymphocyte populations were contributing proportionally to the apoptotic population in both groups of donors, but the percentage of T and B cells involved in this cell death process was significantly increased in HIV-infected persons. To study the relationship between the increased apoptosis in HIV infection and the activation state of the immune system, we analyzed cell surface expression of activation markers on apoptotic and nonapoptotic cells. We found that in the chronic phase of HIV infection, 50 to 60% of the apoptotic cells exhibited an activated phenotype (they were HLA-DR+, CD38+, CD45RO+, and Fas+), and interestingly, the CD45RO+ subset appeared to be more prone to apoptosis in HIV-positive persons. This study also indicates that the activated phenotype found on apoptotic cells was not a distinctive feature of patients' lymphocytes since it was in similar proportion in apoptotic cells from control lymphocytes. However, a significant correlation was found between the intensity of anti-CD3-induced apoptosis in both CD4 and CD8 T cells from HIV-infected persons and their in vivo expression of CD45RO and HLA-DR molecules. Finally, a significant correlation was found between the intensity of spontaneous or anti-CD3-induced apoptosis in total lymphocytes and disease progression; this was confirmed when the CD4 and CD8 T cell subsets were analyzed separately. Altogether these observations indicate that the increased susceptibility to apoptosis of peripheral T cells from HIV-infected persons correlates with disease progression and support the hypothesis that the chronic activation of the immune system occurring throughout HIV infection is the primary mechanism responsible for this cell deletion process.

Apoptosis↗
PubMed1996

Apoptosis associated with ex vivo down-regulation of Bcl-2 and up-regulation of Fas in potential cytotoxic CD8+ T lymphocytes during HIV infection.

In this study, we have investigated whether the enhanced apoptosis of CD4+ and CD8+ T lymphocytes throughout HIV infection was controlled by the bcl-2 proto-oncogene, an inhibitor of programmed cell death (PCD) in mammals. We have analyzed the intracellular expression of the Bcl-2 protein by flow cytometry in freshly isolated peripheral T cells from HIV-infected and noninfected individuals. While no decrease in Bcl-2 expression was detected in the CD4+ T cell subset from the seropositive donors, a reduced level of Bcl-2 was found in a fraction of CD8+ T lymphocytes, with the proportion of these cells increasing as HIV infection progressed. We show that the low Bcl-2-expressing CD8+ T cells were highly susceptible to spontaneous apoptosis upon short term culture. Interestingly, PCD significantly increased when these lymphocytes were cultured in the presence of a Fas-specific mAb, which was related to the high expression of the Fas Ag on their surface. The low Bcl-2 CD8+ subpopulation displayed activation markers CD45RO, HLA-DR, and CD38 and expressed TIA-1-positive, but perforin-negative, granules, while lacking the CD28 Ag. These observations suggest that such low Bcl-2 CD8+ T cells correspond to either immature or end-staged anergic CTLs. Moreover, they indicate that down-regulation of Bcl-2 and up-regulation of Bcl-2 and up-regulation of Fas in CD8+ T lymphocytes, associated with the chronic stimulation of these cells during HIV infection, might render them sensitive to Fas-mediated PCD. Such a Bcl-2/Fas-regulated apoptosis could be responsible for the disappearance of both memory CD45RO+ T cell response and HIV-specific cytotoxic activity occurring in the course of HIV infection and could contribute to AIDS pathogenesis.

Apoptosis↗
PubMed1996

Comparative analysis of apoptosis in HIV-infected humans and chimpanzees: relation with lymphocyte activation.

Programmed-cell death (apoptosis) is a physiological cell death process which appears exacerbated in peripheral lymphocytes from HIV-infected persons. On the contrary, a barely detectable level of apoptosis is found in peripheral lymphocytes from HIV-infected chimpanzees, which support long-term productive infection without developing AIDS. In the present study, we analyzed the relationship between apoptosis and the general state of immune activation in PBMC from HIV-infected humans and chimpanzees. In addition, apoptosis control in the CD8 subset by the bcl-2 proto-oncogene was compared in both human and chimpanzees. Taken together, the results indicate that the degree of apoptosis correlates with the state of activation of the immune system and this observation together with the finding that apoptosis concerns all lymphocyte subsets indicates that the low level of apoptosis in HIV-infected chimpanzees is related to the lack of immune activation in this nonpathogenic model.

Animals↗
PubMed1995

Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis.

In this paper we used a multiparametric approach to analyze extensively the events occurring during apoptotic cell death of thymocytes, and furthermore, we asked whether alterations in mitochondrial structure and function are occurring in early stages of apoptosis. A multiparametric quantitative analysis was performed on normal or apoptotic thymocytes emerging from a few-hour culture performed in culture medium or in the presence of dexamethasone. Simultaneous detection of light scattering properties, integrity of plasma membrane (trypan blue exclusion), chromatin condensation (AO/EB staining of entire cells or PI staining of nuclei), and DNA fragmentation (in situ nick-translation in apoptotic cells) allowed a precise analysis of the preapoptotic and apoptotic stages. Moreover a thorough study of mitochondrial transmembrane potential (delta psi m) assessed following in a time course study the uptake by apoptotic cells of the cationic lipophilic dye DiOC6(3) or the J-aggregate-forming cation JC-1, indicates that a drop in delta psi m occurs very early in thymocyte apoptosis, before DNA fragmentation. This is associated with alteration in mitochondrial structure assessed by cytofluorimetric study of NAO uptake in apoptotic cells. Finally these dramatic alterations in mitochondrial structure and function occurring in early stages of apoptosis were confirmed by confocal and electron microscopy analysis.

Animals↗
PubMed1995

The early intracellular production of a reactive oxygen intermediate mediates apoptosis in dexamethasone-treated thymocytes.

Many conditions that induce an oxidative stress are capable of evoking apoptosis. This has lead to the proposal of oxidative stress as a mediator of apoptosis. We show that, in murine thymocytes, oxidative stress and apoptosis occur in the same cell. We identified four distinct apoptotic subpopulations that appeared sequentially in time. Catalase protected from dex-amethasone-induced death in the initial stages of apoptosis, while iron chelators and Vitamin E did not. Further studies provided evidence supporting the early production of an intracellular oxidative intermediate as an obligatory step for the efficient induction of apoptosis. We propose that at least one of the molecules capable of filling this role is hydrogen peroxide.

Journal Article↗
PubMed1994

The ocular penetration of oral sparfloxacin in humans.

The penetration of sparfloxacin into the aqueous humor after oral administration was studied in 28 patients undergoing cataract surgery. Each patient received a single, oral dose of 400 mg of sparfloxacin. In eight other patients scheduled to undergo vitreal surgery, multiple daily oral doses were administered for a total amount of 1,000 mg. The aqueous levels were (mean +/- SEM) 0.127 +/- 0.036 microgram/ml to 0.404 +/- 0.159 microgram/ml from two to 24 hours after ingestion. In the vitreous, the mean drug level was 0.840 microgram/ml (range, 0.480 to 2.060 microgram/ml), from 4.3 to 8.0 hours after the most recent oral dose. Blood samples obtained at the same time as vitreous and aqueous taps were assayed by high-performance liquid chromatography. These data demonstrate that therapeutic levels of sparfloxacin may be achieved in noninflamed, noninfected eyes undergoing cataract or vitreous surgery.

Administration, Oral↗
PubMed1993

Programmed cell death in AIDS-related HIV and SIV infections.

One of the difficulties in understanding the complex pathology of human immunodeficiency virus (HIV) infection is to explain the progressive depletion of the CD4 helper T cell population and consequently the destruction of the immune system. Although cytopathic effects of HIV are observed in vitro, they cannot in vivo account for CD4 T cell depletion because relatively few cells are productively infected. Thus immunological mechanisms must be envisaged. We have found that peripheral blood lymphocytes (PBLs) from asymptomatic HIV-infected individuals are primed for a suicide process known as apoptosis or programmed cell death (PCD). DNA fragmentation characteristic of apoptosis was enhanced by stimulation of lymphocytes with ionomycin, a known inducer of apoptosis in suitably primed cells. Identification of the T cell subpopulations programmed for apoptosis indicated that both CD4+ and CD8+ cells died when cultured without stimulation or when polyclonally stimulated with ionomycin. Activation-induced cell death was also observed after stimulation with self-MHC class II-dependent superantigens, namely bacterial toxins from Staphylococcus (SEB), Streptococcus (ETA), and Myocoplasma (MAM) and under these conditions the CD4+ T cells were preferentially affected. To explore whether new macromolecular synthesis were required for apoptosis, various known inhibitors of apoptosis such as cycloheximide, cyclosporin A, Zn2+, or EGTA were tested. Activation-induced apoptosis was found sensitive to these inhibitors, indicating an active mechanism, but apoptosis observed in nonstimulated cultures was not, suggesting that these cells already contained the complete machinery for death. Prevention of apoptosis could be obtained in the presence of a mixture of cytokines and the minimal signal necessary for this prevention was IL-1 alpha and IL-2. Finally, a correlation between PCD and AIDS-pathogenesis was suggested by the comparison of lymphocytes from lentivirus-infected primates suceptible (SIV-infected macaques) and resistant (HIV-infected chimpanzees) to AIDS. Altogether our results suggest that, during HIV or SIV infection, PCD may contribute in vivo to the deletion of reactive T cells after antigenic stimulation.

Animals↗
PubMed1990

Concentrations of cefpodoxime in plasma and lung tissue after a single oral dose of cefpodoxime proxetil.

Eighteen patients undergoing thoracotomy for suspected pulmonary neoplasia were given 200 mg cefpodoxime equivalent by mouth, before operation. Plasma samples were obtained before dose administration, and plasma and lung tissue samples were obtained at the time of operation which was 3, 6 or 12 h after the dose. All samples were assayed for cefpodoxime. The mean ratios for lung tissue/plasma concentrations were similar between 3 and 12 h after dose, suggesting that equilibrium between plasma and lung tissue concentrations was reached within 3 h of medication. The mean concentrations of cefpodoxime in lung tissue were 0.63 +/- 0.16, 0.52 +/- 0.09 and 0.19 +/- 0.02 mg/kg at 3, 6 and 12 h after administration, respectively. These observations indicate good, rapid and sustained penetration into lung tissue in concentrations greater than or equal to the MIC90 for most common micro-organisms found in community-acquired pneumonia.

Administration, Oral↗
PubMed1987

[BCG vaccination in France].

First used in 1921 and obligatory since 1950, BCG vaccination is a part of the classical arsenal in the struggle against tuberculosis in France. The progressive reduction in the incidence of tuberculosis leads one to wonder what to expect now and in the future, so much so that the degree of protection conferred by BCG is continually discussed. In animal experiments, BCG vaccination is efficacious but there is no absolute protection conferred. In man, the results of 9 prospective studies performed with control groups have thrice shown an 80% protection, thrice a 30% protection and thrice no protection (in the case of studies from Southern India). On the grounds that there were large differences in the methodology of the 9 studies and that the best methodology was found in the 3 studies which showed good protective efficacy of BCG, it is justifiable to consider that the protection conferred against tuberculosis by a correct BCG vaccination is of the order of 80% and lasts 15 years (direct effect of BCG). Equally, a similar protection has been observed in numerous retrospective studies. But it is not accompanied by a reduction in the transmission of tuberculous bacilli in the population vaccinated with BCG. Since one does not observe any reduction in the incidence of tuberculosis in non-vaccinated subjects who live in contact with the vaccinated population (indirect effect of BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

BCG Vaccine↗
PubMed1985

Resistance of high and low antibody responder lines of mice to the growth of avirulent (BCG) and virulent (H37Rv) strains of mycobacteria.

The resistance to Mycobacterium bovis (BCG) of lines of mice selected for high (H) or low (L) antibody responsiveness was estimated from the rate of BCG multiplication in the organs. During the first 2 weeks after i.v. infection with 5 X 10(6) CFU, BCG multiplied faster in the spleens of H than of L mice. Afterwards there was a more drastic reduction of viable BCG counts in H mice than in L mice so that the residual BCG counts were significantly lower in H mice than in L mice, not only in the spleen but also in the liver and lungs. On the 14th day of infection, the spleen and liver enlargement and the increase of phagocytic activity were similar in the two lines, suggesting an identical T lymphokine release. In contrast with BCG, during the first 2 weeks after infection with 7 X 10(5) CFU, M. tuberculosis (H37Rv) multiplied in the spleens of L mice at a similar or a slightly faster rate than in the spleens of H mice. On the 4th week, the viable H37Rv counts were reduced in H mice whereas L mice did not survive the infection. In mice vaccinated with BCG 5 months before virulent challenge, the multiplication of H37Rv was inhibited in the H and L lines. The protective effect of BCG is therefore stronger in L mice taking into account their higher innate susceptibility to H37Rv. This might be due to the higher level of living BCG found in L mice at the time of challenge.

Animals↗