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Biomedical subjects

G Torpier

Publications and source records attributed to G Torpier.

At least 19 recordsLinked to original sources
PubMed1992

Productive human immunodeficiency virus-1 infection of megakaryocytic cells is enhanced by tumor necrosis factor-alpha.

Recent findings have indicated that megakaryocytes may be susceptible to human immunodeficiency virus (HIV) infection, suggesting a potential role for megakaryocytes as viral reservoirs in HIV-infected patients. We report that the megakaryocytic cell line Dami could be productively infected with the HTLV III-B strain of HIV-1, in 26 different experiments (results of 16 experiments are reported); productive infection lasted up to 30 weeks. Despite a lack of detectable surface expression of the CD4 molecule and very low levels of CD4 mRNA, between 40% and 60% of megakaryocytic cells produced viral proteins after contact with HIV-1. Neither cytopathogenic effects nor syncytial formation was observed. Production of high levels of functional viral particles was indicated by analysis of p24 protein levels, reverse transcriptase activity, ultrastructural studies, and the capacity of supernatants from infected Dami cells to infect the Molt-4 T-lymphocytic cell line. HIV-1 RNA and protein levels in infected Dami cells were enhanced by treatment with tumor necrosis factor-alpha (TNF-alpha), and decreased by treatment with interferon-alpha (IFN-alpha) and IFN-gamma. Transient transfection of the megakaryocytic cells with various constructs of the HIV-1 promoter (LTR) linked to the luciferase reporter gene suggested that the effect of TNF-alpha was related, as in monocytic and T-cell lines, to transactivation of the enhancer region of the HIV-1 LTR. These findings indicate that signals provided by the immune system may modulate HIV-1 expression in cells of the megakaryocytic lineage.

Cell Line↗
PubMed1992

Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals.

In immature thymocytes, T cell receptor for antigen (TCR) mobilization leads to an active T cell suicide process, apoptosis, which is involved in the selection of the T cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals (Ameisen, J.C., and A. Capron. 1991. Immunol. Today. 4:102). Here, we report that the selective failure of CD4+ T cells from 59 clinically asymptomatic HIV-infected individuals to proliferate in vitro to TCR mobilization by major histocompatibility complex class II-dependent superantigens and to pokeweed mitogen (PWM) is due to an active CD4+ T cell death process, with the biochemical and ultrastructural features of apoptosis. Activation-induced cell death occurred only in the CD4+ T cell population from HIV-infected asymptomatic individuals and was not observed in T cells from any of 58 HIV-seronegative controls, including nine patients with other acute or chronic infectious diseases. Activation-induced CD4+ T cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody (mAb). The CD28 mAb not only prevented apoptosis but also restored T cell proliferation to stimuli, including PWM, superantigens, and the tetanus and influenza recall antigens. These findings may have implications for the understanding of the pathogenesis of acquired immune deficiency syndrome and for the design of specific therapeutic strategies.

Antigens, CD↗
PubMed1992

Interleukin 5 messenger RNA expression by eosinophils in the intestinal mucosa of patients with coeliac disease.

Interleukin 5 (IL-5), the major factor involved in eosinophil differentiation, is produced by T cells or mast cells. In the present study, we found that eosinophils infiltrating the mucosa of four patients with active coeliac disease also express the IL-5 mRNA. No positive signal was obtained in normal duodenum tissues and in the cell infiltrate from patients submitted to gluten restriction. The identification of labeled mucosal cells as eosinophils relied on their typical morphology. Moreover, highly purified blood eosinophils from three out of four patients with eosinophilia were also strongly labeled with the IL-5 antisense but not with the corresponding sense probe. Together, these results suggest that eosinophils have the capacity to synthesize IL-5, which could contribute to paracrine interactions with T and B cells and, in autocrine fashion, locally participate, through binding to the IL-5 receptor, to eosinophil differentiation and activation. These data might have implications not only in the pathology of coeliac disease but also in other diseases associated with eosinophil infiltration.

Celiac Disease↗
PubMed1992

Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.

Antigens, Polyomavirus Transforming↗
PubMed1992

Immunopathological study of eosinophils in eosinophilic granuloma of bone: evidence for release of three cationic proteins and subsequent uptake in macrophages.

Eosinophils from two patients with eosinophilic granuloma of bone (EGB) were studied by combined immunohistochemical and immuno-ultrastructural methods with antibodies directed against three eosinophil granule proteins: major basic protein, eosinophil cationic protein, and eosinophil peroxidase. Immunohistostaining showed the presence and distribution of large numbers of eosinophils in the granuloma. Immuno-ultrastructural methods showed alterations of eosinophil fine structure associated with some steps in the release of granule proteins. No granule extrusion was seen, but rather cationic proteins diffused within cytoplasmic tubulo-vesicular structures. Furthermore, the three granule proteins were found within phagolysosomes of surrounding macrophages, suggesting an interaction between eosinophils and phagocytic cells at the destructive stage of EGB.

Adolescent↗
PubMed1992

Activated eosinophils in adult coeliac disease: evidence for a local release of major basic protein.

The eosinophil population is increased in the jejunal mucosa of patients with coeliac disease. Eosinophils may participate in the mucosal damage by releasing their granule components that have cytotoxic properties such as eosinophil cationic protein (ECP) and major basic protein (MBP). This study aimed to assess the presence of ECP and MBP in the jejunal mucosa of 10 adult patients with active coeliac disease who presented with villous flattening. Endoscopic jejunal biopsy specimens were obtained from macroscopically flattened jejunal mucosa and were processed for ultrastructural study and immunogold labelling using anti-MBP, anti-ECP, and anti-IgA antibodies. Numerous eosinophils were found in the upper part of the lamina propria and showed two types of morphological change: some were lytic and others exhibited ultrastructural signs of activation, containing altered granules with fading of the central core. IgA plasma cells were intermingled with eosinophils and had dense deposits on the external side of their cytoplasmic membrane. MBP was detected in central cores of granules but also diffusely in their matrix and in tight association with dense extracellular deposits. Conversely, ECP was detected only in the matrix of eosinophil granules. This study showed that numerous eosinophils are in an activated state in the mucosa of patients with active coeliac disease and release cytotoxic proteins such as MBP, which could contribute to the mucosal damage. The observation that eosinophils and IgA plasmocytes were closely associated in the mucosa supports a role for IgA in eosinophil recruitment and activation in coeliac disease.

Adolescent↗
PubMed1991

Improved permeabilization procedure for flow cytometric detection of internal antigens. Analysis of interleukin-2 production.

A cell membrane permeabilizing treatment is described which involves the use of lysolecithin at low concentration in acidic acetate buffer and paraformaldehyde fixation. It preserved well-separated scatter cytograms of small and large lymphocytes. The accuracy of the immunochemical detection of internal antigens by flow cytofluorography was demonstrated by the linear relationship between the percentage of fluorescent cells detected and the proportion of intracellular antigen-containing cells in mixtures with antigen-negative cell lines. Cell cycle analysis by dual nuclear staining with propidium iodide and FITC-conjugated Ki-67 antibody recognising in vitro stimulated human T lymphocytes verified that the proliferating lymphocytes retained their increased light scatter properties after permeabilization. Enumeration of interleukin-2 (IL-2) producing cells by their cytoplasmic immunofluorescence showed that enlarged lymphocytes were the main IL-2 producing cells. This improved permeabilization procedure, by gating small and enlarged lymphocytes separately, makes it possible to determine by two color fluorescence the immunophenotype of activated T cells committed to interleukin production.

Cell Cycle↗
PubMed1991

Fast double antibody radioimmunoassay of human granulocyte myeloperoxidase and its application to plasma.

The haem enzyme myeloperoxidase (MPO) (EC 1.11.1.7) with a spectral A430/A280 ratio greater than 0.7 and a specific activity of 125 U/mg was purified from isolated human neutrophils. To obtain a radioimmunoassay (RIA) for this enzyme, a specific antiserum against human neutrophil MPO was raised in rabbits and used at an initial dilution of 1/10,000. MPO labelled with 125iodine by a technique of self-labelling in the presence of H2O2, had a specific activity of 24 mCi/mg. After incubation at room temperature (2 h) and separation by double antibody precipitation in the presence of polyethylene glycol, the sensitivity of the RIA was 21 ng/ml. The RIA showed good precision and accuracy with intra- and interassay coefficients of variation of less than 7% for MPO concentrations ranging from 100 to 800 ng/ml, and satisfactory recoveries of known amounts of exogenous MPO in plasma. For the measurement of MPO in blood, the best sampling technique was to collect blood into EDTA. Rapid centrifugation (within 20 min) was necessary for blood collected into heparin. Mean MPO values in normal individuals were 340 +/- 98 ng/ml in EDTA plasma (n = 152) and 332 +/- 82 ng/ml in heparinized plasma (n = 34). When MPO was measured 12-6 h after injury in critically ill patients high values (above 1000 ng/ml) were found in 6/15 patients with multiple injuries. In patients with sepsis (n = 22), MPO values were always above 1000 ng/ml.

Animals↗
PubMed1991

Isolation and characterization of human Lp-B lipoprotein containing apolipoprotein B as the sole apolipoprotein.

A sequential immunoaffinity chromatography procedure was developed to isolate from whole normolipidemic human plasma a subpopulation of apoB containing particles (Lp-B) which is virtually free of non apoB protein. The absence of non apoB protein in Lp-B was assessed by enzyme immunoassay against apolipoproteins A-I, A-II, A-IV, E, C-III and (a). Electron microscopy and fractionation of the isolated particles by gel filtration demonstrated that these particles were heterogeneous in size. However, most of them had diameters between 18 and 26 nm. These particles were found to be rich in cholesterol (molar ratio cholesterol/apoB = 2246 +/- 995) poor in triacylglycerol (molar ratio triacylglycerol/apoB = 555 +/- 518) and had a phospholipids/apoB molar ratio of 713 +/- 348. Most of the cholesterol was esterified (66% +/- 5%). Lp-B particles bound to the apoB, E receptor of HeLa cells with a lower affinity than LDL prepared by ultracentrifugation (1.030 kg/l less than d less than 1.053 kg/l). (KD = 18.9 vs 10.5 nmol/l).

Apolipoproteins B↗
PubMed1991

Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells.

Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo AII (LpAI) and particles containing apo AI and apo AII (LpAI:AII) isolated from native human plasma, and from HDL2 or HDL3, showed that only LpAI were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent Kd values between 10 and 25 micrograms/ml). During this long-term exposure, LpAI:AII demonstrated a concentration-dependent inhibition (10-60 micrograms/ml) of LpAI-mediated cholesterol efflux from adipose cells under conditions where LpAI:AII did not deliver cholesterol to the cells and where no net change in the distribution of apo AI between LpAI and LpAI:AII was observed. The antagonizing and modulating role of LpAI:AII in preventing cholesterol efflux mediated by LpAI appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo AI in LpAI particles and apo AII in LpAI:AII particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux.

Adipose Tissue↗
PubMed1991

[Role of Schistosoma mansoni bilharziasis in male hypogonadism].

In tropical and subtropical areas, schistosomiasis may cause anatomic anomalies of the genital organs responsible for permanent or reversible infertility. Furthermore, it has been suggested that this parasitic infection may have adverse consequences on the endocrine system. To specify the effects of schistosomiasis on endocrine function, production of pituitary gonadotropins and testosterone in rats and hamsters experimentally infected with Schistosoma mansoni was studied. Prior to infection, hormone levels were within the normal range for our laboratory in all animals studied. Hormone levels (FSH, LH and testosterone) fell significantly in all experimentally infected animals, as compared with uninfected controls. These data suggest that the inhibitory effect (documented by the fall in all the hormone parameters studied) of Schistosoma mansoni on gonadal function is due to an action at the hypothalamic or pituitary level. The potential role of parasite ecdysteroids in the mechanism of action of schistosomes deserves to be studied.

Animals↗
PubMed1990

Toxoplasma gondii: characterization and localization of antigens secreted from tachyzoites.

Since we had previously demonstrated the protective role played by Toxoplasma excreted-secreted antigens, the aim of the present work was to produce monoclonal antibodies directed against these antigens in order to determine if their localization in the parasite is compatible with a mechanism of excretion or secretion. Western immunoblotting analysis revealed three monoclonal antibodies (TG17-179, TG17-43, and TG17-113) raised against excreted-secreted antigens of 28.5, 27, and 21 kDa, respectively. The TG17-179 which reacts with antigens isolated by Concanavalin A affinity chromatography is directed against a glycosylated 28.5-kDa component. Colloidal immunogold labeling showed the ultrastructural localization of the 21-, 27-, and 28.5-kDa antigens in the matrix of the dense granules of tachyzoites and associated with the microvilli network of the parasitophorous vacuole, after host cell invasion. These observations suggest the following mechanism of Toxoplasma secretion: secreted antigens are first stored in tachyzoite-dense granules and are then released inside the parasitophorous vacuole. Among the secretory molecules characterized here, the native 27-kDa antigen recognized by TG17-43 is a calcium-binding protein found to be intermixed with the 21- and 28.5-kDa antigens inside the dense granules and hence could play a role in the packaging of secretory products. In addition, the 21- and 28.5-kDa antigens were also located beneath the parasite plasma-lemma. This particular location could reflect a transient step characteristic of T. gondii secretion.

Animals↗
PubMed1990

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase.

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.

Amino Acid Sequence↗
PubMed1990

Structural homology of tropomyosins from the human trematode Schistosoma mansoni and its intermediate host Biomphalaria glabrata.

Molecular mimicry has been considered as a possible way for parasites to escape host immune responses. This work concerns the characterization of protein determinants shared by Schistosoma mansoni and its intermediate host Biomphalaria glabrata. Parasite (Sm39) and mollusc (Bg 39) cross-reactive proteins were identified and shown to induce in rabbit and mouse, antibodies specific for invertebrate determinants. Ultrastructural studies demonstrated that antibodies to Sm39 specifically bound to muscular structures of parasite and mollusc. Molecular cloning and sequencing indicated that Bg39 corresponded to a muscular isoform of tropomyosin. The mollusc sequence showed a 51-65% homology with seven different muscular tropomyosins from vertebrate and invertebrate species. The highest score of homology was observed with S. mansoni tropomyosin, suggesting that cross-reactive determinants could be specific for the trematode and its intermediate host. In miracidia, Sm39 epitopes were also shown to be contained in the vesicles present in epidermal ridges and cellular bodies. Such vesicles are involved in the formation of a protective tegument around sporocysts, suggesting a possible role of cross-reactive tropomyosins in miracidia and/or sporocyst-snail interactions.

Amino Acid Sequence↗
PubMed1989

Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.

A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes.

Amino Acid Sequence↗