Biomedical subjects

J C Chermann

Publications and source records attributed to J C Chermann.

At least 55 records · Page 3Linked to original sources

PubMed1994

Involvement of HIV nef protein in abnormal hematopoiesis in AIDS: in vitro study on bone marrow progenitor cells.

In previous studies (1-4), we described disrupted hematopoiesis in long-term human bone marrow cultures infected with HIV. Conditioned medium (CM) from HIV-1 non-productively infected liquid cultures inhibits the proliferation of granulo-monocytic progenitor cells (CFU-GM) in clonogenic assays. A growth-inhibitory soluble factor was therefore suspected and we report here that this factor has been identified as the nef gene product. These nef-containing supernatants are specifically neutralized by anti-nef antibodies and recombinant nef has the same growth inhibitory properties. We used a nef-deficient virus to confirm that nef protein is responsible for the in vitro growth inhibition of CFU-GM.

Acquired Immunodeficiency Syndrome↗

PubMed1993

Binding of human immunodeficiency virus type-1 (HIV-1) to partially purified membrane vesicles of lymphoblastoid cell line CEM.

Binding of human immunodeficiency virus type 1 (HIV-1) to membrane of its target cells was studied by a quantitative and non-isotopic method called the viral membrane trapping method (VMTM). Membranes prepared from the CD4 positive lymphoblastoid cell line CEM and adsorbed to a solid support retained the ability to bind HIV-1. Similar results were obtained by Western dot blot and ELISA modification of VMTM, when membrane fraction was bound to nitrocellulose or polystyrene, respectively. In ELISA modification, viral association with 1 microgram of membranes coated on 96-well microplate was linear within a range of 3.75 micrograms to 60 micrograms of p24gag protein. The use of anti-CD4 mAbs, OKT4A and 13B8.2, identified CD4 molecule as a major HIV-1 binding component of membrane fraction. The procedure will allow the study of virus binding to this and to other possible additional receptor(s).

Antibodies, Monoclonal↗

PubMed1993

The gag precursor contains a specific HIV-1 protease cleavage site between the NC (P7) and P1 proteins.

The predicted protease cleavage site (p7/p1; [J. Virol. 66 (1992) 1856-1865]) within the nucleocapsid precursor protein (p15) of human immunodeficiency virus, type 1, was confirmed using an in vitro assay employing recombinant HIV-1 protease and a chemically synthesized 72 amino acid polypeptide containing the p7 and p1 protein domains of the native gag polyprotein. The cleavage occurred between amino acid 55 (N) and amino acid 56 (F) of the polypeptide, as determined by N-terminal sequencing. The hydrolysis was optimal at pH 6.0 and at high salt concentration. The kinetic parameters Km, kcat and kcat/Km were 99 microM (+/- 8), 0.152 s-1 (+/- 0.002) and 1.56 mM-1.s-1 (+/- 0.11), respectively. Reconstituted as well as denatured polypeptides were cleaved at approximately the same rate, demonstrating that the conformation of the p7 protein, as a result of the Zn(2+)-binding, had no significant effect on the rate of hydrolysis of the p7/p1 cleavage.

Amino Acid Sequence↗

PubMed1993

Evidence that membrane proteins of rhabdomyosarcoma cell line RD bind human immunodeficiency virus type 1 (HIV-1).

Membrane proteins (MP) obtained from the human mesenchymal rhabdomyosarcoma cell line RD were coated on 96-well polystyrene microplates and tested for their ability to bind human immunodeficiency virus type 1 (HIV-1). The virus bound to MP was detected by solid phase assay. Anti-human CD4 monoclonal antibodies directed against the HIV-1 gp120 binding site of the CD4 receptor did not inhibit viral binding to MP. HIV-1 specific polypeptides were recovered from coated MP to microplates by a modification of the solid phase immunoisolation technique and shown by immunoblotting analysis using a high titer of biotinylated human anti-HIV-1 IgG. Together these findings provide evidence that HIV-1 binding to RD cell surfaces can proceed via a mechanism other than those mediated by the CD4 receptor.

Animals↗

PubMed1993

New HIV plaque titration; application to the assay of neutralizing antibody.

A simple, sensitive and accurate plaque assay was developed using HPB-Ma, a variant of the human T-cell line HPB-ALL, which becomes adherent to the substratum after infection with an amphotropic murine sarcoma virus (MSVa). The simplicity of this novel plaque assay allowed us to examine a large number of serum samples from patients with HIV infection for neutralizing antibody activity against two human immunodeficiency virus type-1 (HIV-1) strains. During the progression of clinical disease, the neutralizing activity in the sera from two individual patients remained unchanged or increased. A patient with a known time of HIV infection produced cross-neutralizing antibody at 25-34 weeks. The neutralizing activity in the sera from 17 asymptomatic carriers, four patients with AIDS-related complex and four AIDS patients was also examined and was found to be unrelated to the clinical stage.

Cell Line↗

PubMed1993

Standardized nested polymerase chain reaction-based assay for detection of human immunodeficiency virus type 1 DNA in whole blood lysates.

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.

Base Sequence↗

PubMed1992

Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines.

Cellular proteins associated with immunodeficiency viruses were identified by determination of the amino acid sequence of the proteins and peptides present in sucrose density gradient-purified human immunodeficiency virus (HIV)-1, HIV-2, and simian immunodeficiency virus (SIV). beta 2 microglobulin (beta 2m) and the alpha and beta chains of human lymphocyte antigen (HLA) DR were present in virus preparations at one-fifth the concentration of Gag on a molar basis. Antisera to HLA DR, beta 2 m, as well as HLA class I precipitated intact viral particles, suggesting that these cellular proteins were physically associated with the surface of the virus. Antisera to class I, beta 2m, and HLA DR also inhibited infection of cultured cells by both HIV-1 and SIV. The specific, selective association of these cellular proteins in a physiologically relevant manner has major implications for our understanding of the infection process and the pathogenesis of immunodeficiency viruses and should be considered in the design of vaccines.

AIDS Vaccines↗

PubMed1992

HIV-1 protease inhibitors containing statine: inhibitory potency and antiviral activity.

Several series of chemically different inhibitors of the HIV-1 aspartyl protease have been described. Nevertheless despite the high in vitro potency showed, in most cases these inhibitors are unable to inhibit viral replication in infected cells. Penetration of the inhibitors across the cell membrane might account for their low antiviral activity. The relationship between inhibitory potency, antiviral activity and chemical structures of a series of oligopeptides containing statine or statine derivatives are presented here.

Amino Acid Sequence↗

PubMed1992

Statine based tripeptides as potent inhibitors of HIV-1 replication.

Starting from highly potent HIV-1 protease pepstatine analog inhibitors, we have tried to find the minimum consensus sequence which is necessary to conserve anti-protease potency and antiviral activity. We describe here some statine based tripeptides which exhibit high affinity for the protease and are able to inhibit the reproductive cycle of HIV-1 in MT-4-infected cells.

Amino Acid Sequence↗

PubMed1992

HIV-1 infectivity of human carcinoma cell lines lacking CD4 receptors.

Human immunodeficiency virus type 1 (HIV-1) prototype, HIV1 LAV, and a Zairian virus HIV1 NDK, an isolate highly cytopathic for CD4+ lymphocytes, were used to infect eleven different CD4 negative non-lymphoid human cell lines. Eight of the lines were derived from carcinomas wherein human papillomavirus was thought to have been etiologic. All these cell lines lacked CD4 receptor and CD4 specific mRNA. After cocultivation with sensitive CEM cells, HIV-1 LAV was rescued from six infected cell lines and HIV-1 NDK from nine. Shedding of free virus into the culture medium was observed in three cell lines infected by HIV-1 NDK and in only one cell line infected by HIV-1 LAV. The infectibility of CD4 negative cell lines indicates that both HIV-1 strains were able to use a CD4 independent mechanism to infect the cells; however, HIV-1 NDK showed the higher efficiency of infection. This virus was also able to overcome the intracellular block of viral reproduction. These results suggest that a broader spectrum of cell types of non-lymphoid origin lacking the CD4 receptor can serve as a viral reservoir. In some cases they are direct producers of infectious HIV-1 particles. This suggests, that in addition to immunosuppressive mechanisms, HIV-1 could play a more direct role in induction of neoplastic changes.

CD4 Antigens↗

PubMed1992

Structural variability of env and gag gene products from a highly cytopathic strain of HIV-1.

The glycoprotein precursor of the highly cytopathic Zairian virus HIV1-NDK synthesized in CEM leukemic cells displayed a molecular mass of 140 kDa (gp140) as compared to the 160 kDa of gp160 of HIV1-LAV prototype strain. This precursor was cleaved to produce a smaller than prototype extra-cellular envelope glycoprotein (gp100) and a transmembrane component with a usual size (gp41). Immunoprecipitates from tunicamycin-treated infected cells demonstrated the presence of a non-glycosylated precursor of 100 kDa for HIV1-LAV prototype strain and 90 kDa for HIV1-NDK. Digestion of labeled precipitates with a mixture of endoglycosidase F and glycopeptidase F reduced the size of HIV1-LAV gp160 and gp120 to 100 and 60 kDa, respectively, while HIV1-NDK gp140 and gp100, after treatment with the same enzymes, displayed an apparent molecular mass of 90 kDa and 55 kDa, respectively. From these data we conclude that HIV1-LAV gp120 and HIV1-NDK gp100 differ both in their proteic moiety (60 kDa and 55 kDa, respectively) and in their carbohydrate moiety (60 kDa and 45 kDa, respectively). These differences could not be deduced from the available gene sequences of the two viruses. A chimeric virus containing the first 124 amino acid residues of the envelope glycoprotein coded by HIV1-LAV sequence and the rest by HIV1-NDK displayed normal size envelope glycoproteins, demonstrating the involvement of this N-terminal sequence in the alteration of the molecular mass characteristic of HIV1-NDK gp140 and gp100. Finally, characterization of the gag gene products from both strains demonstrated that HIV1-NDK p18 and p15 have a slower electrophoretic mobility as compared to its HIV1-LAV counterparts. Therefore, structural properties of HIV1-NDK env and gag products, reflected by their unusual electrophoretic mobilities, may be responsible for HIV1-NDK biological properties.

Amidohydrolases↗

PubMed1992

HIV1 cytopathogenicity-genetic difference between direct cytotoxic and fusogenic effect.

Formation of large syncytia, rapid cell killing, and early onset of replication are characteristics of the highly cytopathic Zairian virus strain HIV1 NDK compared with the HIV1 LAV prototype. Recombinant provirus molecules derived from cloned infectious DNAs of HIV1 LAV and NDK were constructed by reciprocal exchange of genetic material using conserved restriction sites. Different regions of the HIV1 genome were responsible for variability of the direct single-cell cytotoxic and fusogenic effects. A minimal, provisionally defined portion of genetic information responsible for the higher cytotoxicity of HIV1 NDK compared to the HIV1 LAV prototype was localized in the fragment Spel1042/EcoRl4183, containing the 3'-terminal half of gag and a majority of the pol gene. This region also determined the rapid replication properties of HIV1 NDK. The increased fusogenic potential of HIV1 NDK was associated with the simultaneous presence of HIV1 NDK fragments BssHll255/Spel1042 and EcoRl5278/Xhol8401 which contained the splicing donor, packaging sequence, p18 gag protein, and the HIV env gene. The increase in the direct killing effect but not in the syncytium forming ability of HIV1 NDK correlated with the early onset of replication and rapid spread of HIV1 NDK in cell cultures. The HIV1 NDK fragments BssHll/Spel and EcoRl/Xhol were by themselves necessary but not sufficient to induce formation of large syncytia.

Cell Death↗

PubMed1992

Distinctive pattern of infection and replication of HIV1 strains in blood-derived macrophages.

The macrophage-tropic virus HIV1-PAR, isolated from cerebrospinal fluid of HIV1-seropositive man, induced cytopathic effect accompanied by different magnitude of the virus production in blood-derived macrophages (BDM) obtained from different donors. HIV1-PAR-specific RNA was detected by in situ hybridization in 15 and 66% of BDM producing low and high levels of virus, respectively. In contrast with HIV1-PAR, infection of BDM with two laboratory strains adapted to T-cell lines, HIV1-LAV prototype and HIV1-NDK, a Zairian virus that is highly cytopathic for T-lymphocytes, resulted in a low production of HIV1 p24gag in culture fluid. Expression of HIV1-LAV and HIV1-NDK RNA was detected by in situ hybridization in a maximum of 1% of macrophages. Only HIV1-NDK, and not HIV1-LAV, induced ultrastructural alterations in BDM. In contrast with a striking difference in the production of macrophage-tropic and T-lymphotropic viruses, no significant differences were found in the proportion of macrophages containing retrotranscribed genomes of HIV1-. HIV1 DNA was detected by in situ hybridization in 93, 100, and 80% of macrophages infected with HIV1-PAR, HIV1-LAV, and HIV1-NDK, respectively. A higher level of HIV1 DNA was detected by polymerase chain reaction in the BDM infected with HIV1-PAR than in that infected with HIV1-LAV and HIV1-NDK. The results indicate that both macrophage-tropic as well as T-lymphotropic viruses can enter and retrotranscribe their genomes in a vast majority of macrophages.

DNA, Viral↗

PubMed1992

Modulation of normal human erythropoietic progenitor cells in long-term liquid cultures after HIV-1 infection.

Long-term bone marrow cultures (LTBMC) have been infected by two isolates of human immunodeficiency virus type 1 (HIV-1) (HIV-1 LAV and HIV-1 NDK) at multiplicities of infection ranging from 10(2) to 2.10(6) tissue culture infectious units (TCIU) per 10(6) bone marrow mononuclear cells (BMMNC). These infected cells are nonproducer cells and the viruses can be rescued by coculture with peripheral blood lymphocytes, cord blood lymphocytes, or BMMNC and not by the CEM cell line. HIV-1 clearly is not cytopathic for these cells. Following production and growth of erythroid burst-forming units (BFU-E) and erythroid colony-forming units (CFU-E) for at least 6 weeks after infection with HIV-1 NDK, colony assays displayed a 50% inhibition of BFU-E production during 3 weeks of LTBMC. This was followed by a stimulation phase. On the contrary, HIV-1 LAV induces a 150% stimulation of BFU-E production, followed by 50% inhibition. Production of CFU-E was inhibited by 80-100% with the two isolates of HIV-1 after four weeks of LTBMC. Stimulatory and inhibitory activities were recovered from supernatants of infected LTBMC and lymphoid CEM cell lines, suggesting that HIV-1 induces release of a humoral factor responsible for disruption of hemopoietic progenitor cell production in vitro and consequently for hematologic abnormalities in AIDS patients.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Severe in vitro inhibition of erythropoiesis and transient stimulation of granulopoiesis after bone-marrow infection with eight different HIV-2 isolates.

OBJECTIVE: To correlate severe anaemia and frequent neutropenia in HIV-2-infected patients with an inhibitory effect on bone-marrow progenitors common to several HIV-2 isolates. DESIGN: The effects of eight HIV-2 isolates on early (BFU-E) and late (CFU-E) erythroid progenitors and on granulomonocytic (CFU-GM) progenitors, produced in long-term bone-marrow cultures (LTBMC), were studied. METHODS: Absolute numbers of BFU-E, CFU-E and CFU-GM per culture flask were calculated weekly for each HIV-2-infected LTBMC using semi-solid clonogenic assays, and compared with those obtained in mock-infected LTBMC. Levels of significance for comparisons were determined by an analysis of variance (ANOVA). RESULTS: Pooled data from 24 series of LTBMC (three series for each HIV-2 isolate) revealed 80 and 100% inhibition of BFU-E and CFU-E on days 6 and 12 of LTBMC, respectively, while transient stimulation of CFU-GM was observed between days 14 and 20 of LTBMC, followed by total inhibition on day 30. CONCLUSIONS: These results confirm a direct inhibitory effect of HIV-2 on in vitro haematopoiesis. The similar pattern of erythroid progenitor inhibition obtained from seven out of eight isolates suggests that the inhibitory effect on erythropoiesis is a feature common to a large number of HIV-2 isolates, which correlates with clinical findings.

Blotting, Southern↗

PubMed1992

The effects of HIV on hematopoiesis.

Hematologic abnormalities are commonly associated with infection by the human immunodeficiency virus (HIV) that is the etiologic agent of the acquired immunodeficiency syndrome (AIDS). Bone marrow examinations reveal frequent dysplasia, dyserythropoïesis, lymphocytic infiltration and plasmocytosis. These marrow morphologic characteristics are strongly associated with peripheral blood cytopenia of one or more lineage: anemia, granulocytopenia or thrombocytopenia are frequently described. The pathophysiology of these observed deficiencies is still unclear, although several mechanisms have been postulated as possible explanations for the hematological features of AIDS patients. The hematologic manifestations observed in patients with AIDS or AIDS-related complex (ARC) and the postulated pathogenetic mechanisms are discussed in this review.

Anemia↗

PubMed1992

Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion.

Selected strains of the human immunodeficiency virus (HIV) types 1 and 2 are able to infect human colon epithelial cells in vitro, suggesting a mechanism for the anal route of HIV transmission. In some cases, HIV is not produced by infected colon cells but can be rescued after coculture with T-lymphoid cells. One of the HIV strains (HIV1-NDK) replicated well in colonic cells. A transmission electron microscope study demonstrated two major structural perturbations in producer colon cells: an unusual number of secretion bodies and the appearance of intracellular lumina with disorganized microvilli, indicating a defect in brush border assembly and differentiation. Either abnormality could account for HIV-induced enteropathy consisting of chronic diarrhea and malabsorption in the absence of enteric pathogens. Moreover, HT29 cells infected with HIV provide a unique model for selection of enterotropic HIV strains.

Cell Differentiation↗

PubMed1992

The human immunodeficiency virus (HIV) gag gene product p18 is responsible for enhanced fusogenicity and host range tropism of the highly cytopathic HIV-1-NDK strain.

Formation of large syncytia and rapid cell killing are characteristics of the Zairian human immunodeficiency virus type 1 isolate HIV-1-NDK, which is highly cytopathic for CD4+ lymphocytes in comparison with the HIV-1-LAV prototype. Chimeric viruses containing different combinations of HIV-1-NDK genetic determinants corresponding to the splice donor, the packaging signal, and the coding sequence of the p18gag protein together with the HIV-1-NDK EcoRI5278-XhoI8401 fragment were obtained by polymerase chain reaction-directed recombination. Phenotypic analysis of recombinant viruses indicated that 75 amino acids from the N-terminal part of HIV-1-NDK p18gag protein together with the HIV-1-NDK envelope glycoprotein are responsible for enhanced fusogenicity of HIV-1-NDK in CD4+ lymphocytes as well as for enhanced infectivity of HIV-1-NDK in some CD4- cells lines. The HIV-1-NDK splice donor/packaging sequence and the sequence encoding the gag protein p25 were not important for the variation observed in HIV-1 fusogenicity.

Amino Acid Sequence↗