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Biomedical subjects

J C Chermann

Publications and source records attributed to J C Chermann.

At least 73 records · Page 4Linked to original sources
PubMed1992

Transient stimulation of granulopoiesis and drastic inhibition of erythropoiesis in HIV-2-infected long-term liquid bone marrow cultures.

Impaired hematopoiesis is commonly associated with human immunodeficiency virus HIV-1 and HIV-2-related AIDS. HIV-1 infection of hematopoietic progenitors has been studied, whereas HIV-2 infection of these cells is less well documented. In this work, we studied myeloid and erythroid progenitor production and differentiation in long-term bone marrow (LTBM) cultures after HIV-2 infection. A nonadherent fraction from these cultures containing the hematopoietic progenitors is nonproductively infected with HIV-2, whereas stroma cells replicate the virus only weakly. HIV-2 can be rescued from nonadherent T-depleted bone marrow cells, and its replication in stroma cells is amplified by cocultivation with HIV permissive cells. Colony assays performed weekly during the 6 weeks of LTBM cultures revealed a 100% inhibition of erythroid colony-forming unit (CFU)-E and erythroid burst-forming unit (BFU)-E production after 12 days of culture, whereas granulomonocytic colony forming units (CFU-GM) production was stimulated until day 20 and then disappeared on day 30. Stimulatory and inhibitory activities were recovered from supernatants of infected LTBM cultures and an infected lymphoid CEM cell line, suggesting that release of viral factor(s) may be responsible for HIV-2-induced impairment of hematopoietic progenitor production in vitro. Based on these results, an indirect effect of HIV-2 infection on the commitment of myeloid and erythroid progenitors, resulting in a dysregulated hematopoiesis, is postulated.

Bone Marrow↗
PubMed1991

Human immunodeficiency virus can infect the apical and basolateral surfaces of human colonic epithelial cells.

The gastrointestinal tract is considered to be a major route of infection for the human immunodeficiency virus (HIV). To understand the interaction of HIV with epithelial cells of the intestinal mucosa, we have studied the infection of a human colon cancer cell clone HT-29-D4. The enterocyte-like differentiation of this clone can be modulated in vitro according to the concentration of glucose. We show that: (i) undifferentiated HT-29-D4 cells can be infected by HIV types 1 and 2 (HIV-1 and HIV-2) strains with no subsequent effect on cell growth; (ii) undifferentiated HT-29-D4 cells express a CD4-related antigen bearing epitopes of the immunoglobulin-like variable (V) region domains V1 and V2 of CD4 but lacking the epitope known to be involved in HIV envelope recognition; (iii) differentiated HT-29-D4 cells can be infected by HIV after an interaction with either the apical brush border membrane (luminal side) or the basolateral side (serosal side); (iv) the CD4-like molecule is restricted to the basolateral domain of differentiated cells; and (v) the infection is not inhibited by anti-CD4 monoclonal antibodies (mAbs) OKT4, OKT4A, Leu-3a, Bl4, 13-B-8-2, S-T4 or S-T40. We conclude that epithelial intestinal cells may represent a major site of entry for HIV. Infection of these epithelial cells may occur via the basolateral membrane by HIV-bearing lymphocytes or macrophages of the lamina propria and via the apical membrane by HIV present in the bowel lumen. This infection may remain silent for up to 9 months, and the virus can be rescued by cocultivation with lymphoid cells. These data may give an explanation for the long latent seronegative state that may occur in a HIV-infected individual.

Adenocarcinoma↗
PubMed1991

Characterization of monoclonal antibodies identifying type and strain-specific epitopes of human immunodeficiency virus type 1.

Several hybridoma cell lines were raised against the highly cytopathic Zaïrian isolate of Human Immunodeficiency Virus (HIV), HIV1-NDK. The specificity of the secreted monoclonal antibodies (mAb) was demonstrated by immunoblotting, radioimmunoprecipitation and immunofluorescence. Two hybridoma cell lines secreted mAb reacting with independent epitopes of the NDK p17 capsid protein and its precursors. One, RL16.24.5, is specific for the NDK isolate whereas the other, RL16.45.1, along with anti-p25 RL16.30.1 mAb, bind all HIV1 isolates but not HIV2. Together with the previously described mAb RL4.72.1 those reagents define lentivirus subfamily (HIV1, HIV2, SIV) type/subtype (HIV1) and strain (HIV1-NDK) specific epitopes expressed on HIV1-NDK core proteins. The last mAb RL16.76.1 binds the env gene products gp160 and gp120.

Animals↗
PubMed1991

Expression of an immunogenic region of HIV by a filamentous bacteriophage vector.

Vectors derived from the Escherichia coli filamentous phage, fd-tet, expressing parts of the human immunodeficiency virus (HIV) gag genes were constructed and analyzed. The immunoreactive domain of HIV Gag antigens was produced in the form of a fusion protein, with a phage minor coat protein, called protein III, playing an important role in phage infectivity. A micropanning procedure, utilizing the strong affinity of biotinylated antibody to streptavidin, was applied for the selection of clones. A simple preparation procedure consisting of polyethyleneglycol precipitation of the recombinant phage from the E. coli supernatant allowed us to detect HIV antigens by enzyme-linked immunosorbent assay (ELISA). Cloned FUSE-gag, as isolated using anti-Gag RL4.72.1 monoclonal antibody (mAb), contained a nucleotide sequence coding for 91 amino acids of HIV Gag p24. It specifically reacted with the mAb in the ELISA. Construction of the mAb-selectable phages permitted localization of epitopes for mAb. Infectivity of the phage clone was specifically neutralized by the anti-HIV mAb. Immunoelectroblotting analysis of recombinant phages revealed the presence of an about 65-kDa band reacting with anti-HIV mAb. This Mr corresponded to the size of the fused form of the FUSE 1 protein III. Human sera from HIV-infected and uninfected individuals reacted with recombinant protein III, as well as the original form of protein III.

Amino Acid Sequence↗
PubMed1991

Productive infection of CD4+ cells by selected HIV strains is not inhibited by anti-CD4 monoclonal antibodies.

Differential susceptibility of four diverse HIV strains to inhibition of infection of CD4+ CEM cells by anti-CD4 monoclonal antibodies was studied. The highly cytopathic HIV-1 246 and NDK strains were able to infect CEM cells and undergo several cycles of replication at saturating doses of LEU3-A, OKT4-A, and 13B8-2 monoclonal antibodies, whereas propagation of reference HIV-1 BRU and weakly cytopathic strain HIV-1 PAS was inhibited. Postadsorption treatment by anti-CD4 antibodies had stronger inhibitory effect than did treatment during the virus adsorption period. In parallel experiments, the same monoclonal antibodies successfully blocked syncytium formation between uninfected MT4 cells and CEM cells infected by all four HIV-1 virus strains tested. To explain these seemingly contradictory data we have postulated that anti-CD4 antibodies efficiently inhibit cell-to-cell but not virus-to-cell infection.

Antibodies, Monoclonal↗
PubMed1991

Selected human immunodeficiency virus replicates preferentially through the basolateral surface of differentiated human colon epithelial cells.

We have used HIV1-NDK-infected HT29 cells grown on permeable substratum to study the polarity of virus maturation in human intestinal cells. When cultured in glucose-containing medium, these cells are mostly undifferentiated. The removal of glucose from the medium allowed the emergence of a selected differentiated subpopulation which continued to produce viral particles in the culture supernatant. The polarity of viral production was evaluated by harvesting virus from the two sides of the monolayer. Seventy-five percent of released HIV was found on the basolateral side of the monolayer. Mature viral particles were observed by electron microscopy near the apical (luminal) and the basolateral (serosal) membrane. These data suggest that epithelial cells of the colon productively infected by a selected strain of HIV are able to produce the virus through both sides of the epithelium but mainly through the serosal side.

Cell Differentiation↗
PubMed1991

Surface CD4 density remains constant on lymphocytes of HIV-infected patients in the progression of disease.

In an attempt to question the influence of circulating virus, soluble gp120 or CD4 self-reacting antibodies upon results of CD4+ T-cell immunophenotyping in AIDS patients, five anti-CD4 mAb defining several epitopes of the V1 and V2 domains of the CD4 molecule were used to analyse the epitopic density of CD4 on lymphocytes of seropositive patients taken at stages II, III and IV of HIV infection, according to the Centers for Disease Control (CDC, Atlanta) classification. Our results demonstrate that each CD4 epitopic density measured on circulating lymphocytes remains constant at a mean level of 46,000 epitopes per cell whatever the stage of the disease and whatever the serum p25 concentration. These data provide evidence that antibody accessibility to several CD4 epitopes is not altered by putative interactions between CD4 molecules and circulating virus, soluble gp120 or anti-CD4 autoantibodies. If such binding events, as expected, do occur in vivo, they are of too low a magnitude to influence the immunophenotyping. Furthermore, we show that mAb specific for different epitopes in the V1 and V2 domains of the CD4 molecule can be used interchangeably for the biological followup of the CD4+ cell population in blood samples of HIV-infected patients.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Fetal human immunodeficiency virus type 1 infection of different organs in the second trimester.

Human immunodeficiency virus type 1 (HIV-1) infection in utero was examined by isolating the virus and detecting the HIV-1 DNA sequence from different fetal tissues. The brain, thymus, lung, liver, spleen, and placenta tissues from fetuses (10-23 weeks of gestation) born to HIV-1-infected asymptomatic mothers were examined. HIV-1 was isolated from 2 of 7, 1 of 7, and 1 of 7 cocultures of splenic, thymic, and trypsin-resistant cells from the liver and placenta, respectively, with peripheral blood mononuclear cells; 20-30% and 40-60% of splenic and of thymic cells were CD4+ lymphoid cells and 40-80% of trypsin-resistant cells were mononuclear phagocytes. The HIV-1 DNA sequence was detected in 4 of 7, 3 of 7, 1 of 7, 1 of 7, 2 of 7, and 2 of 6 samples from the spleen, thymus, brain, lung, liver, and placenta, respectively, using the polymerase chain reaction. In one case, the intensity of the HIV-1 DNA sequence appeared to be correlated with the success of viral isolation. We indicate that fetal HIV-1 infection may frequently occur in the second trimester and the cells responsible for the infection may be CD4+ lymphoid cells and mononuclear phagocytes.

Adult↗
PubMed1991

Susceptibility of cultured human trophoblasts to infection with human immunodeficiency virus type 1.

Primary cultures of essentially pure human term trophoblasts were studied to determine their ability to support the expression of complete proviral clones of human immunodeficiency virus (HIV) and their permissiveness to this virus. Transient expression of molecular clones derived from two biologically distinct strains, BRU and NDK, resulted in the release of comparable amounts of infectious virions, which were rescued by cocultivation with permissive CEM-SS cells. Trophoblasts were inoculated with three HIV-1 isolates, RF, 3B and NDK, which differ in their cytopathogenicity on T lymphoblastoid cells. Infection of cells by all three strains was demonstrated by the presence of virus-specific proteins in the trophoblasts and the detection of virus gag gene-related DNA sequences by the polymerase chain reaction (PCR), but cells were more susceptible to infection with the RF and NDK strains than with the 3B strain. The virus was readily transmitted to the CEM-SS cells with simultaneous formation of syncytia between the two cell types. Flow cytometry and direct radioimmunoassay revealed no trace of the CD4 receptor on the surface of the cultured trophoblasts and CD4 mRNA could not be detected by Northern blot hybridization, although a minimal amount of CD4-associated mRNA was detected by PCR. Our data suggest that infection of trophoblasts occurs independently of the pathway mediated by CD4.

Base Sequence↗
PubMed1991

Human transformed trophoblast-derived cells lacking CD4 receptor exhibit restricted permissiveness for human immunodeficiency virus type 1.

We investigated the nature of interaction of the malignantly transformed cell lines of trophoblast origin BeWo, JAR, and JEG-3 with three different human immunodeficiency virus type 1 (HIV-1) isolates (RF, 3B, and NDK). After inoculation with cell-free virus, the persistence of infection was determined for 1 month by monitoring the presence of viral DNA in the cells by the polymerase chain reaction (PCR). Furthermore, the infectious virus in the culture supernatant was assayed with CEM-SS cells, and attempts to rescue the virus by cocultivation with CEM-SS cells were made. Appraised on the basis of the relative amount of viral DNA and the frequency of positive cocultivation. JEG-3 was the most permissive and BeWo was the least permissive cell line. However, when the cells were transfected with two biologically active molecular clones of HIV-1, the BRU and NDK isolates, all three cell lines turned out to support the production of mature virus progeny to the same extent. The abundance of viral DNA sequences in the infected cells varied with the isolate, showing an overall decline from RF to NDK. The amount of viral DNA in the cells and its expression decreased during the period of observation; this decrease was mirrored in an erosion of the virus recovery rate at cocultivation from 71% recovery on day 8 to failure of isolation on day 32. None of the cell lines expressed detectable amounts of cell surface CD4 molecules when assayed by flow microfluorometry and direct radioimmunoassay. Northern (RNA) blot hybridization analysis of both the total RNA and the mRNA did not reveal any CD4-specific message: nonetheless, by using the PCR, sequences specifically related to the CD4 gene were uncovered. The data demonstrate that the trophoblast-derived cell lines are susceptible to infection with HIV and that they support transient viral replication in the initial phases of infection. However, the latent form of infection may persist over a period of several weeks.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Characterization of monoclonal antibodies directed against distinct conserved epitopes of human immunodeficiency virus type 1 core proteins.

Two mouse hybridoma cell lines secreting antibodies to the Human Immunodeficiency Virus (HIV) p25 major core protein and its precursors p55 and p41, were developed after immunization with the highly cytopathic Zaïrian HIV-1 isolate, NDK. These monoclonal antibodies also react with the gag gene products from HIV-1-BRU prototype and present cross reaction with HIV-2-ROD, and SIV-AGM. They map into topographically distinct areas of p25 and define epitopic regions topographically separated from those recognized by four other anti-p25 mAb suggesting the existence of at least 6 spatially distinct epitopic regions on HIV-1-p25 core protein.

Animals↗
PubMed1990

Differential inhibitory effects of various flavonoids on the activities of reverse transcriptase and cellular DNA and RNA polymerases.

Four flavonoids, 5,6,7-trihydroxyflavone (baicalein), 3,3',4',5,7-pentahydroxyflavone (quercetin), 3,3',4',5,6,7-hexahydroxyflavone (quercetagetin) and 3,3',4',5,5',7-hexahydroxyflavone (myricetin), were found to be potent inhibitors of reverse transcriptases from Rauscher murine leukemia virus (RLV) and human immunodeficiency virus (HIV). Under the reaction conditions employed, any one of these flavonoids almost completely inhibited the activity of RLV reverse transcriptase at a concentration of 1 microgram/ml. HIV reverse transcriptase was inhibited by 100%, 100%, 90% and 70% in the presence of 2 micrograms/ml quercetin, myricetin, quercetagetin and baicalein, respectively. The mode of inhibition of these flavonoids was competitive (RLV reverse transcriptase) or partially competitive (HIV reverse transcriptase) with respect to the template.primer complex, (rA)n.(dT), and noncompetitive with respect to the triphosphate substrate, dTTP. The Ki values for RLV reverse transcriptase were found to be 0.37 microM and 0.08 microM for baicalein and quercetin, respectively and those for HIV reverse transcriptase were 2.52 microM, 0.52 microM, 0.46 microM and 0.08 microM for baicalein, quercetin, quercetagetin and myricetin, respectively. Comparative studies with other flavonoids (hydroxyflavones, dihydroxyflavones and polyhydroxyflavones and flavanones) carried out to clarify the structure/activity relationships, revealed that the presence of both the unsaturated double bond between positions 2 and 3 of the flavonoid pyrone ring, and the three hydroxyl groups introduced on positions 5, 6 and 7, (i.e. baicalein) were a prerequisite for the inhibition of reverse transcriptase activity. Removal of the 6-hydroxyl group of baicalein required the introduction of three additional hydroxyl groups at positions 3, 3' and 4' (quercetin), to afford a compound still capable of inhibiting the reverse transcriptase activity. Quercetagetin which contains the structures of both baicalein and quercetin, and myricetin which has the structure of quercetin with an additional hydroxyl group on the 5' position also proved strong inhibitors of reverse transcriptase activity. The inhibition by baicalein of reverse transcriptase is highly specific, whereas quercetin and quercetagetin were also strong inhibitors of DNA polymerase beta and DNA polymerase I, respectively. Myricetin was also a potent inhibitor of both DNA polymerase alpha and DNA polymerase I.

Animals↗
PubMed1990

Differential inhibition by an antitumoral drug 10-[gamma-diethylaminopropylamino]-6-methyl-5H-pyrido[3',4': 4,5]pyrrolo [2,3-G]isoquinoline (BD-40), a pyrido-pyrrolo-isoquinoline derivative, of in vitro DNA synthesis catalyzed by various DNA polymerases.

The effects of BD-40, a pyrido-pyrrolo-isoquinoline analogue of ellipticines, and its 2-acetylated derivative (BD-84) and in vitro DNA synthesis catalyzed by purified preparations of various DNA polymerases were examined. The major conclusions are: (1) Both BD-40 and BD-84 strongly inhibit the DNA synthesis by DNA polymerase or reverse transcriptase with poly(rA).oligo(dT) as the template.primer. (2) Both compounds moderately inhibit the DNA synthesis by DNA polymerase alpha or E. coli DNA polymerase I with activated DNA. However, the DNA synthesis by DNA polymerase beta is resistant to inhibition by BD-40 and slightly sensitive to that by BD-84. (3) BD-84 is more inhibitory than BD-40 in DNA syntheses by various DNA polymerases except in those by DNA polymerase alpha and terminal deoxyneuclotidyltransferase to which both compounds are similarly inhibitory. (4) Kinetic analyses revealed that the observed inhibitions are due to competition between the drug or the drug-bound template.primer and the free template.primer for the same binding site of the enzyme.

Animals↗