Biomedical subjects

N J Roberts

Publications and source records attributed to N J Roberts.

At least 19 recordsLinked to original sources

PubMed1999

A nod factor binding lectin with apyrase activity from legume roots.

A lectin isolated from the roots of the legume, Dolichos biflorus, binds to Nod factors produced by rhizobial strains that nodulate this plant and has a deduced amino acid sequence with no significant homology to any lectin reported to date. This lectin also is an enzyme that catalyzes the hydrolysis of phosphoanhydride bonds of nucleoside di- and triphosphates; the enzyme activity is increased in the presence of carbohydrate ligands. This lectin-nucleotide phosphohydrolase (LNP) has a substrate specificity characteristic of the apyrase category of phosphohydrolases, and its sequence contains four motifs characteristic of this category of enzymes. LNP is present on the surface of the root hairs, and treatment of roots with antiserum to LNP inhibits their ability to undergo root hair deformation and to form nodules on exposure to rhizobia. These properties suggest that this protein may play a role in the rhizobium-legume symbiosis and/or in a related carbohydrate recognition event endogenous to the plant.

Amino Acid Sequence↗

PubMed1998

QacR is a repressor protein that regulates expression of the Staphylococcus aureus multidrug efflux pump QacA.

The Staphylococcus aureus QacA protein is a multidrug transporter that confers resistance to a broad range of antimicrobial agents via proton motive force-dependent efflux of the compounds. Primer extension analysis was performed to map the transcription start points of the qacA and divergently transcribed qacR mRNAs. Each gene utilized a single promoter element, the locations of which were confirmed by site-directed mutagenesis. Fusions of the qacA and qacR promoters to a chloramphenicol acetyl transferase reporter gene were used to demonstrate that QacR is a trans-acting repressor of qacA transcription that does not autoregulate its own expression. An inverted repeat overlapping the qacA transcription start site was shown to be the operator sequence for control of qacA gene expression. Removal of one half of the operator prevented QacR-mediated repression of the qacA promoter. Purified QacR protein bound specifically to this operator sequence in DNase I-footprinting experiments. Importantly, addition of diverse QacA substrates was shown to induce qacA expression in vivo, as well as inhibit binding of QacR to operator DNA in vitro, by using gel-mobility shift assays. QacR therefore appears to interact directly with structurally dissimilar inducing compounds that are substrates of the QacA multidrug efflux pump.

Amino Acid Sequence↗

PubMed1998

Genetic evaluation of suspected cases of transient HIV-1 infection of infants.

Detection of human immunodeficiency virus-type 1 (HIV-1) on only one or a few occasions in infants born to infected mothers has been interpreted to indicate that infection may be transient rather than persistent. Forty-two cases of suspected transient HIV-1 viremia among 1562 perinatally exposed seroreverting infants and one mother were reanalyzed. HIV-1 env sequences were not found in specimens from 20; in specimens from 6, somatic genetic analysis revealed that specimens were mistakenly attributed to an infant; and in specimens from 17, phylogenetic analysis failed to demonstrate the expected linkage between the infant's and the mother's virus. These findings argue that transient HIV-1 infection, if it exists, will only rarely be satisfactorily documented.

DNA, Viral↗

PubMed1997

The generation of 1H-NMR-detectable mobile lipid in stimulated lymphocytes: relationship to cellular activation, the cell cycle, and phosphatidylcholine-specific phospholipase C.

Mobile lipids detected using 1H-NMR in stimulated lymphocytes were correlated with cell cycle phase, expression of the interleukin-2 receptor alpha and proliferation to assess the activation status of the lymphocytes. Mobile lipid levels, IL-2R alpha expression and proliferation increased after treatment with PMA and ionomycin. PMA or ionomycin stimulation alone induced increased IL-2R alpha expression but not proliferation. PMA- but not ionomycin-stimulation generated mobile lipid. Treatment with anti-CD3 antibody did not increase IL-2R alpha expression or proliferation but did generate increased amounts of mobile lipid. The cell cycle status of thymocytes treated with anti-CD3, PMA or ionomycin alone indicated an accumulation of the cells in the G1 phase of the cell cycle. The generation of mobile lipid was abrogated in anti-CD3 antibody-stimulated thymic lymphocytes but not in splenic lymphocytes, using a phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor which blocked cells in the G1/S phase of the cell cycle. This suggests that the 1H-NMR-detectable mobile lipid may be generated in anti-CD3 antibody-stimulated thymic lymphocytes by the action of PC-PLC activity via the catabolism of PC, in the absence of classical signs of activation.

Animals↗

PubMed1994

Analysis of human antiviral cytotoxic T-lymphocyte responses for vaccine trials using cryopreserved mononuclear leukocytes: demonstration of feasibility with influenza virus-specific responses.

The feasibility of measuring virus-specific human cytotoxic T-lymphocyte (CTL) activity by using cryopreserved mononuclear leukocytes to support clinical vaccine trials was addressed. Autologous fresh and cryopreserved cells from the same sample of peripheral blood were used as sources of CTL precursors and were tested for influenza virus-specific activity. The data indicated that virus-specific CTL activity could be measured by using cryopreserved cells; this could also be done in assays that are designed to characterize the responsible effector cell population.

Cryopreservation↗

PubMed1993

Use of FITC-labeled influenza virus and flow cytometry to assess binding and internalization of virus by monocytes-macrophages and lymphocytes.

The binding of influenza virus to the surface of cells and the internalization of virus particles by all or a subset of cells are key points in the pathogenesis of viral infection. The current studies established a method for discrimination of surface-bound from internalized influenza virus. Fluorescein isothiocyanate (FITC) was attached to the viral hemagglutinin and neuroaminidase proteins; the fluorescent virus retained infectivity. A flow cytometric technique was then adapted for study of virus-cell interactions, with addition of ethidium bromide to quench green fluorescence associated with FITC-labeled virus that was cell-bound but remained external. Ethidium bromide was excluded by intact cell membranes, and internalized virions retained green fluorescence. Cells could be examined by fluorescence microscopy or flow cytometry, with flow cytometry allowing rapid, kinetic assessment of large numbers of cells and subsets of virus-exposed cells. The data showed that, whereas a majority of both monocytes-macrophages and lymphocytes bound influenza virus, a large percentage of monocytes-macrophages but only a very small percentage of lymphocytes internalized the virus. This procedure provides a simple and effective method to distinguish surface-bound from internalized influenza virus, and allows precise kinetic analyses on large numbers of cells.

Adult↗

PubMed1993

Human macrophage responses to vaccine strains of influenza virus: synthesis of viral proteins, interleukin-1 beta, interleukin-6, tumour necrosis factor-alpha and interleukin-1 inhibitor.

Interactions between influenza viruses and human macrophages were examined to detect potential mechanisms for enhanced febrile reactions previously associated with administration of an avian-human H1N1 reassortant vaccine. Cells exposed to that strain were compared with cells exposed to wild-type and cold-adapted H1H1 and H3H2 strains and an avian-human H3N2 strain. Cells exposed to the avian-human H1N1 virus showed increased synthesis of viral neuraminidase, previously reported to induce fever-producing cytokines, but no detectable increase in production of interleukin-1 beta, interleukin-6 and tumour necrosis factor-alpha measured by immunoassay, or decrease in interleukin-1 inhibitor activity by bioassay.

Fever↗

PubMed1993

Persisting human immunodeficiency virus type 1 gp160-specific human T lymphocyte responses including CD8+ cytotoxic activity after receipt of envelope vaccines.

Mononuclear leukocytes (MNL) were obtained from vaccina-naive, non-human immunodeficiency virus (HIV) infected subjects who were vaccinated with HIV-1-derived recombinant (r) live vaccina-gp160, 4 of whom were boosted 1-2 years later with purified rgp160. MNL obtained after receipt of the vaccinia-gp160 alone showed persisting (> or = 1 year) gp160-specific lymphocyte proliferative responses and production of immune-specific interferon (IFN)-gamma. All 4 subjects who were boosted with rgp160 responded to the boost, including 2 whose cellular responses had waned prior to the boost. MNL from these 4 exhibited gp160-specific proliferative responses, IFN-gamma production, and cytotoxic T lymphocyte activity. The gp160-specific cytolysis was severely reduced or abolished by depletion of CD8+ cells and was not detected using HLA class I-mismatched target cells. Persisting (> or = 15 months after boost) HIV gp160-specific T cell recognition and functional responses can be induced by HIV-derived envelope vaccines.

AIDS Vaccines↗

PubMed1993

Cross-reactive response to human immunodeficiency virus type 1 (HIV-1) gp120 and HLA class I heavy chains induced by receipt of HIV-1-derived envelope vaccines.

Autoantibodies specific to HLA class I antigens were detected in the sera of persons vaccinated with human immunodeficiency virus type 1-derived recombinant vaccines by using synthetic peptides representing the amino acid sequences recognized by an HLA class I/gp120 cross-reactive monoclonal antibody. Study subjects received recombinant vaccinia gp160 (vacc-env) alone, vacc-env followed by one dose of recombinant gp160 (rgp160, 640 micrograms), or four doses of rgp160 alone (640 or 80 micrograms). All sera from vacc-env/rgp160-vaccinated subjects contained HLA/gp120 cross-reactive antibodies, as did all sera from recipients of the rgp160 alone at 640 micrograms/dose. In contrast, none of the sera from subjects who received either the vacc-env alone or the 80 micrograms/dose rgp160 alone contained detectable HLA cross-reactive antibodies, and these same sera showed little or no envelope reactivity on Western blot. The results showed a striking correlation between immunogenicity and the induction of cross-reactive antibodies by the rgp160 vaccine.

AIDS Vaccines↗

PubMed1992

Recent observations regarding the pathogenesis of recurrent respiratory syncytial virus infections: implications for vaccine development.

Respiratory syncytial virus (RSV) and influenza virus are common pathogens for all age groups. Currently licensed influenza virus vaccines generally provide protection from clinically detectable disease caused by antigenically matched challenging viruses. In contrast, vaccine development for RSV has been hampered by the inability of candidate vaccines to induce protective immunity to naturally occurring infection. The precise mechanism(s) responsible for the RSV vaccine failures have not been determined. We raise the possibility that infection by RSV is associated with attenuation of both proliferative and non-proliferative RSV-specific responses by human mononuclear leucocytes that results in the suppression or delay of host anamnestic defences, allowing development of recurrent clinical illness despite pre-existing immunity.

Humans↗

PubMed1992

The limited role of the human interferon system response to respiratory syncytial virus challenge: analysis and comparison to influenza virus challenge.

The respiratory syncytial virus (RSV)-induced production of interferon (IFN) by human macrophages and mononuclear leukocytes (MNL) and the sensitivities of RSV to subtypes of IFN-alpha were examined and compared to IFN production induced by influenza virus. Influenza virus induced high titers of total IFN bioactivity, transcription of the IFN-alpha 1 and IFN-beta gene products and production of IFN-gamma. In contrast, RSV induced minimal or no detectable total IFN activity, and the absence of IFN bioactivity could not be attributed to inhibitors of IFN activity. There was no detectable transcription of IFN-alpha or IFN-beta gene products by the cells exposed to RSV. RSV-exposed MNL did produce small amounts of IFN-gamma, consistent with prior sensitization of the cell donors to the virus, but titers were substantially lower than those induced by influenza virus. RSV showed minimal but equivalent susceptibility to several subtypes of IFN-alpha. The data raise the possibility that the IFN system has a limited direct role in early host defense against RSV infection, but results should not be extrapolated directly to in vivo events.

Cells, Cultured↗

PubMed1992

Vaccination of vaccinia-naive adults with human immunodeficiency virus type 1 gp160 recombinant vaccinia virus in a blinded, controlled, randomized clinical trial. The AIDS Vaccine Clinical Trials Network.

The safety and immunogenicity of a human immunodeficiency virus type 1 (HIV-1) gp160 recombinant vaccinia virus (HIVAC-1e) vaccine was evaluated in vaccinia-naive, healthy adults at low risk for acquiring HIV-1 infection. Volunteers (n = 36) were randomized to receive HIVAC-1e or control vaccinia virus at two dosages by bifurcated needle puncture at 0 and 2 months; 12 HIVAC-1e and 6 control vaccinia virus recipients received either 10(6) or 10(7) pfu/mL at each inoculation. There was no significant difference in lesion size, level of viral replication, or systemic symptoms after vaccination with HIVAC-1e or control vaccinia virus. Of 22 HIVAC-1e recipients with lesion formation, 16 developed low-titer gp160-specific antibody responses detectable by Western blot. The peak response occurred between days 70 and 120 and was still detectable at day 365 in 9 of 18 vaccinees. gp160-specific lymphoproliferative responses were detected in 5 of 10 vaccinees. Vaccination with HIVAC-1e was safe in vaccinia-naive, healthy adults and could induce both humoral and cell-mediated gp160-specific immune responses.

AIDS Vaccines↗

PubMed1992

Sulfuric acid aerosol exposure in humans assessed by bronchoalveolar lavage.

Epidemiologic and experimental evidence suggests that exposure to acidic aerosols may affect human health. Brief exposures to acidic aerosols alter mucociliary clearance and increase airway responsiveness, but effects on host defense mechanisms at the alveolar level have not been studied in humans. Twelve healthy, nonsmoking volunteers between 20 and 39 yr of age were exposed for 2 h to aerosols of approximately 1,000 micrograms/m3 sulfuric acid (H2SO4) or sodium chloride (NaCl [control]), with intermittent exercise, in a randomized, double-blind fashion. Each subject received both exposures, separated by at least 2 wk. Bronchoalveolar lavage (BAL) was performed 18 h after exposure in order to detect evidence of an inflammatory response, changes in alveolar cell subpopulations, or changes in alveolar macrophage (AM) function, which is important in host defense. When compared with NaCl, exposure to H2SO4 did not increase polymorphonuclear leukocytes in BAL fluid. The percentage of T lymphocytes decreased in association with H2SO4 exposure, but the difference was not statistically significant (14.9% after NaCl, 11.5% after H2SO4; p = 0.14). Antibody-mediated cytotoxicity of AM increased in association with H2SO4 exposure (percent lysis 19.1 after NaCl, 23.6 after H2SO4; p = 0.16). No significant change was seen in release of superoxide anion or inactivation of influenza virus in vitro. Brief exposures to H2SO4 aerosol at 1,000 micrograms/m3 do not cause an influx of inflammatory cells into the alveolar space, and no evidence was found for alteration in antimicrobial defense 18 h after exposure.

Adult↗

PubMed1991

Interleukin-1-inhibitor activity induced by respiratory syncytial virus: abrogation of virus-specific and alternate human lymphocyte proliferative responses.

Respiratory syncytial virus (RSV) infection has been shown to induce human mononuclear leukocyte (MNL) production of net interleukin-1 (IL-1)-inhibitor activity. In the current studies of IL-1-inhibitor effects, RSV-exposed cells were compared with autologous MNL that were sham-exposed or exposed to inactivated RSV or influenza virus (which induces net IL-1 activity and commonly elicits effective homotypic immunity). Exposure of MNL to influenza virus or inactivated RSV resulted in increased expression of human leukocyte antigen-DR, the IL-2 receptor, and the transferrin receptor and increased progression through the cell cycle by 3 days. In contrast, exposure to infectious RSV resulted in decreased marker expression and cell cycle arrest, with abrogation of proliferation in response to the virus or other stimuli. These data raise the possibility that a contributing mechanism for recurrence of RSV infection is early suppression of the clonal expansion of virus-specific lymphocytes due to net IL-1-inhibitor activity.

Cell Cycle↗

PubMed1991

Human immunodeficiency virus (HIV-1) gp160-specific lymphocyte proliferative responses of mononuclear leukocytes from HIV-1 recombinant gp160 vaccine recipients.

The lymphocyte proliferative responses were studied of 12 volunteers enrolled in a phase I trial of a baculovirus-expressed recombinant human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (rgp160) vaccine. Six subjects received rgp160 and three subjects each received recombinant hepatitis B vaccine or placebo at 0, 1, and 6 months. rgp160 and a control preparation, baculovirus-expressed recombinant HIV-1 p24, were used as in vitro antigens. At day 56, all rgp160 recipients had stimulation indexes (rgp160/rp24) greater than 3.0, and five of six had differences in counts per minute (cpm) greater than 1000. Stimulation indexes were less than 2.0 and cpm differences were less than 150 in all six who did not receive rgp160. Lymphocyte proliferative responses were first noted 2 weeks to 5 months before initial Western blot reactivity and persisted for greater than or equal to 540 days, even among subjects who lost detectable antibody. Thus, the HIV-1 rgp160 vaccine induces persistent cellular immune recognition as demonstrated by lymphocyte proliferation.

Adult↗

PubMed1991

In vitro interactions between bovine papillomavirus and human monocytes and macrophages.

Limited data suggest that macrophages play a role in the pathogenesis of bovine papillomavirus (BPV) infections. In the present study, interactions between these cells and BPV-1 were explored by exposing in vitro human blood monocytes and monocyte-derived macrophages to purified virions. Immediately or up to 28 days after exposure, cell culture supernatants as well as cell lysates were collected. Interleukin 1 activity was detected in the supernatants of monocytes early after exposure to BPV (0-3 days), but little was found after exposure of macrophages to BPV. In addition, both monocyte and macrophage cell lysates contained episomal BPV DNA which, after an initial decrease in copy number, increased 14-28 days later. Concomitantly, there was progressive disappearance of detectable BPV major capsid protein in cell lysates. These observations support the concept that monocytes and macrophages play a role in the pathogenesis of papillomavirus infections.

Animals↗

PubMed1991

Characterization of human alveolar macrophage Fc gamma receptor III: a transmembrane glycoprotein that is shed under in vitro culture conditions.

Three classes of Fc gamma receptors (FcR) have been identified on blood leukocytes: FcRI, FcRII, and FcRIII. Two forms of FcRIII have recently been characterized; a phosphatidylinositol linked form is found on neutrophils, whereas a transmembrane form of the molecule is found on a subset of peripheral blood lymphocytes. Peripheral blood monocytes express low levels of FcRIII on their surface, whereas FcRIII is readily expressed by tissue macrophages. The purpose of this investigation was to characterize the form of FcRIII expressed by normal human alveolar macrophages (AM) obtained from normal subjects by bronchoalveolar lavage. We found FcRIII expressed by AM has a molecular mass of 50 to 60 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis and migrates as a single band with a molecular mass of 35 kD after digestion with endoglycosidase F. Macrophage FcRIII was resistant to cleavage by phosphatidylinositol-specific phospholipase C. These results demonstrate that FcRIII expressed by AM is a transmembrane glycoprotein similar to the molecule found on peripheral blood lymphocytes. Scatchard binding analysis using 125I-labeled mAb 3G8 showed that AM express similar numbers of FcRIII as found on neutrophils (73,300 +/- 16,300 versus 69,300 +/- 8,500 receptor sites/cell, respectively; P = 0.73), whereas fewer binding sites were found on FcRIII-positive peripheral blood lymphocytes (35,300 +/- 13,900; P = 0.04). Of note, we found expression of FcRIII by AM was selectively and dramatically reduced during short term in vitro incubation at 37 degrees C. Receptor shedding as a result of proteolytic cleavage is probably responsible for the reduced expression that occurs during short-term in vitro culture.

Antibodies, Monoclonal↗

PubMed1991

Suppressed expression of ICAM-1 and LFA-1 and abrogation of leukocyte collaboration after exposure of human mononuclear leukocytes to respiratory syncytial virus in vitro. Comparison with exposure to influenza virus.

Human mononuclear leukocytes (MNL) exposed to respiratory syncytial virus (RSV) produce net IL-1 inhibitor bioactivity with the anticipated consequences of cell cycle arrest, suppressed virus-specific proliferation, and reduced expression of activation markers. These studies were undertaken to investigate effects of exposure and resultant net IL-1 inhibitor activity on the expression of the intercellular adhesion molecule-1 (ICAM-1), and its ligand the lymphocyte function-associated antigen (LFA-1). MNL collected at 1, 4, and 24 h after exposure to influenza virus (which induces net IL-1 bioactivity) showed enhanced expression of ICAM-1 and LFA-1 relative to sham-exposed MNL and exhibited cell clustering. In contrast, exposure to RSV was associated with suppressed expression of both ICAM-1 and LFA-1 and with minimal detectable cell clustering throughout the culture period. Influenza virus-exposed MNL produced significantly more IL-1 and IFN-gamma (which require cell-cell collaboration for optimal production) than did RSV-exposed MNL. These data raise the possibility that exposure of MNL to RSV fails to elicit or blocks the early events necessary for cellular collaboration, contributing to early suppression of the clonal expansion of RSV-specific lymphocytes.

Adult↗