Biomedical subjects

N J Roberts

Publications and source records attributed to N J Roberts.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1991

Mechanisms of nitrogen dioxide toxicity in humans.

These studies were undertaken to evaluate short-term respiratory effects and identify markers of nitrogen dioxide toxicity during exposures designed to approximate realistic conditions. With the development of bronchoalveolar lavage as a clinical investigative technique, the evaluation focused on the assessment of effects induced at the alveolar level. The exposure protocols were designed to assess the duration of nitrogen dioxide-induced effects and determine exposure-response relationships. Groups of normal, nonsmoking volunteers of both sexes between the ages of 18 and 40 years, without airway hyperreactivity, constituted the study population. The exposure protocols required a total of three to five days for each subject, depending on the timing of bronchoalveolar lavage. Subjects were exposed to nitrogen dioxide or air for three hours in a double-blind, randomized fashion in a 45-m3 environmental chamber, with intermittent exercise sufficient to quadruple minute ventilation. Pulmonary function was measured during and after exposure, and airway reactivity to carbachol was assessed before and after exposure. Lavaged cells were examined for their capacity to inactivate influenza virus and secrete IL-1 in vitro. Cell-free lavage fluid was analyzed for total protein, albumin, alpha 2-macroglobulin, arylsulfatase, and alpha 1-protease inhibitor. The studies were undertaken in three phases, each of approximately one year's duration. In Phase 1, 15 subjects were exposed to a background concentration of 0.05 parts per million2 (ppm) nitrogen dioxide and to three 15-minute peaks of 2.0 ppm, and underwent bronchoalveolar lavage 3.5 hours after nitrogen dioxide exposure. During Phase 2, 8 subjects were exposed to continuous 0.60 ppm nitrogen dioxide and underwent bronchoalveolar lavage 18 hours later. Finally, in Phase 3, 15 subjects were exposed to continuous 1.5 ppm nitrogen dioxide and underwent bronchoalveolar lavage 3.5 hours after exposure. No significant symptomatic or pulmonary function changes could be detected in response to any of the nitrogen dioxide exposures. However, a small but significant increase in airway reactivity was observed in normal subjects after exposure to 1.5 ppm nitrogen dioxide. Following the highest dose of carbachol (10 mg/mL), the forced expiratory volume in one second decreased 7.5 +/- 1.1 percent after nitrogen dioxide exposure compared to 4.8 +/- 1.1 percent after exposure to air (p less than 0.05). No symptoms were induced in any of the groups by the carbachol exposures. Analyses of cells recovered by bronchoalveolar lavage during all three phases revealed no differences in total cell recovery, cell viability, or differential cell counts.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1990

Human alveolar macrophage FcR-mediated cytotoxicity. Heteroantibody- versus conventional antibody-mediated target cell lysis.

Human alveolar macrophage have three distinct receptors for IgG: FcRI, FcRII, and FcRIII. In order to compare the ability of these receptors to mediate target cell lysis, three different assay systems were examined. First, we studied lysis of chicken E (CE) opsonized with heteroantibodies, which are synthetic antibodies composed of Fab fragments with anti-FcR activity covalently linked to Fab fragments with anti-CE activity. We found alveolar macrophage readily lysed heteroantibody-opsonized CE via each of the three FcR classes (FcRI, 20 +/- 5%; FcRII, 27 +/- 7%; and FcRIII, 13 +/- 13%, p less than 0.05). Non-FcR-dependent lysis of anti-beta 2-microglobulin x anti-CE heteroantibody-opsonized CE was not detected. Second, lysis of hybridoma cell lines bearing anti-FcR antibodies on their cell surface was examined to assess killing of "tumor-like" target cells. Whereas peripheral blood monocytes and lymphocytes were able to lyse hybridoma cell lines bearing surface anti-FcR mAb, alveolar macrophages were not. Third, activity of alveolar macrophage FcR was examined in a conventional antibody-dependent cellular cytotoxicity assay by using O+ (R1,R2) human RBC opsonized with human anti-D and anti-CD serum as target cells. We found lysis of anti-D and anti-CD opsonized human RBC was mediated exclusively via FcRI. No activity of FcRII or FcRIII was detected in these latter assays even if performed under conditions that impair FcRI activity. Thus, all three FcR present on alveolar macrophage mediate lysis of heteroantibody-opsonized CE; in contrast, with the use of a conventional antibody-dependent cellular cytotoxicity assay, only FcRI activity was detected. We were unable to demonstrate lysis of anti-FcR-bearing hybridoma cell lines by alveolar macrophages.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1990

Role of the monocyte-macrophage in influenza virus infection of lymphocytes: implications for HIV infection.

Knowledge of the pathogenesis of viruses which are less virulent than human immunodeficiency virus (HIV) may provide valuable insights into the pathogenesis of HIV infection. Influenza virus, an enveloped RNA virus, infects monocyte-macrophages, although the infection is brief and abortive. Isolated purified lymphocytes are completely resistant to infection. In contrast, mixtures of lymphocytes and macrophages can synthesize all virus proteins. Infection requires physical association of monocyte-macrophages and lymphocytes in "clusters." These studies with influenza virus suggest that the pathogenesis of virus infections in mixed cell cultures may be very different from that observed in purified cell populations, and they suggest that similar studies should be performed with HIV.

HIV Infections↗

PubMed1989

Interleukin-1 inhibitor production by human mononuclear leukocytes and leukocyte subpopulations exposed to respiratory syncytial virus: analysis and comparison with the response to influenza virus.

Net interleukin-1 (IL-1) inhibitor activity is induced by exposure of purified human monocytes-macrophages to respiratory syncytial virus (RSV). Furthermore, IL-1 inhibitor activity was produced by monocytes-macrophages exposed to RSV in the presence of lymphocytes, that is, by unseparated mononuclear leukocytes (MNL). Purified RSV-exposed lymphocytes, as well as the lymphocytes exposed within MNL preparations, produced net IL-1 inhibitor activity. In contrast, net IL-1 activity was produced when purified monocytes-macrophages or unseparated MNL were exposed to influenza virus. The RSV-induced IL-1 inhibitors demonstrated antiproliferative effects on mitogen-stimulated human lymphocytes as well as on the mouse thymocytes used in standard assays. The results raise the possibility that such antiproliferative activity is mediated, at least in part, by monocytes-macrophages. The data also suggest that IL-1 inhibitors produced by MNL after exposure to RSV may contribute along with other factors to the recurrence of RSV infection in immune individuals.

Humans↗

PubMed1989

Regulation of lymphocyte proliferation after influenza virus infection of human mononuclear leukocytes.

Previous studies demonstrated that in vitro infection with influenza A viruses altered several functions of human monocytes-macrophages but did not detectably alter functions of human lymphocytes. However, both types of cells were infected, as determined by production and surface expression of viral antigens. In the current studies, human mononuclear leukocytes were infected in vitro and assayed for both influenza virus-induced proliferation and mitogen (phytohemagglutinin [PHAl)-induced proliferation, as well as for ability to stimulate proliferative responses by normal autologous leukocytes. The leukocytes showed proliferation in response to the infectious virus, but concomitant depressed proliferative responses to PHA. Coculture experiments suggested suppression of PHA-induced responses by the virus-infected cells. However, upon coculture with fresh autologous leukocytes (without PHA stimulation), both virus-infected macrophages and virus-infected lymphocytes induced autologous lymphocyte proliferative responses. Altered proliferative responses to mitogen stimulation after exposure to the virus were not due to diminished interleukin-1 production or diminished expression of HLA-DR by monocytes-macrophages. The expression of influenza virus antigens and resulting induction of autologous proliferative responses, combined with depressed mitogen-induced proliferation, may be important in human antiviral defense.

Adult↗