RESUMEN
The addition of IFN-gamma to cultures of peripheral blood mononuclear cells (PBMCs) obtained from asymptomatic HIV-infected patients increased cell proliferation in response to HIV envelope synthetic peptides (Env), influenza A virus (VIRUS), and allogeneic lymphocytes (ALLO) but not to phytohaemagglutinin (PHA) stimulation. F(Ab)2 fragments of IgG purified from the sera of HIV-seropositive patients specifically interfered with IFN-gamma-induced cell proliferation in response to recall antigens. Neutralization of the lymphokine activity was found to be sustained by specific IFN-gamma antibodies. Data obtained demonstrate that IFN-gamma can restore the cell-mediated immunity of a number of asymptomatic HIV+ individuals in vitro, while IFN-gamma antibodies present in sera of patients with AIDS interfere with the activity of the lymphokine.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Anticuerpos Monoclonales/inmunología , Infecciones por VIH/inmunología , Seronegatividad para VIH/inmunología , Interferón gamma/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Anti-VIH/inmunología , Proteína gp120 de Envoltorio del VIH/farmacología , Humanos , Inmunidad Celular , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Virus de la Influenza A/inmunología , Isoantígenos/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Proteínas RecombinantesRESUMEN
Individual cells capable of interferon-gamma (IFN-gamma) synthesis are easily detected by immunofluorescence and flow cytometric analysis using an anti-IFN-gamma monoclonal antibody as specific reagent. By IFN-gamma flow cytometry assay, we demonstrated that HIV-seropositive patients, starting at the early stage of viral infection, generally have an increased percentage of lymphocytes potentially able to produce IFN-gamma, compared with healthy blood donors. IFN-gamma expression in patient lymphocytes was observed to increase with the progressive stages of HIV infection, with the highest figures occurring in stage C patients. Such increased IFN-gamma expression involved both CD4+ and CD8+ T cell subsets. Most interestingly, we found patients at the same stage of HIV infection who had similar numbers of total and CD4+ lymphocytes but highly different percentages of lymphocytes potentially capable of producing IFN-gamma.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Interferón gamma/biosíntesis , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Seropositividad para VIH/complicaciones , Seropositividad para VIH/metabolismo , Humanos , Inmunofenotipificación , Ionomicina/farmacología , Ionóforos/farmacología , Activación de Linfocitos/efectos de los fármacos , Abuso de Sustancias por Vía Intravenosa/complicaciones , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
MAb IGMB-15, an anti-hIFN-gamma MAb, neutralizes the antiproliferative activity of hIFN-gamma without affecting that of hIFN-alpha or hIFN-beta. The neutralizing capacity of MAb IGMB-15 is wide: it has been assessed on cell lines whose origin and sensitivity to hIFN-gamma differ. The binding of hIFN-gamma to its receptor and its subsequent internalization into the target cell were not influenced by the antibody. MAb IGMB-15 has been found to interact with hIFN-gamma in solution but not when the lymphokine was associated with its cell surface receptor, showing that the growth of certain cell lines can be inhibited at the cell membrane level. This finding is consistent with the existence of an accessory factor responsible for the antiproliferative activity of hIFN-gamma.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Interferón gamma/inmunología , Antígenos CD/inmunología , Sitios de Unión de Anticuerpos , División Celular , Reactivos de Enlaces Cruzados , Humanos , Pruebas de Neutralización , Receptores de Interferón/inmunología , Proteínas Recombinantes , Células Tumorales Cultivadas , Receptor de Interferón gammaRESUMEN
CD28 interaction with B7 molecules, expressed on the membranes of antigen-presenting cells, co-stimulates cytokine production, T-cell proliferation and generation of cytotoxic lymphocytes. The expression of CD28 markers on CD4+ and CD8+ lymphocytes was studied in a group of subjects at various stages of HIV infection. A reduction in the percentage of CD28-bearing CD4+ and CD8+ cell subsets was observed during the asymptomatic stage of the disease. This reduction was more pronounced in AIDS than in non-AIDS patients. At the same time, an increase in the absolute CD8+CD28- cell number (greater in stage A than in stage B and C subjects) was observed in HIV-infected patients. The finding of an altered pattern of CD28 expression on T cells might per se explain certain early defects in the cytokine pattern and in the immune response peculiar to HIV-infected patients.
Asunto(s)
Antígenos CD28/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , Citometría de Flujo , Humanos , InmunofenotipificaciónRESUMEN
Cells infected with human immunodeficiency virus type 1 (HIV-1) develop viral antigens which can be detected by immunofluorescence. We developed a flow cytometric immunofluorescence assay (FIFA) which provides a quantitative analysis of HIV-1 p24 using a monoclonal antibody (mAb) as a specific reagent. The reduction of HIV p24 antigen expression in viral infected cells was then used to determine HIV neutralizing antibody titers in human sera. Results obtained by FIFA for detecting neutralizing antibodies against HIV-1, when compared with results obtained by indirect immunofluorescence assay (IFA), showed an overall index of agreement of 94.1%. The correlation between the neutralizing antibody titers obtained with each method was found to be highly significant (ro = 0.8; p < 0.01). The simple methodology and the adaptability of this FIFA to various assay conditions make it suitable for routine purposes and for assessing the efficacy of vaccination strategies.
Asunto(s)
Citometría de Flujo/métodos , Técnica del Anticuerpo Fluorescente , Anticuerpos Anti-VIH/sangre , Proteína p24 del Núcleo del VIH/análisis , Infecciones por VIH/inmunología , VIH-1/inmunología , Anticuerpos Monoclonales , Línea Celular , Separación Celular , Humanos , Pruebas de Neutralización , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The induction of HLA-DR antigen expression on U937 cells by interferon-gamma (IFN-gamma) is positively influenced by amount of fetal calf serum (FCS) added to the tissue culture medium. A transient alkalinization of FCS before its addition to the medium, dramatically decreased the immunomodulating activity of IFN-gamma. FCS was also found to be a dose-dependent enhancer of the IFN-gamma-induced 2',5'-oligoadenylate (2-5A) synthetase production. Our findings suggest the need for a serum factor(s), labile at basic pH values, to support at least two of the multiple IFN-gamma activities.
Asunto(s)
Sangre Fetal/fisiología , Antígenos HLA-DR/análisis , Interferón gamma/farmacología , 2',5'-Oligoadenilato Sintetasa/biosíntesis , Animales , Bovinos , División Celular/efectos de los fármacos , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Monocitos/inmunologíaRESUMEN
This study was undertaken to assess the capability of Bacillus subtilis spores to modify the peripheral-blood lymphocyte (PBL) subsets or determine the de novo expression of activation markers. The data we obtained show that spores of B. subtilis are able to increase the expression of certain cell activation markers and that such activation is dose-dependent. In fact, doses of 2 x 10(9) spores did not give rise to changes in any of the parameters evaluated, while doses of 6 x 10(9) increased the HLA-DR antigen expression on T-lymphocytes. At the highest dosage used (12 x 10(9), B. subtilis spores caused the appearance of cells bearing the CD25 and CD71 activation markers. Therefore, such cell activation markers may prove useful for monitoring the activity of B. subtilis spores, and possibly of other immunomodulating agents, in the course of clinical research.
Asunto(s)
Bacillus subtilis/inmunología , Subgrupos Linfocitarios/inmunología , Administración Oral , Adulto , Antígenos de Diferenciación de Linfocitos T/metabolismo , Femenino , Antígenos HLA-DR/metabolismo , Humanos , Inmunidad Celular , Activación de Linfocitos , Masculino , Esporas Bacterianas/inmunologíaRESUMEN
1. The effects of retinoic acid, gamma-interferon, cytosine arabinoside, nerve growth factor, tumor necrosis factor, and 12-O-tetradecanoylphorbol 13-acetate on the human neuroblastoma cell line, LAN-5, were studied. Intracellular levels of acetylcholinesterase, neuron-specific enolase, catecholamines and related neurotransmitters, vasointestinal peptide, and substance P were evaluated after induction. 2. Cell morphology was strongly affected by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. The main effects of retinoic acid and gamma-interferon were the loosening of cell clusters and the extension of long neurites; cytosine arabinoside induced cell body swelling and marked neuritogenesis. Following 12-O-tetradecanoylphorbol 13-acetate treatment, the cells became small, round, and neuritic. Conversely, modifications induced by nerve growth factor and tumor necrosis factor were mild. Cell proliferation rate was reduced by retinoic acid, gamma-interferon, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate, while nerve growth factor and tumor necrosis factor were devoid of effects. 3. Acetylcholinesterase activity was significantly stimulated by retinoic acid and by gamma-interferon. Neuron-specific enolase activity was unaffected by all treatments except 12-O-tetradecanoylphorbol 13-acetate, which enhanced it by 1.6-fold. 4. The cellular catecholamine and related metabolite content was lowered by retinoic acid and gamma-interferon, while cytosine arabinoside and, even more, 12-O-tetradecanoylphorbol 13-acetate showed a stimulatory activity on their intracellular accumulation. 5. Finally, the cell-associated vasointestinal peptide level was strikingly increased by gamma-interferon and, to a lesser extent, by retinoic acid, cytosine arabinoside, and 12-O-tetradecanoylphorbol 13-acetate. 6. It is concluded that the most relevant biochemical changes associated with LAN-5 cells differentiation involve the repertoire of neurotransmitters and neuropeptides. These events vary in quality and in quantity, likely due to the pattern complexity of gene expression triggered by each inducer in determining the diversity of neuronal phenotypes.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Neurotransmisores/metabolismo , Biomarcadores de Tumor/metabolismo , Catecolaminas/metabolismo , Diferenciación Celular/efectos de los fármacos , Citarabina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Factores de Crecimiento Nervioso/farmacología , Neuroblastoma/metabolismo , Neuropéptidos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
HIV-1 antigens generate in man both a humoral and cellular immune reaction. However, in ARC/AIDS patients, the cellular response is inhibited by HIV-1 which induces an antiproliferative (suppressive) effect on activated T cells. To overcome this inhibition and up-regulate the cellular response, we designed a new vaccine strategy directed both against HIV-1 and immunosuppression and we used an immunizing preparation composed of HIV-1 antigens combined with immunoregulatory peptides prepared in a biologically inactivated but immunogenic form. In mice, this preparation induced anti-HIV-1 antibodies and a cell-mediated cytotoxicity directed against H2 restricted cells carrying HIV-1 antigens.
Asunto(s)
VIH/inmunología , Terapia de Inmunosupresión/métodos , Vacunas Virales/inmunología , Animales , Combinación de Medicamentos , Femenino , Antígenos VIH/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Péptidos/administración & dosificaciónRESUMEN
The effects of 5-hydroxytryptamine (5-HT) and of a number of 5-HT receptor agonists and antagonists on the release of endogenous aspartate were investigated in rat cerebellum slices and synaptosomes depolarized with high K+. The release of endogenous aspartate evoked from slices by 35 mmol/l KCl and from synaptosomes by 15 mmol/l KCl was strongly (about 90%) calcium-dependent. In slices the release of aspartate was inhibited by exogenous 5-HT (0.1-100 nmol/l) in a concentration-dependent manner. The indoleamine was very potent, producing 30% inhibition at 0.1 nmol/l. The effect of 10 nmol/l 5-HT was partly but maximally counteracted by ketanserin (300-1000 nmol/l), a 5-HT2 receptor antagonist, but fully blocked by 300 nmol/l of the mixed 5-HT1/5-HT2 receptor antagonist methiothepin. The 5-HT1A receptor agonist 5-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) inhibited the K(+)-evoked release of endogenous aspartate in a concentration-dependent manner. The effect of 8-OH-DPAT was antagonized by methiothepin, but not by ketanserin which fully antagonized the inhibition produced by DOI. In cerebellar synaptosomes the release of endogenous aspartate evoked by 15 mmol/l K+ was inhibited by exogenous 5-HT and by 8-OH-DPAT, but not by DOI. Methiothepin (100-300 nmol/l) antagonized the inhibitory effects of 100 nmol/l 5-HT or 8-OH-DPAT. However, 1000 nmol/l of various 5-HT receptor antagonists [ketanserin, methysergide, (--)-propranolol, spiperone or ICS 205-930] did not counteract the effect of 100 nmol/l 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Ácido Aspártico/metabolismo , Cerebelo/metabolismo , Receptores de Serotonina/fisiología , Sinaptosomas/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin , Anfetaminas/farmacología , Animales , Calcio/fisiología , Cerebelo/anatomía & histología , Ketanserina/farmacología , Masculino , Metiotepina/farmacología , Potasio/fisiología , Ratas , Ratas Endogámicas , Receptores de Serotonina/clasificación , Serotonina/farmacología , Antagonistas de la Serotonina/farmacología , Tetrahidronaftalenos/farmacologíaRESUMEN
Slices and synaptosomes from human cerebral cortex (which had to be removed to reach deeply located tumours) and, for comparison, synaptosomes from guinea-pig and rat cerebral cortex were preincubated with [3H]5-hydroxytryptamine and superfused with physiological salt solution containing an inhibitor of 5-hydroxytryptamine uptake. The effects of alpha-adrenoceptor agonists and antagonists on the electrically (slices) or potassium-evoked (synaptosomes) tritium overflow were studied. In human cerebral cortical slices, the electrically-evoked [3H] overflow was inhibited by noradrenaline (pIC25 value: 6.35); the non-selective alpha-adrenoceptor antagonist phentolamine, at a concentration of 0.32 mumol/l, strongly antagonized the inhibitory effect of noradrenaline (apparent pA2 value: 8.19) but did not affect the evoked overflow by itself. In synaptosomes from humans, guinea-pigs and rats, noradrenaline also inhibited the K(+)-evoked [3H] overflow in a concentration dependent manner; the alpha 2-adrenoceptor clonidine (1 mumol/l), but not the alpha 1-adrenoceptor agonist methoxamine (1 mumol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 mumol/l) was abolished by the alpha 2-adrenoceptor antagonist idazoxan (0.5 mumol/l), but not by the alpha 1-adrenoceptor antagonist prazosin (1 mumol/l). It is concluded that release-inhibiting adrenoceptors of the alpha 2-subtype exist on 5-hydroxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.