RESUMEN

The safety and tolerance of interferon alfa-n3 (IFN-alpha n3) was tested in 20 adults with asymptomatic human immunodeficiency virus type 1 (HIV-1) infection (> 400 CD4 lymphocytes/mm3). IFN-alpha n3 was self-injected three times per week for 3-6 months: 5 patients received 1 mega-IU (MIU)/dose, 10 received 5 MIU/dose, and 5 escalated to their maximum tolerated dose. Subjects were evaluated every 2-4 weeks through 2 months after cessation of treatment. Neuropsychological tests were given at 3-month intervals. Markers of IFN activity, anti-IFN neutralizing antibodies, and antiviral response were measured monthly. IFN-alpha n3 was safe and well tolerated: influenza-like symptoms were uncommon, laboratory toxicity was minimal, no adverse neurobehavioral side effects were evident, and no patient developed neutralizing antibodies against IFN. IFN-alpha n3 induced IFN-specific biologic responses and dose-related antiviral activity against HIV-1. Subjects showed stabilization of CD4 cells for > 20 months. IFN-alpha n3 should be studied in combination with other antiretroviral agents and in persons with more advanced HIV-1 infection.

Asunto(s)

Antivirales/uso terapéutico , Proteínas de Unión al GTP , Infecciones por VIH/terapia , VIH-1 , Interferón-alfa/uso terapéutico , Adulto , Antivirales/administración & dosificación , Antivirales/efectos adversos , Antivirales/análisis , Recuento de Linfocito CD4 , Eritrocitos/inmunología , Femenino , Antígenos de Histocompatibilidad Clase I/análisis , Humanos , Interferón-alfa/administración & dosificación , Interferón-alfa/efectos adversos , Leucocitos/inmunología , Masculino , Persona de Mediana Edad , Proteínas de Resistencia a Mixovirus , Pruebas Neuropsicológicas , Proteínas/análisis , ARN Viral/sangre

RESUMEN

IFN-alpha is an antiviral cytokine detected in plasma of HIV-1-infected patients during acute viremia and during late-stage disease. Monocytes produced IFN-alpha in response to HIV-1:1) IFN-alpha was produced predominantly by adherent cells; 2) depleting CD14+ cells nearly abolished HIV-1-induced IFN-alpha production; and 3) intracytoplasmic IFN-alpha was detected in CD14+ cells. During cell culture, monocytes differentiated into macrophages and lost their ability to produce IFN-alpha when challenged with HIV-1. These cells remained capable of producing IFN-alpha in response to other stimuli such as poly(I:C), a synthetic dsRNA. Thus, we examined two negative-stranded RNA viruses that have dsRNA intermediates, Newcastle disease virus and Sendai virus, and a DNA virus, herpes simplex virus type I (HSV-1). Macrophages lost their ability to produce IFN-alpha in response to HSV-1, but not to Sendai virus or to Newcastle disease virus. Thus, HIV-1 and other viruses were capable of inducing IFN-alpha through a mechanism that was independent of dsRNA. In conclusion, these data suggest that there are dsRNA-dependent and -independent mechanisms for the induction of IFN-alpha production, and that as monocytes differentiate into macrophages, they selectively lose their ability to produce IFN-alpha through the dsRNA-independent mechanism.

Asunto(s)

VIH-1/inmunología , Interferón-alfa/biosíntesis , Macrófagos/inmunología , Monocitos/inmunología , Diferenciación Celular/inmunología , Células Cultivadas , Herpesvirus Humano 1/inmunología , Humanos , Técnicas In Vitro , Macrófagos/citología , Monocitos/citología , Virus de la Enfermedad de Newcastle/inmunología , Virus de la Parainfluenza 1 Humana/inmunología , Poli I-C/inmunología , ARN Bicatenario/inmunología , ARN Viral/inmunología

RESUMEN

HIV-infected monocytes form highly invasive network on basement membrane matrix and secrete high levels of 92-kd metalloproteinase (MMP-9), an enzyme that degrades basement membrane proteins. In the present study, using matrigel as a model basement membrane system, we demonstrate that treatment of human immunodeficiency virus (HIV)-infected monocytes with interferon-gamma at 50 U/ml inhibited the ability of infected monocytes to form an invasive network on matrigel and their invasion through the matrigel matrix. These effects were associated with a significant reduction in the levels of MMP-9 produced by HIV-infected monocytes treated with interferon-gamma 1 day prior to infection with HIV as compared with that of untreated HIV-infected monocytes. Monocytes treated with interferon-gamma 1 day after HIV infection showed the presence of integrated HIV sequences; however, the levels of MMP-9 were substantially lower than those produced by monocytes inoculated with live HIV, heat-inactivated HIV, or even the control uninfected monocytes. Exposure of monocytes to heat-inactivated HIV did not result in increased invasiveness or high MMP-9 production, suggesting that regulation of metalloproteinase by monocytes was independent of CD4-gp120 interactions and required active virus infection. Furthermore, addition of interferon-gamma to monocytes on day 10 after infection inhibited MMP-9 production by more than threefold with no significant reduction of virus replication. These results indicate that the mechanism of interferon-gamma-induced down-regulation of MMP-9 levels and reduced monocyte invasiveness may be mediated by a mechanism independent of antiviral activity of IFN-gamma in monocytes. Down-regulation of MMP-9 in HIV-infected monocytes by interferon-gamma may play an important role in the control of HIV pathogenesis.

Asunto(s)

Antivirales/farmacología , VIH/efectos de los fármacos , Interferón gamma/farmacología , Monocitos/efectos de los fármacos , Membrana Basal/patología , Células Cultivadas , Colagenasas/biosíntesis , VIH/patogenicidad , Humanos , Metaloproteinasa 9 de la Matriz , Monocitos/patología , Monocitos/virología

RESUMEN

Murine macrophages supported exponential intracellular growth of Francisella tularensis LVS in vitro with a doubling time of 4 to 6 h. LVS was internalized and remained in a vacuolar compartment throughout its growth cycle. The importance of endosome acidification to intracellular growth of this bacterium was assessed by treatment of LVS-infected macrophages with several different lysosomotropic agents (chloroquine, NH4Cl, and ouabain). Regardless of the agent used or its mechanism of action, macrophages treated with agents that blocked endosome acidification no longer supported replication of LVS. Over several experiments for each lysosomotropic agent, the number of CFU of LVS recovered from treated macrophage cultures was equivalent to the input inoculum (approximately 10(4) CFU) at 72 h. In contrast, over 10(8) CFU was consistently recovered from untreated cultures. Pretreatment of macrophages with these endosome acidification inhibitors did not alter their ingestion of bacteria. Further, the effects of the inhibitors were completely reversible: inhibitor-pretreated LVS-infected macrophages washed free of the agent and cultured in medium fully supported LVS growth over 72 h. Endosome acidification is an important cellular event essential for release of iron from transferrin. The growth-inhibitory effects of both chloroquine and NH4Cl were completely reversed by addition of ferric PPi, a transferrin-independent iron source, at a neutral pH but not by addition of excess holotransferrin. Thus, intracellular localization in an acidic vesicle which facilitates the availability of iron essential for Francisella growth is a survival tactic of this bacterium, and iron depletion is one mechanism that macrophages use to inhibit its growth.

Asunto(s)

Francisella tularensis/crecimiento & desarrollo , Hierro/metabolismo , Macrófagos Peritoneales/microbiología , Cloruro de Amonio/farmacología , Animales , Compartimento Celular , Deferoxamina/farmacología , Endosomas/microbiología , Endosomas/fisiología , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica

RESUMEN

We have previously shown that the treatment of monocytes with interferon-gamma (IFN-gamma) prior to exposure with human immunodeficiency virus type-1 (HIV) results in complete inhibition of HIV infection of monocytes. In the present report, we have extended this study to obtain information on the mechanism(s) underlying IFN-gamma-induced inhibition of HIV infection of monocytes. To examine the effect of IFN-gamma on HIV entry, the first event in the infectious cycle of the virus, we amplified HIV-gag sequences in the genomic DNA and RNA of IFN-gamma treated monocytes, and found no evidence for the presence of either proviral DNA or HIV RNA sequences. These results were consistent with the absence of intracellular HIV particles either in the latent or actively replicating state as determined by flow-cytometric analysis of these cells. Furthermore, no HIV-induced cytopathic effects, such as multinucleated giant cell formation or cell death, were observed in IFN-gamma-treated monocytes after their exposure to HIV. Stimulation of IFN-gamma-treated monocytes 6 days postinfection with tumor necrosis factor-alpha (TNF-alpha), which is known to augment HIV replication in the infected cells, did not result in the induction of the HIV indicating the absence of latent HIV infection in IFN-gamma-treated monocytes. Treatment of monocytes with IFN-gamma, TNF-alpha, or with a combination of the two agents which is known to induce antimicrobial free radical nitric oxide (NO2- in the murine system did not induce NO2- production human monocytes suggesting the antiviral activity of IFN-gamma to be independent of NO2(-)-mediated killing of HIV or HIV-infected monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Antígenos CD4/biosíntesis , Regulación hacia Abajo , VIH-1/patogenicidad , Interferón gamma/farmacología , Monocitos/virología , Células Cultivadas , Efecto Citopatogénico Viral , Proteína p24 del Núcleo del VIH/biosíntesis , VIH-1/fisiología , Humanos , Cinética , Monocitos/inmunología , Óxido Nítrico/metabolismo , Reacción en Cadena de la Polimerasa , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Replicación Viral

RESUMEN

This report presents the results of studies using long-term bone marrow cultures (LTBMC) of human bone marrow cells to investigate the effect of HIV-1 on in vitro hematopoiesis. Confluent stromal cell layers established from human bone marrow cells were irradiated to eliminate residual hematopoietic progenitor cells and exposed to HIV-1ADA or to HIV-1IIIB, monocytotropic and lymphocytotropic strains of HIV-1, respectively. A productive infection did not develop in cultures exposed to HIV-1IIIB but did for cultures exposed to HIV-1ADA as there was a progressive increase in HIV-1 p24 antigen. Stromal cell layers infected with HIV-1ADA were also cocultured with autologous CD34+ bone marrow cells. Four days, 1, 2, and 3 weeks later, the number of colony-forming units granulocyte/macrophage (CFU-GM) in non- and HIV-infected LTBMC was determined. The number of CFU-GM increased during the first week in both non- and HIV-infected LTBMC. One week after the coculture of CD34+ cells with stromal cell layers infected with HIV-1ADA, the number of CFU-GM in six out of eight experiments was reduced compared to noninfected control LTBMC. In those six experiments, the number of CFU-GM was 53 +/- 6% standard error of the mean (SEM) of the number in noninfected LTBMC. A reduced number of CFU-GM was observed in the nonadherent fraction of HIV-infected LTBMC for at least 2 weeks. These results demonstrate that some cells in the stromal cell layers of LTBMC were targets for HIV-1 and that HIV-infected stromal cell layers suppressed or delayed the production of CFU-GM.

Asunto(s)

Células de la Médula Ósea , Infecciones por VIH/fisiopatología , Hematopoyesis , Células Madre Hematopoyéticas/citología , Antígenos CD/análisis , Antígenos CD34 , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Sustancias de Crecimiento/farmacología , Humanos , Técnicas In Vitro , Factores de Tiempo

RESUMEN

Monocytes treated with 500 IU/ml human recombinant interferon-gamma (rIFN-gamma) 1 day before and continuously after human immunodeficiency virus (HIV) infection showed no evidence of virus replication 7 days after addition of the viral inoculum. There was no HIV-associated cytopathic effect, no reverse transcriptase (RT) activity or p24 detected in culture fluids, and no HIV RNA or DNA in cell lysates. Furthermore, no evidence of HIV infection was evident in replicate cultures in which all IFN-gamma was removed at 7 days and the cells were cultured for an additional 3 weeks without IFN-gamma. The 50% inhibitory dose for reduction of maximum RT activity in HIV-infected monocyte cultures was about 1 IU/ml IFN-gamma. No increase in HIV replication was evident in monocytes treated with IFN-gamma at any concentration (0 to 5000 IU/ml) or at any time (7 days before to 10 days after HIV infection). In side-by-side experiments with identical monocytes and HIV-1 stock, rIFN-gamma was 10 to 20 times more effective than rIFN-alpha 2b for induction of antiviral activity. With both interferons, significant antiviral activity was evident with monocytes treated 1 day before, at the time of, or up to 3 days after infection. At 7 to 10 days after infection (a time at which less than 20% of total cells were infected with HIV) addition of even high concentrations of IFN-alpha or IFN-gamma had no effect on virus replication. These data suggest that the principal action of IFN-alpha and IFN-gamma was directed against the fluid-phase virus. Cell-cell spread of infection within the HIV-infected monocyte culture and extent of virus replication in HIV-infected cells were not affected by interferon treatment.

Asunto(s)

VIH-1/genética , VIH-1/aislamiento & purificación , Interferón gamma/farmacología , Monocitos/microbiología , Síndrome de Inmunodeficiencia Adquirida/patología , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Síndrome de Inmunodeficiencia Adquirida/prevención & control , Secuencia de Bases , Southern Blotting , Células Cultivadas , ADN Viral/genética , Humanos , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/efectos de los fármacos , Reacción en Cadena de la Polimerasa , ARN Viral/genética , Replicación Viral/efectos de los fármacos

RESUMEN

Alveolar macrophages (AMs) were analyzed for ability to support replication of the intracellular bacterium Francisella tularensis live vaccine strain (LVS). AM supported in vitro growth (2 to 3 logs over 5 days) of LVS with a doubling time of 8 +/- 0.8 h. AMs were analyzed for responsiveness to rIFN-gamma for destruction of this lung pathogen. AM treated with 50 U/ml rIFN-gamma allowed early growth of bacteria (six doublings over 48 h) but between 48 and 96 h rIFN-gamma-treated AM eliminated 1.5 logs of LVS. AMs were sensitive to effects of rIFN-gamma; as little as 5 U/ml rIFN-gamma stimulated AM antimicrobial activity, with half-maximal activity 0.3 U/ml. rIFN-gamma-induced antimicrobial effects in AM correlated with amount of nitrites produced, but nitric oxide played only a minimal role in antibacterial effects induced in AM, because NG-MMLA (specific inhibitor of L-arginine-dependent nitric oxide production) failed to block antimicrobial activity of IFN-gamma-stimulated AM. IL-10, TGF-beta 1, and IFN-alpha (cytokines known to regulate effector functions of activated macrophages) also did not block anti-F. tularensis activity of IFN-gamma-stimulated AM. Reactive oxygen metabolites, depletion of tryptophan, and sequestration of iron did not contribute to anti-F. tularensis activity because addition of superoxide dismutase or catalase, excess iron, or tryptophan to IFN-gamma-treated AM did not reverse the anti-F. tularensis activity observed in these cells. Regulation of AM effector activity differed from that of other macrophage populations, in that while rIFN-gamma-stimulated AM produced TNF-alpha (100 U/ml at 72 h), TNF-alpha was not required as a costimulator for induction of antimicrobial activities by rIFN-gamma because anti-TNF-alpha treatment of rIFN-gamma-stimulated AM blocked TNF-alpha but had no effect on either production of nitrites or anti-F. tularensis activity.

Asunto(s)

Francisella tularensis/inmunología , Macrófagos Alveolares/inmunología , Óxido Nítrico/fisiología , Tularemia/inmunología , Animales , Citotoxicidad Inmunológica , Interferón gamma/farmacología , Interleucina-1/fisiología , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Recombinantes , Factor de Necrosis Tumoral alfa/fisiología

RESUMEN

Cellular mechanisms that control susceptibility to opportunistic infection in human immunodeficiency virus (HIV)-infected individuals remain poorly understood. HIV may induce certain cellular genes that restrict HIV replication and protect cells against other superinfecting viral pathogens. Indeed, HIV-infected monocytes resist infection by vesicular stomatitis virus (VSV). HIV-induced VSV interference in monocytes increases with time after HIV infection. Such interference was evident 6 h after HIV infection and reached maximal levels at 14 days. Monocytotropic but not T cell-tropic HIV strains elicited these effects, signaling a requirement for viral entry and/or replication. Viral interference was independent of interferon (IFN) and was unaffected by addition of neutralizing IFN-alpha and -beta antibodies. The well-described IFN-alpha-inducible antiviral pathways were examined to determine their relationship to the cellular mechanism(s) underlying VSV interference. HIV and IFN-alpha both induced the expression of 2-5A synthetase and Mx gene. In contrast, the guanylate-binding protein (GBP), 6-16, and 9-27 cellular genes were up-regulated by IFN-alpha but not HIV. MxA was detected in HIV-infected monocytes but not in uninfected monocytes. The association between Mx expression and resistance to VSV, coupled with previously described anti-VSV activities by human MxA, suggested that Mx may be an effector molecule for the HIV-induced anti-VSV activities. These results, taken together, suggest that HIV can induce antiviral cellular gene expression, independent of IFN.

Asunto(s)

Proteínas de Unión al GTP , Regulación de la Expresión Génica/efectos de los fármacos , Infecciones por VIH/fisiopatología , VIH/aislamiento & purificación , Interferón-alfa/farmacología , Monocitos/citología , Monocitos/microbiología , 2',5'-Oligoadenilato Sintetasa/análisis , Antivirales/análisis , Antivirales/genética , Antivirales/fisiología , Secuencia de Bases , Northern Blotting , Células Cultivadas , Cicloheximida/farmacología , ADN/análisis , ADN/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/fisiología , VIH/fisiología , Humanos , Interferones/metabolismo , Interferones/fisiología , Datos de Secuencia Molecular , Monocitos/enzimología , Proteínas de Resistencia a Mixovirus , Reacción en Cadena de la Polimerasa/métodos , Proteínas/análisis , Proteínas/genética , Proteínas/fisiología , ADN Polimerasa Dirigida por ARN , Regulación hacia Arriba , Virus de la Estomatitis Vesicular Indiana/fisiología

RESUMEN

Various lines of defense against infection are present in all living creatures. The balance between symbiosis and parasitism is determined by the mechanisms through which the host resists infection and by the extent of injury induced by the parasite: both factors contribute to disease. Lines of host defense can be arbitrarily divided into three components: 1) barrier functions of skin and mucous membranes and their innate physical and secretory antimicrobial components; 2) elements of host defense that do not necessarily require prior exposure to an infectious agent or immunologic memory (mast cells, granulocytes, macrophages, NK cells, gamma/delta T cells); and 3) immune responses directed against specific epitopes on the infectious agent induced by prior exposure and immunologic memory (alpha/beta T cells, B cells). Analysis of such host defense mechanisms repeatedly documents tremendous redundancy and overlap between these lines of defense. Further, there is open communication, so that a change at any one level ripples throughout the system. Acquired nonspecific resistance to infection is an example of such a ripple. Host response to one infection alerts the immune system, so that the general level of resistance to other infectious agents is increased. This response is initiated by an immune response (third line of defense) but effected by nonspecific elements (second line of defense). The survival value of such responses is obvious. There are numerous examples in both mouse and man of the operation of these systems in response to infection. Further, the menus of antimicrobial components available to both mouse and man for resistance to infection are very similar, but not identical. Indeed, it is said that the genetic basis for differences between mice and man revolve around a difference of less than 10% in DNA sequences. But there are differences! Mouse macrophages produce IFN-beta in response to infection, human cells produce IFN-alpha. Mouse macrophages effect antimicrobial activity principally through induction of NO synthase and the generation of toxic nitrogen oxides. This pathway has yet to be described with human macrophages. In both man and mouse, F. tularensis is an obligate intracellular parasite of macrophages that requires an essential component provided by the cell for its replication. That mouse and man are not so different is well illustrated by the effector mechanisms induced by IFN-gamma for antimicrobial activity against F. tularensis.(ABSTRACT TRUNCATED AT 400 WORDS)

Asunto(s)

Activación de Macrófagos/inmunología , Animales , Bacterias/inmunología , Citotoxicidad Inmunológica , Humanos , Interferón gamma/inmunología , Ratones , Modelos Biológicos , Óxido Nítrico/inmunología , Transducción de Señal/inmunología

Asunto(s)

Francisella tularensis/patogenicidad , Macrófagos/microbiología , Animales , Citocinas/inmunología , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Francisella tularensis/crecimiento & desarrollo , Francisella tularensis/inmunología , Humanos , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Tularemia/etiología

Asunto(s)

Síndrome de Inmunodeficiencia Adquirida/inmunología , Infecciones por VIH/inmunología , VIH/fisiología , Interferones/fisiología , Monocitos/fisiología , Replicación Viral , Síndrome de Inmunodeficiencia Adquirida/microbiología , Animales , Antivirales/toxicidad , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Células Cultivadas , Citocinas/fisiología , VIH/efectos de los fármacos , Infecciones por VIH/microbiología , Duplicado del Terminal Largo de VIH , VIH-1/inmunología , VIH-1/fisiología , Humanos , Interferón-alfa/fisiología , Interferón-alfa/toxicidad , Interferón gamma/fisiología , Monocitos/microbiología , FN-kappa B/metabolismo , Factor de Transcripción Sp1/metabolismo , Replicación Viral/efectos de los fármacos

RESUMEN

Natural IFN-alpha n3, a purified mixture of many different natural IFN alpha species, was 10- to 100-fold more effective than equal concentrations of human rIFN-alpha 2b or rIFN-alpha 2a for inhibition of HIV replication in primary human monocytes. This difference was highly reproducible in multiple side-by-side experiments using the identical HIV-1 inoculum and the same monocyte target cells: natural IFN-alpha n3 was more effective than rIFN-alpha 2b at lower concentrations for protection against a constant HIV-1 inoculum; cells treated with natural IFN-alpha n3 were protected against a greater HIV-1 challenge than were cells treated with the same concentration of rIFN-alpha 2b. Fractionation of natural IFN-alpha n3 by reversed-phase high-pressure liquid chromatography (RP-HPLC) showed that most antiviral activity for HIV localized to discrete and reproducible peaks. The RP-HPLC peak that contained purified natural IFN-alpha 2b was the least effective fraction. These data suggest heterogeneity among IFN-alpha species for antiviral activity against HIV and may provide a molecular basis for more effective IFN-alpha therapy.

Asunto(s)

VIH-1/efectos de los fármacos , Interferón-alfa/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , VIH-1/fisiología , Humanos , Interferón alfa-2 , Interferón-alfa/administración & dosificación , Interferón-alfa/aislamiento & purificación , Monocitos/efectos de los fármacos , Monocitos/microbiología , Proteínas Recombinantes , Replicación Viral/efectos de los fármacos

RESUMEN

IFN-alpha is in plasma of HIV-1 infected patients during early and late-stage disease and may play a role in control of virus replication. The stimulus for IFN-alpha production, the cells that produce this cytokine, and the effectiveness of this IFN-alpha response for control of virus infection are not yet defined. Culture fluids from freshly isolated PBMC of HIV-1 seronegative donors contained high levels of IFN-alpha after exposure to 100 to 1000 infectious HIV-1 particles per culture. Levels of IFN-alpha induced by HIV-1 were directly dependent on the number of monocytes in cell preparations: No IFN-alpha was detected from T cell-enriched PBMC. In monocyte cultures, induction of IFN-alpha by HIV-1 was relatively specific: Levels of IL-1 beta, IL-6, IFN-gamma, and TNF-alpha remained at baseline. Capacity of HIV-1 virions to induce IFN-alpha was not dependent on virus replication. IFN-alpha was induced by (a) heat-inactivated HIV-1, (b) virions from 8E5 cells, a cell line that releases noninfectious HIV-1, (c) HIV-1-infected cells fixed in paraformaldehyde, and (d) T cell-tropic HIV-1 that binds to but does not infect monocytes. Capacity of HIV-1 virions and HIV-1 infected cells to induce IFN-alpha was completely inhibited by soluble rCD4 or mAb against CD4 or gp120. Antibodies against CD4, however, did not induce monocytes to produce IFN-alpha. HIV-1-induced IFN-alpha production was inhibited by antibodies against both V3 loop determinants and the CD4 binding site of gp120. Further, sera and purified immunoglobulin from HIV-1 infected patients also inhibited HIV-1-induced IFN-alpha production. These observations suggest that potentially protective antiviral responses associated with IFN-alpha production in HIV-1 infected patients are inhibited by the development of antibodies against gp120.

Asunto(s)

Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/inmunología , VIH-1/inmunología , Interferón-alfa/biosíntesis , Monocitos/inmunología , Humanos , Técnicas In Vitro , Replicación Viral

RESUMEN

To investigate the expression of human immunodeficiency virus (HIV) genes in human monocytes, a DNA transfection system was developed and characterized using cultured primary monocytes. Monocytes that were cultured 6-7 days in an adherent monolayer were efficiently recovered and transfected by electroporation with an expression vector containing the Escherichia coli lacZ gene under control of the cytomegalovirus immediate-early promoter. Successful transfection was detected by expression of beta-galactosidase activity and by histochemical staining for beta-galactosidase in cells that were allowed to readhere to plastic following transfection. Over 30% of the surviving adherent monocytes expressed the transfected beta-galactosidase gene. In the same manner, monocytes were transfected with HIV provirus clones pIIIB and pIIB/PB. The provirus pIIIB/PB differs from pIIIB only in that it contains a small sequence from the env gene of a macrophage tropic HIV-1. Virus derived from pIIIB will not replicate in monocytes whereas virus derived from pIIIB/PB will. Monocytes transfected with either provirus DNA expressed high levels of p24 antigen within 1 day of transfection, and cell-free supernatants contained virus that was infectious for T cells. In contrast, only supernatants from pIIIB/PB transfections contained virus capable of infecting monocytes. Thus, proviral DNA of T cell tropic HIV efficiently completes the retroviral life cycle in monocytes in a manner indistinguishable from that of macrophage tropic HIV, and progeny virus retain their T cell tropism.

Asunto(s)

VIH-1/genética , Monocitos/microbiología , Provirus/genética , Transfección/métodos , ADN Viral , Regulación Viral de la Expresión Génica , VIH-1/crecimiento & desarrollo , Humanos , Provirus/crecimiento & desarrollo , beta-Galactosidasa/sangre

RESUMEN

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.

Asunto(s)

Arginina/metabolismo , Interferón gamma/fisiología , Mycobacterium bovis/inmunología , Óxido Nítrico/metabolismo , Tularemia/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Arginina/análogos & derivados , Arginina/farmacología , Interferón gamma/inmunología , Activación de Macrófagos , Masculino , Ratones , Ratones Endogámicos C3H , Organismos Libres de Patógenos Específicos , Tularemia/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , omega-N-Metilarginina

Asunto(s)

Infecciones por VIH/microbiología , VIH-1 , Enfermedades Pulmonares/microbiología , Macrófagos Alveolares/microbiología , Susceptibilidad a Enfermedades , Infecciones por VIH/fisiopatología , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Interferón-alfa/fisiología , Enfermedades Pulmonares/fisiopatología , Macrófagos Alveolares/fisiología , Monocitos/microbiología , Monocitos/fisiología , Fenotipo , Replicación Viral/fisiología

RESUMEN

An IgM monoclonal antibody (mAb) recognized surface antigens specific to Francisella tularensis wild-type (Schu4) and live vaccine strain (LVS), and reacted with both in ELISA and slide agglutination tests. This mAb also reacted with LVS microorganisms in tissues of infected mice as assessed by an indirect fluorescence technique. Western blot analysis showed the mAb to react with antigens associated with F. tularensis LPS.

Asunto(s)

Anticuerpos Monoclonales , Antígenos Bacterianos/inmunología , Francisella tularensis/inmunología , Inmunoterapia , Tularemia/terapia , Animales , Antígenos Bacterianos/análisis , Vacunas Bacterianas , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Francisella tularensis/aislamiento & purificación , Inmunoglobulina M/uso terapéutico , Hígado/microbiología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Tularemia/inmunología , Tularemia/patología

RESUMEN

Levels of human immunodeficiency virus (HIV) DNA, RNA, or p24 antigen and reverse transcriptase activity in T-cell cultures treated with 500 IU of recombinant alpha interferon (rIFN alpha) per ml were comparable to those in control cultures. Radioimmunoprecipitation analysis of proteins in lysates of IFN-treated T cells documented a marked accumulation of HIV proteins. Localization of gp120 by immunofluorescence showed a diffuse pattern in IFN-treated cells quite distinct from the ring pattern in untreated control cells. That large quantities of gp120 in aberrant cell compartments might affect HIV morphogenesis was confirmed in infectivity studies: virions from IFN-treated cells were 100- to 1,000-fold less infectious than an equal number of virions from control cells. Direct examination of IFN-treated and control HIV-infected cells by transmission electron microscopy showed little difference in the number or distribution of viral particles. However, quantitation of gp120 by immunogold particle analysis revealed a marked depletion of envelope glycoprotein in virions released from IFN-treated cells. This defect in gp120 assembly onto mature viral particles provides a molecular basis for this loss of infectivity.

Asunto(s)

Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/fisiología , Interferón Tipo I/farmacología , Linfocitos T/inmunología , Proteínas Virales/metabolismo , Virión/fisiología , Células Cultivadas , ADN Viral/análisis , ADN Viral/metabolismo , Proteína p24 del Núcleo del VIH/análisis , Proteína p24 del Núcleo del VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Transcriptasa Inversa del VIH , VIH-1/patogenicidad , Humanos , ARN Viral/análisis , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes , Linfocitos T/efectos de los fármacos , Linfocitos T/microbiología , Proteínas Virales/aislamiento & purificación , Virión/efectos de los fármacos , Virión/patogenicidad

RESUMEN

The hamster IgM mAb 5D3 is specific for an 73-kDa LPS receptor on murine leukocytes. This mAb inhibits binding of radiolabeled LPS to splenocytes and acts as an agonist for induction of LPS-mediated changes in macrophage function. Resident peritoneal macrophages treated with IFN-gamma and mAb 5D3 developed potent cytotoxic activity against tumor cells. Cells treated with IFN-gamma or mAb 5D3 alone were inactive. Macrophage cytotoxic activity induced by IFN-gamma and mAb 5D3 was inhibited by NGMMLA and coincident with high levels of NO2-released into culture fluids. These data show that mAb 5D3 serves as an effective trigger signal for induction of cytotoxic activity with IFN-gamma-primed macrophages. Indeed, mAb 5D3 exactly mimicked the effects of LPS in these same systems. Unlike LPS, effects of mAb 5D3 on induction of macrophage cytotoxic activity and production of nitrogen oxides was abrogated after boiling, and not affected by addition of polymyxin B. The effects of LPS and mAb 5D3 as a trigger signal for IFN-gamma-primed macrophages were associated with production of TNF activity in culture fluids and inhibited by mAb against rTNF-alpha. Expression of class II MHC on macrophages induced by IFN-gamma treatment was suppressed by both LPS and mAb 5D3. These suppressive effects of LPS and mAb 5D3 were not affected by NGMMLA or mAb against rTNF-alpha. Finally, macrophages treated with LPS or mAb 5D3 before exposure to IFN-gamma and LPS or mAb 5D3 did not develop cytotoxic activity or high levels of NO2- in the culture fluids. These same cells developed both effector activities after addition of rTNF-alpha. These results in toto identify the 73-kDa protein as a receptor that mediates LPS-induced changes in macrophage effector function. The mAb 5D3 serves as a specific and defined reagent agonist for analysis of LPS receptor-linked change.

Asunto(s)

Citotoxicidad Inmunológica , Interferón gamma/farmacología , Macrófagos/inmunología , Óxidos de Nitrógeno/toxicidad , Receptores Inmunológicos/fisiología , Animales , Arginina/análogos & derivados , Arginina/farmacología , Antígenos de Histocompatibilidad Clase II/metabolismo , Calor , Inmunidad Celular , Receptores de Lipopolisacáridos , Lipopolisacáridos/administración & dosificación , Ratones , Ratones Endogámicos C3H , Polimixina B/farmacología , Proteínas Recombinantes , omega-N-Metilarginina