RESUMEN
We have developed a murine model to study the involvement of dendritic cells (DC) in human immunodeficiency virus (HIV) routing from an inoculation site to the lymph nodes (LN). Murine bone marrow-derived DC migrate to the draining LN within 24 h after subcutaneous injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the virus recovered in the draining LN was still able to productively infect human T cells. After a vaginal challenge with Hi HIV-1, the virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be detected in peripheral LN as soon as 30 min after injection. The specific depletion of a myeloid-related LN DC population, previously shown to take up blood macromolecules and to translocate them into the LN, prevented HIV transport to LN. Together, our data demonstrate the critical role of DC for HIV routing to LN after either a vaginal or an intravenous challenge, which does not require their infection. Therefore, despite the fact that the mouse is not infectable by HIV, this small animal model might be useful to test preventive strategies against HIV.
Asunto(s)
Células Dendríticas/inmunología , VIH-1/patogenicidad , Ganglios Linfáticos/virología , Vagina , Animales , Secuencia de Bases , Línea Celular , Técnicas de Cocultivo , Cartilla de ADN , Femenino , VIH-1/inmunología , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos , Ratones Transgénicos , Modelos BiológicosRESUMEN
Many antiviral drugs must be metabolized to their active form by cellular enzymes. Their antiviral activity may therefore be limited by an inefficient metabolism, leading to low intracellular concentration of the active form or to the accumulation of toxic intermediate metabolites. Gene transfer might be used to overcome such limitations by transducing a gene able to increase intracellular drug metabolism. To prove such a concept, we chose the well-studied paradigm of zidovudine (AZT) metabolism and anti-HIV activity. AZT-triphosphate is the active form of AZT, acting through inhibition of HIV reverse transcription. In human cells, the rate-limiting step for AZT phosphorylation is catalyzed by the thymidylate kinase. We thus tested the capacity of herpes simplex virus type 1 thymidine kinase, which possesses a thymidylate kinase activity, to improve AZT metabolism and antiviral activity. Our results show enhanced AZT phosphorylation in HSV-1 TK-expressing lymphoid and monoblastoid cells, which correlated with significantly improved antiviral activity against different strains of HIV-1. The antiviral activity of Foscarnet, another reverse transcriptase inhibitor that does not require phosphorylation, remained unchanged. These results suggest that gene transfer might be envisioned for genetic pharmacomodulation of antiviral drugs.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/metabolismo , Herpesvirus Humano 1/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Zidovudina/administración & dosificación , Zidovudina/metabolismo , Animales , Células Cultivadas , Foscarnet/administración & dosificación , Humanos , Linfocitos/metabolismo , Linfocitos/virología , Ratones , Monocitos/metabolismo , Monocitos/virología , Fosforilación , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaAsunto(s)
Células Dendríticas/virología , VIH-1/patogenicidad , Ganglios Linfáticos/virología , Animales , Células de la Médula Ósea/fisiología , Células de la Médula Ósea/virología , Movimiento Celular , Células Dendríticas/fisiología , Infecciones por VIH/etiología , Infecciones por VIH/virología , Humanos , Técnicas In Vitro , Ratones , Ratones Endogámicos DBA , Ratones TransgénicosAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/genética , Terapia Genética , Neoplasias/genética , Nucleósidos/metabolismo , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Síndrome de Inmunodeficiencia Adquirida/terapia , Regulación de la Expresión Génica , Genes Virales , Humanos , Neoplasias/metabolismo , Neoplasias/terapia , Nucleósidos/uso terapéutico , Timidina Quinasa/genética , Proteínas Virales/genéticaRESUMEN
The human colon epithelial line HT29 represents a semipermisive cellular system for human immunodeficiency virus type 1 (HIV-1). It could be productively infected with HIV-1 NDK, a Zairian virus isolate highly cytopathic for CD4 positive lymphocytes, whereas infection with the prototype virus HIV-1 LAV was nonproductive. Recombinant viruses derived from HIV-1 LAV and HIV-1 NDK were used to determine the genetic control, step of virus/cell cycle, and molecular mechanism responsible for productive versus nonproductive infection of intestinal cells. Both parental viruses and all recombinants retrotranscribed their genomes with a similar kinetics and were able to complete HIV-1 DNA synthesis, HIV-1 LAV provirus present in preintegration complexes could be rescued by cocultivation with T-lymphocytes. However, it was aborted during prolonged cultivation of HT29 cells. Our results suggest that (i) gag/pol region of HIV-1 genome (fragment BssHII255-EcoRI4183) genetically controlled productive infection of intestinal cells and that (ii) the difference between productive and abortive infection occurred before synthesis of HIV-1 mRNA, at the integration level.
Asunto(s)
Colon/virología , Genes gag/genética , Genes pol/genética , VIH-1/fisiología , Replicación Viral/genética , Línea Celular , Colon/citología , ADN Viral/biosíntesis , Productos del Gen gag/genética , Genoma Viral , Transcriptasa Inversa del VIH , VIH-1/genética , VIH-1/patogenicidad , Humanos , Cinética , Regiones Promotoras Genéticas/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Ribonucleasa H/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samples requires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nested polymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysates rather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase capture and counting of the final amplified products allowed us to define precise interpretive criteria to determine the positivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tat regulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individuals demonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of the seropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulness of this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions of blood lysates from 26 HIV-1-seropositive patients.
Asunto(s)
ADN Viral/genética , VIH-1/genética , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Linfocitos T CD4-Positivos/microbiología , ADN Viral/sangre , Estudios de Evaluación como Asunto , Genes gag , Seropositividad para VIH/diagnóstico , Seropositividad para VIH/microbiología , VIH-1/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Sensibilidad y EspecificidadRESUMEN
The HIV1-PAR strain, isolated from the cerebrospinal fluid of an HIV1-seropositive man suffering from encephalopathy, replicated well in cord blood lymphocytes, poorly in peripheral blood mononuclear cells, and to different levels in blood-derived macrophage (BDM) cultures prepared from different blood donors. In marked contrast to its replication in primocultures, it did not grow in CEM and U937 cell lines. HIV1-PAR production in BDM was inhibited by more than 90% after treatment with OKT4A or 13B8.2 monoclonal antibodies (mAb) binding to adjacent epitopes of the D1 domain of the CD4 molecules. A lower but significant inhibitory effect was observed after BDM treatment with BL4 and OKT4 mAb, directed to the D2 and D3 domain of the CD4 molecule, respectively. The entire HIV1-PAR envelope glycoprotein gene was amplified by polymerase chain reaction and sequenced. The deduced amino acid sequence of HIV1-PAR gp160 revealed the presence of 847 amino acids and 86% homology with the HIV1 LAV virus prototype. An alignment of the amino acid sequence of the envelope glycoprotein of HIV1-PAR and HIV1-LAV showed that the differences were mostly clustered within the five variable regions. Five CD4-binding domains, the gp120/gp41 cleavage site, the putative gp41 fusion domain and 21 out of the 22 cysteine residues were conserved in both isolates. The results further confirm the macrophage-tropic character of the HIV1-PAR virus.
Asunto(s)
Antivirales/fisiología , Antígenos CD4/fisiología , Productos del Gen env/química , VIH-1/fisiología , Leucocitos Mononucleares/microbiología , Precursores de Proteínas/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos CD4/inmunología , Línea Celular , Proteína gp120 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH , Seropositividad para VIH/líquido cefalorraquídeo , VIH-1/química , VIH-1/clasificación , VIH-1/aislamiento & purificación , Humanos , Masculino , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Replicación ViralRESUMEN
The processing of the human immunodeficiency virus (HIV) gag and gag-pol precursor proteins by the virus-encoded protease is an essential step in maturation of infectious virus particles. Like most retroviral proteases, the HIV protease belongs to the aspartyl-protease family and can be inhibited by specific inhibitors. Twenty-four synthetic peptides known to be inhibitors of human renin were tested for inhibition of HIV replication in tissue cultures. One of them, a synthetic peptide analogue, SR41476, which has been shown to be a specific inhibitor of purified recombinant HIV1 protease in vitro, totally blocked infection with different isolates including the HIV1 LAV prototype, the highly cytopathic Zairian isolate HIV1 NDK, and HIV2 ROD, both in primary blood lymphocytes (PBL) and in the lymphoid cell lines MT4 and CEM, for at least 3 weeks. It also significantly reduced virus replication in chronically infected CEM cells, without any effect on cell proliferation. Radioimmunoprecipitation assay revealed that the inhibitor blocked processing of polyprotein precursors p55 gag and p40 gag into a mature form of gag proteins, p25 and p18. Synthetic peptide analogue SR 41476, when added before infection, efficiently inhibited formation of HIV DNA provirus and successfully suppressed synthesis of HIV-specific proteins. These results imply that the HIV protease inhibitor not only inhibited virus maturation in the late phase of the HIV replication cycle, but also interfered in the early phase, before the provirus was formed. This mechanism of antiviral activity provides new possibilities and strategies for AIDS chemotherapy.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , VIH-1/efectos de los fármacos , Péptidos/farmacología , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Células Cultivadas , ADN Viral/biosíntesis , Productos del Gen gag/metabolismo , VIH-1/fisiología , Humanos , Péptidos y Proteínas de Señalización Intercelular , Linfocitos/microbiología , Datos de Secuencia Molecular , Proteínas Virales/biosíntesis , Replicación Viral/efectos de los fármacosRESUMEN
The possible early role of endogenous glucocorticosteroids on the further ultra-structural development of the fetal rat lung epithelium was investigated in vitro using a potent antiglucocorticoid drug, RU 486. Lung primordia were explanted on day 13 of gestation and cultured for 4-6 days in the presence of fetal calf serum, with or without RU 486 in excess. The results obtained show that osmiophilic lamellar bodies specific for morphologically differentiated type-II cells did appear in a number of epithelial cells, even though the glucocorticosteroids possibly present in the culture medium, or transferred at explantation were antagonized by RU 486. The number of lamellar bodies stored in these pneumocytes was not different from that in controls. In contrast, their total surface area per cell profile was transiently decreased. Taken together the reported data strongly suggest that endogenous glucocorticosteroids are not necessary for the initiation of type-II cell differentiation.
Asunto(s)
Desarrollo Embrionario y Fetal/efectos de los fármacos , Glucocorticoides/antagonistas & inhibidores , Pulmón/embriología , Mifepristona/farmacología , Animales , Bovinos/sangre , Bovinos/embriología , Diferenciación Celular/efectos de los fármacos , Técnicas de Cultivo , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/embriología , Pulmón/citología , Pulmón/efectos de los fármacos , Microscopía Electrónica , Ratas , Ratas EndogámicasRESUMEN
The role of endogenous glucocorticoids in the control of surfactant system maturation was investigated in the fetal rat using an antiglucocorticoid molecule synthesized by Roussel-UCLAF, RU 486. The drug was administered to the mother from day 16 of gestation on. In a preliminary step, the transplacental transfer of RU 486 and its antiglucocorticoid effects on fetal target tissues were verified by evidencing RU 486-receptor complexes in fetal liver and lung, by measuring liver glycogen content, and by evaluating fetal blood corticosterone. The maturational state of fetal lungs was assessed biochemically on days 19, 20, and 21 of gestation by measuring their glycogen content, the phospholipid content of whole lung tissue and isolated surfactant fraction, and the incorporation of [methyl-3H]choline into DSPC. Morphological development was studied by analyzing electron micrographs of type II cells. The measured parameters clearly indicated a slowing of maturational processes in lungs of fetuses from RU 486-treated mothers, thereby demonstrating that endogenous glucocorticoids are actually involved in the control of lung maturation. In addition, the obtained results showed that endogenous corticosteroids specifically acted on the surfactant system of the fetal lung.