RESUMO
To identify the mechanisms by which human immunodeficiency virus type 1 (HIV-1) might penetrate the epithelial barrier during sexual transmission to women and the mechanisms of vaccine-associated protection against entry, we characterized early epithelial responses to vaginal inoculation of simian immunodeficiency virus strain mac251 (SIVmac251) in naive or SIVmac239Δnef-vaccinated rhesus macaques. Vaginal inoculation induced an early stress response in the cervicovaginal epithelium, which was associated with impaired epithelial integrity, damaged barrier function, and virus and bacterial translocation. In vaccinated animals, early stress responses were suppressed, and the maintenance of epithelial barrier integrity correlated with prevention of virus entry. These vaccine-protective effects were associated with a previously described mucosal system for locally producing and concentrating trimeric gp41 antibodies at the mucosal interface and with formation of SIV-specific immune complexes that block the stress responses via binding to the epithelial receptor FCGR2B and subsequent inhibitory signaling. Thus, blocking virus entry may be one protective mechanism by which locally concentrated non-neutralizing Ab might prevent HIV sexual transmission to women.
Assuntos
Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Internalização do Vírus , Administração Intravaginal , Animais , Epitélio/fisiologia , Epitélio/virologia , Feminino , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Estresse Fisiológico , Vacinação , Vagina/fisiologia , Vagina/virologiaRESUMO
A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.
Assuntos
Doença de Alzheimer/genética , Encéfalo/metabolismo , Scrapie/genética , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Clonagem Molecular , Cricetinae , DNA/genética , Humanos , Camundongos , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Scrapie/patologia , OvinosRESUMO
In lymphoid tissue, where human immunodeficiency virus-type 1 (HIV-1) is produced and stored, three-drug treatment with viral protease and reverse transcriptase inhibitors markedly reduced viral burden. This was shown by in situ hybridization and computerized quantitative analysis of serial tonsil biopsies from previously untreated adults. The frequency of productive mononuclear cells (MNCs) initially diminished with a half-life of about 1 day. Surprisingly, the amount of HIV-1 RNA in virus trapped on follicular dendritic cells (FDCs) decreased almost as quickly. After 24 weeks, MNCs with very few copies of HIV-1 RNA per cell were still detectable, as was proviral DNA; however, the amount of FDC-associated virus decreased by >/=3.4 log units. Thus, 6 months of potent therapy controlled active replication and cleared >99.9 percent of virus from the secondary lymphoid tissue reservoir.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Células Dendríticas/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Tonsila Palatina/virologia , Adulto , Linfócitos T CD4-Positivos/virologia , DNA Viral/análise , Células Dendríticas/citologia , Quimioterapia Combinada , Infecções por HIV/virologia , Inibidores da Protease de HIV/uso terapêutico , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Hibridização In Situ , Cinética , Lamivudina/uso terapêutico , Leucócitos Mononucleares/citologia , Macrófagos/virologia , Provírus/genética , RNA Viral/análise , Inibidores da Transcriptase Reversa/uso terapêutico , Ritonavir/uso terapêutico , Carga Viral , Replicação Viral/efeitos dos fármacos , Zidovudina/uso terapêuticoRESUMO
In sexual transmission of simian immunodeficiency virus, and early and later stages of human immunodeficiency virus-type 1 (HIV-1) infection, both viruses were found to replicate predominantly in CD4(+) T cells at the portal of entry and in lymphoid tissues. Infection was propagated not only in activated and proliferating T cells but also, surprisingly, in resting T cells. The infected proliferating cells correspond to the short-lived population that produces the bulk of HIV-1. Most of the HIV-1-infected resting T cells persisted after antiretroviral therapy. Latently and chronically infected cells that may be derived from this population pose challenges to eradicating infection and developing an effective vaccine.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/transmissão , HIV-1/fisiologia , Ativação Linfocitária , Síndrome de Imunodeficiência Adquirida dos Símios/transmissão , Vírus da Imunodeficiência Símia/fisiologia , Animais , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Ciclo Celular , Colo do Útero/virologia , Células Epiteliais/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Linfonodos/virologia , Macaca mulatta , RNA Viral/análise , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Fatores de Tempo , Replicação ViralRESUMO
Cervicovaginal epithelium plays a critical role in determining the outcome of virus transmission in the female reproductive tract (FRT) by initiating or suppressing transmission-facilitating mucosal immune responses in naïve and SIVmac239Δnef-vaccinated animals, respectively. In this study, we examined the very early responses of cervical epithelium within 24 h after vaginal exposure to SIV in naive and SIVmac239Δnef-vaccinated rhesus macaques. Using both ex vivo and in vivo experimental systems, we found that vaginal exposure to SIV rapidly induces a broad spectrum of pro-inflammatory responses in the epithelium associated with a reciprocal regulation of NF-kB and glucocorticoid receptor (GR) signaling pathways. Conversely, maintenance of high-level GR expression and suppression of NF-kB expression in the epithelium were associated with an immunologically quiescent state in the FRT mucosa and protection against vaginal challenge in SIVmac239Δnef-vaccinated animals. We show that the immunologically quiescent state is induced by FCGR2B-immune complexes interactions that modify the reciprocal regulation of NF-kB and GR signaling pathways. Our results suggest that targeting the balance of NF-kB and GR signaling in early cervicovaginal epithelium responses could moderate mucosal inflammation and target cell availability after vaginal infection, thereby providing a complementary approach to current prevention strategies.
Assuntos
Vacinas contra a AIDS/imunologia , Colo do Útero/patologia , Células Epiteliais/fisiologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Inflamação/imunologia , NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Vagina/patologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/metabolismo , Ácido Aspártico Endopeptidases/genética , Transmissão de Doença Infecciosa , Células Epiteliais/virologia , Feminino , Imunidade nas Mucosas , Inflamação/virologia , Macaca mulatta , Vacinas contra a SAIDS/genética , Transdução de Sinais , VacinaçãoRESUMO
In the SIV (simian immunodeficiency virus)-rhesus macaque model of HIV-1 (human immunodeficiency virus type I) transmission to women, one hallmark of the mucosal response to exposure to high doses of SIV is CD4 T-cell recruitment that fuels local virus expansion in early infection. In this study, we systematically analyzed the cellular events and chemoattractant profiles in cervical tissues that precede CD4 T-cell recruitment. We show that vaginal exposure to the SIV inoculum rapidly induces chemokine expression in cervical epithelium including CCL3, CCL20, and CXCL8. The chemokine expression is associated with early recruitment of macrophages and plasmacytoid dendritic cells that are co-clustered underneath the cervical epithelium. Production of chemokines CCL3 and CXCL8 by these cells in turn generates a chemokine gradient that is spatially correlated with the recruitment of CD4 T cells. We further show that the protection of SIVmac239Δnef vaccination against vaginal challenge is correlated with the absence of this epithelium-innate immune cell-CD4 T-cell axis response in the cervical mucosa. Our results reveal a critical role for cervical epithelium in initiating early mucosal responses to vaginal infection, highlight an important role for macrophages in target cell recruitment, and provide further evidence of a paradoxical dampening effect of a protective vaccine on these early mucosal responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Epitélio/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Macrófagos/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vagina/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Movimento Celular , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Epitélio/virologia , Feminino , Humanos , Imunidade nas Mucosas , Interleucina-8/genética , Interleucina-8/metabolismo , Macaca mulatta , Macrófagos/virologia , VacinaçãoRESUMO
Lactate dehydrogenase-elevating virus (LDV) induces poliomyelitis in immunosuppressed C58 mice resulting in fatal paralysis. We have synthesized and cloned cDNA complementary to the LDV genome, and used the cDNA clones as in situ hybridization probes for the detection of LDV RNA in tissue sections. Direct fluorescent antibody staining using IgG from chronically infected mice was used for the detection of LDV antigens. Using these methods, we have detected LDV RNA and antigens in anterior horn neurons of paralyzed mice. The appearance of LDV RNA and antigen positive motor neurons and their location in the spinal cord correlated with the development of paralytic symptoms. No positive neurons were detected in LDV-infected, susceptible mice without signs of paralysis, but some glial cells of the white and gray matter in the spinal cords of these mice were found to contain LDV RNA. These analyses broaden the host cell range of LDV to include neuronal and other cells in the CNS and support the hypothesis of LDV replication in neurons as the cause of poliomyelitis and paralysis.
Assuntos
Antígenos Virais/análise , Vírus Elevador do Lactato Desidrogenase/isolamento & purificação , Neurônios Motores/microbiologia , Paralisia/etiologia , RNA Viral/análise , Viroses/microbiologia , Animais , DNA , DNA Viral , Eletroforese em Gel de Ágar , Imunofluorescência , Vírus Elevador do Lactato Desidrogenase/genética , Vírus Elevador do Lactato Desidrogenase/imunologia , Camundongos , Hibridização de Ácido Nucleico , Poliomielite/microbiologia , Medula Espinal/microbiologiaRESUMO
Unconventional agents and conventional viruses provide model systems to investigate the pathogenesis of Alzheimer's disease (AD). The essay which follows examines the hypothetical role of herpes simplex in AD and presents some generally applicable experimental approaches to detecting genes in brain tissues. The concluding section, on parallels between AD and diseases of the brain caused by unconventional viruses, defines strategies for isolating genes related to pathology.
Assuntos
Doença de Alzheimer/etiologia , Príons/fisiologia , Simplexvirus/fisiologia , Idoso , Doença de Alzheimer/genética , DNA Viral/análise , Regulação da Expressão Gênica , Humanos , Hibridização de Ácido Nucleico , Simplexvirus/genética , Fenômenos Fisiológicos ViraisAssuntos
Doença de Alzheimer/genética , Astrócitos/patologia , Proteínas do Tecido Nervoso/genética , Scrapie/genética , Doença de Alzheimer/patologia , Amiloide/metabolismo , Animais , Apolipoproteínas E/biossíntese , Apolipoproteínas E/genética , Astrócitos/metabolismo , Química Encefálica , Catepsina D/biossíntese , Catepsina D/genética , DNA/genética , DNA Recombinante/genética , Expressão Gênica , Biblioteca Gênica , Genes , Humanos , Proteínas do Tecido Nervoso/biossíntese , Hibridização de Ácido Nucleico , Proteínas PrPSc , Príons/biossíntese , Príons/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Scrapie/patologia , Ovinos/genéticaRESUMO
Tissue macrophages derive from monocytes of bone marrow origin. Because monocytes from patients with chronic myelogenous leukemia (CML) contain the Philadelphia chromosome (Ph), it seemed probable that tissue macrophages in CML would originate from the malignant clone. Using powerful molecular techniques, we studied pulmonary alveolar macrophages (PAM) from two patients with CML. PAM from Patient 1, a patient in chronic phase studied before bone marrow transplantation (BMT), contained the Ph by Southern blot analysis. Patient 2, an accelerated phase patient, was studied after post-BMT relapse. PAM from this patient not only contained the Ph, but also expressed the BCR/ABL message documented by a new splice junction in situ hybridization technique. This new technique allows detection of BCR/ABL mRNA and determination of splice useage in individual cells. These data confirm the continued replenishing of PAM from peripheral blood monocytes in non-BMT settings and represent the first direct evidence that tissue macrophages are derived from the malignant clone in patients with CML.
Assuntos
Medula Óssea/patologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Macrófagos Alveolares/patologia , Splicing de RNA , RNA Mensageiro/genética , Adulto , Sequência de Bases , Southern Blotting , Transplante de Medula Óssea , DNA de Neoplasias/genética , Humanos , Hibridização In Situ , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/cirurgia , Pulmão/patologia , Macrófagos Alveolares/fisiologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/metabolismoRESUMO
In slow infections caused by scrapie and other unconventional agents, and in Alzheimer's disease (AD), the formation of neuritic plaques and the increase in astrocytes and astrocyte-specific protein, glial fibrillary acidic protein (GFAP), are pathological changes common to both conditions. With the rationale that these parallels imply convergent pathogenetic mechanisms, we identified a gene whose expression increases in both. We now report the results of a more extensive analysis of this gene and show that by sequence analysis it is highly homologous and likely identical to GFAP. GFAP mRNA accumulates late in the course of scrapie in subpial and periventricular astrocytes and in cells in foci in the hippocampus. The increased abundance of GFAP mRNA is accompanied by an increase in the corresponding protein. GFAP mRNA is localized by in situ hybridization to the cell body and processes of astrocytes. In AD, the latter pattern predominates, consistent with induction of GFAP mRNA in the sites of synthesis in glial processes in the neuritic plaque.
Assuntos
Doença de Alzheimer/patologia , Astrócitos/patologia , Genes Virais , Proteína Glial Fibrilar Ácida/genética , RNA Mensageiro/genética , Scrapie/patologia , Doença de Alzheimer/genética , Animais , Sequência de Bases , Northern Blotting , Cricetinae , Proteína Glial Fibrilar Ácida/análise , Humanos , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Scrapie/genéticaRESUMO
Subacute sclerosing panencephalitis (SSPE) is a slow infection caused by measles virus in which several years separate recovery from typical acute measles and the development of a slowly progressive neurological disease. We have investigated replication of measles virus in brain tissue obtained after the onset of neurological disease and in the terminal phase. With a hybridization tomographic technique that combines in situ hybridization with macroradioautographic screening of large areas of tissue, we analyzed the spatial and temporal distribution of virus genes in vivo, using region- and strand-specific probes for the nucleocapsid and matrix genes. We show that early in the course of SSPE there is a global repression in the synthesis and expression of the genome. In the final stage of SSPE most infected cells still have depressed levels of plus- and minus-strand viral RNA and contain nucleocapsid protein but lack matrix protein. These findings provide further evidence for a unified view of slow infections of the nervous system, where the general constraints on virus gene expression provide an explanation for persistence of virus in the face of the host's immune response, and the slow evolution of pathological change. In the final phases of SSPE the more specific block in virus replication accounts for the cell-associated state of the virus and the difficulty in virus isolation.
Assuntos
Genes Virais , Vírus do Sarampo/genética , Panencefalite Esclerosante Subaguda/microbiologia , Encéfalo/metabolismo , Encéfalo/microbiologia , Humanos , Vírus do Sarampo/metabolismo , Hibridização de Ácido Nucleico , RNA Viral/genética , Panencefalite Esclerosante Subaguda/etiologia , Panencefalite Esclerosante Subaguda/metabolismo , Proteínas Virais/metabolismoRESUMO
Visna virus is the prototypic member of a subfamily of retroviruses responsible for slow infections of animals and humans. As a part of our investigation of the functions of viral gene products in virus replication, we have isolated three infectious molecular clones and determined the complete nucleotide sequences of two of the clones. We have also characterized the progeny of the biologically cloned viral stocks and of the infectious clones and document considerable heterogeneity in plaque size and antigenic phenotype of the former that is reduced to near homogeneity in the progeny of the infectious clones. It thus should now be possible to trace the emergence of antigenic variants of visna virus as well as ascribe defined functions to structural and regulatory genes of the virus in determining neurovirulence and the slow tempo of infection.
Assuntos
Genes Virais , Replicação Viral , Vírus Visna-Maedi/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Plexo Corióideo , Deleção Cromossômica , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Viral/genética , DNA Viral/isolamento & purificação , Dados de Sequência Molecular , Fases de Leitura Aberta , Ovinos , Ensaio de Placa Viral , Vírus Visna-Maedi/genéticaRESUMO
Potent combinations of antiretroviral drugs diminish the turnover of CD4+ T lymphocytes productively infected with HIV-1 and reduce the large pool of virions deposited in lymphoid tissue (LT). To determine to what extent suppression of viral replication and reduction in viral antigens in LT might lead correspondingly to repopulation of the immune system, we characterized CD4+ T lymphocyte populations in LT in which we previously had quantitated viral load and turnover of infected cells before and after treatment. We directly measured by quantitative image analysis changes in total CD4+ T cell counts, the CD45RA+ subset, and fractions of proliferating or apoptotic CD4+ T cells. Compared with normal controls, we documented decreased numbers of CD4+ T cells and increased proliferation and apoptosis. After treatment, proliferation returned to normal levels, and total CD4+ T and CD45RA+ cells increased. We discuss the effects of HIV-1 on this subset based on the concept that renewal mechanisms in the adult are operating at full capacity before infection and cannot meet the additional demand imposed by the loss of productively infected cells. The slow increases in the CD45RA+ CD4+ T cells are consistent with the optimistic conclusions that (i) renewal mechanisms have not been damaged irreparably even at relatively advanced stages of infection and (ii) CD4+ T cell populations can be partially restored by control of active replication without eradication of HIV-1.