RESUMO
Avaliou-se o efeito de dieta com torta de girassol, oriunda da produção de biodiesel, sobre o comportamento ingestivo e a resposta fisiológica de 32 cabritos ½ sangue Boer, não castrados, com peso médio inicial de 15,3±3,2kg e idade média de 135 dias. Utilizou-se delineamento inteiramente ao acaso, com quatro tratamentos 0; 8; 16 e 24% de inclusão da torta de girassol e oito repetições. O tempo despendido com ruminação, ócio e mastigação total não foi influenciado pela dieta, e observou-se efeito linear crescente sobre o tempo de ingestão em min/período e em min/dia. A eficiência de ingestão e ruminação da matéria seca e da fibra em detergente neutro também não diferiu entre dietas. Quanto às respostas fisiológicas, as frequências respiratória e cardíaca e as temperaturas retal e superficial não sofreram influência da dieta. Concluiu-se que a torta de girassol pode ser incluída até 24% da matéria seca em dietas de cabritos ½ sangue Boer sem comprometer o comportamento ingestivo e os parâmetros fisiológicos desses animais. O fornecimento de dietas com até 24% de matéria seca de torta de girassol não interfere no estresse calórico de cabritos.
The effect of diets with sunflower cake originated from biodiesel production on the ingestive behavior and physiological responses of 32 crossbred Boer goats, noncastrated, with initial weight of 15.3±3.2 kg and mean age of 135 days was evaluated. A completely randomized design with four treatments (0, 8, 16 and 24% of sunflower cake inclusion) and eight replicates was used. The rumination and idling times and the total chewing time were not affected by diets, but the ingestion time (min/period and min/day) had an increasing linear effect. The rumination and ingestion efficiencies of dry matter and neutral detergent fiber also did not differ among diets. Regarding the physiological responses of animals, the heart and respiratory frequencies and the surface and rectal temperatures were not influenced by diets. Sunflower cake can be included up to 24% DM in diets of crossbred Boer goats without compromising the ingestive behavior and physiological parameter of these animals. Under the climatic conditions evaluated the supply of diets with up to 24% DM of sunflower cake does not mitigate or enhance the heat stress in kids.
Assuntos
Animais , Ingestão de Alimentos/fisiologia , Ruminantes , Administração Retal , Dieta/veterinária , HelianthusRESUMO
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is a major target for antiretroviral strategy to block or curtail HIV infection. A suitable RT-SHIV/macaque model is urgently needed for the evaluation of HIV/AIDS therapies and microbicides specifically targeting HIV-1 RT. METHODS: Fifteen cynomolgus macaques (Macaca fascicularis) were divided into three groups (n = 5) and intravaginally inoculated with 4800, 1200, or 300 TCID(50) of RT-SHIVtc. Systemic infections of RT-SHIVtc exposed macaques were determined by both virological and immunologic parameters during 24 weeks post-challenge. RESULTS: Within 2 weeks post-inoculation, 13 of 15 macaques became infected as confirmed by virus isolation, plasma viral RNA, proviral DNA, declined CD4(+)T cell counts in peripheral blood and seroconversion. CONCLUSIONS: Results serve to validate the infectivity and pathogenicity of RT-SHIVtc following vaginal exposure in M. fascicularis. This RT-SHIVtc/macaque model could be suitable for the pre-clinical evaluation of non-nucleoside RT inhibitor-based anti-HIV microbicides.
Assuntos
Modelos Animais de Doenças , Infecções por HIV , HIV-1 , Macaca fascicularis , Vírus da Imunodeficiência Símia , Administração Intravaginal , Animais , Anticorpos Antivirais/sangue , Contagem de Linfócito CD4 , Linhagem Celular , Suscetibilidade a Doenças , Avaliação de Medicamentos , Feminino , Transcriptase Reversa do HIV , HIV-1/imunologia , Humanos , RNA Viral/sangue , Vírus da Imunodeficiência Símia/imunologiaRESUMO
A model of vertical HIV transmission was developed using oral HIV-2(287) exposure of newborn Macaca nemestrina. The minimal Animal Infectious Dose for this oral route was found to be 10-fold higher than that for atraumatic viral transmission across other mucosal membranes (vaginal/rectal) of juvenile macaques. However, once infection was established, viral replication was rapid and plasma viremia could be detected by reverse-transcriptase polymerase chain reaction and viral co-culture within 1 week following exposure. No animal was resistant to infection and all macaques initially exposed to a subinfectious viral inoculum were subsequently infected by re-exposure of mucosal membranes. Higher viral load during primary infection correlated with a more rapid CD4 depletion; however, all HIV-2(287)-infected animals developed CD4 depletion during the observation period. This animal model can now be used to study early viral replication in the presence and absence of anti-retroviral agents to help identify conditions to reduce vertical HIV transmission in human newborns.
Assuntos
Infecções por HIV/fisiopatologia , Infecções por HIV/transmissão , HIV-2/patogenicidade , Transmissão Vertical de Doenças Infecciosas/veterinária , Macaca nemestrina/virologia , Animais , Animais Recém-Nascidos , Antivirais/uso terapêutico , Contagem de Linfócito CD4 , DNA Viral/análise , Modelos Animais de Doenças , Feminino , Mucosa Bucal/virologia , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carga Viral , ViremiaRESUMO
To study mechanisms involved in mother-to-fetus transmission of human immunodeficiency virus (HIV) in utero, we have developed a chronically catheterized pregnant macaque model that permits simultaneous and sequential determination of virus in maternal and fetal blood and amniotic fluid during pregnancy. In this report, we have characterized this model using three groups of pregnant macaques designed to sample: (1) maternal blood, fetal blood, and amniotic fluid (n = 6); (2) maternal blood and amniotic fluid (n = 6); or (3) maternal blood only (n = 2). After inoculation with the highly pathogenic HIV-2(287), all pregnant macaques developed brief but intense viremias followed by precipitous CD4+ T-cell declines within 2-3 weeks. While all the infants born to dams of the three groups were HIV positive, the degree of infection and outcome of HIV infection varied. All infants were shown to be HIV-RNA-positive by reverse transcriptase-polymerase chain reaction (RT-PCR). However, HIV-infected cells were detected only in the blood of those born to dams enrolled in groups 1 and 2: most of these infants progressed to CD4+ T-cell depletion. The infants in group 3 exhibited HIV-RNA in plasma, although neither HIV-infected cells nor CD4+ T-cell depletion was detectable. However, all infants developed HIV-2-specific antibody at various levels by 2 months of age. Together, the data suggest that, while the degree of instrumentation may modulate intensity of virus transmission to fetus, the highly pathogenic HIV-2(287) exhibited a high frequency of virus transmission from the mother to fetus.
Assuntos
Infecções por HIV/transmissão , HIV-2/patogenicidade , Transmissão Vertical de Doenças Infecciosas/veterinária , Troca Materno-Fetal , Líquido Amniótico , Animais , Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/veterinária , Linfócitos T CD4-Positivos , Cateterismo/veterinária , Modelos Animais de Doenças , Feminino , Infecções por HIV/veterinária , Humanos , Macaca , Reação em Cadeia da Polimerase/veterinária , Gravidez , RNA Viral/análise , Manejo de Espécimes , Viremia/veterináriaRESUMO
Nonhuman primate models of human AIDS have been used successfully to evaluate candidate vaccines and infection intervention therapies. Successes of pathogenicity studies in primate models have been limited because of the varied infection outcomes and characteristic low number of study animals. The acutely pathogenic HIV-2(287)--Macaca nemestrina model has shown promise both in antiviral drug evaluation and in pathogenicity studies. Here we describe virus replication, spread, and host responses during the first 28 days of HIV-2(287) infection. Focusing on 18 macaques from a larger 27-macaque study, we report changing virus loads, CD4(+) cell depletions, and antibody responses both systemically and in the mucosa of the small intestine. After intravenous inoculation, blood and intestinal tissue were collected from pairs of macaques at 12 hr and 1, 2, 4, 6, 10, 14, 21, and 28 days postinfection. Specimens were examined for evidence of infection by quantitative cultures, in situ hybridization, lymphocyte subset monitoring, and antibody production. The data were presented serially as though all samples were collected from a single macaque. The highest blood virus loads were detected between days 10 and 14 and subsequently decreased through day 28. This coincided with a significant increase in ileum mucosa virus loads on day 10, which became undetectable by day 28. The lowest levels of CD4(+) cells were observed on days 21 and 28 in blood and ileum mucosa. CD4(+):CD8(+) cell ratios in blood and ileum dropped dramatically after day 10 to lowest levels by day 28. Intestinal virus loads were inversely correlated with CD4(+) cell and virus-specific antibody levels in the ileum after day 6. These results underscore the suitability of this model for pathogenicity studies as well as the importance of the intestinal lymphoid tissues as an initial site of virus replication and cell destruction during the acute, asymptomatic stage of AIDS development.
Assuntos
Modelos Animais de Doenças , Infecções por HIV/imunologia , HIV-2 , Mucosa Intestinal/imunologia , Macaca nemestrina , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Anticorpos Anti-HIV/análise , Anticorpos Anti-HIV/sangue , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-2/imunologia , HIV-2/isolamento & purificação , HIV-2/fisiologia , Humanos , Íleo/imunologia , Íleo/virologia , Mucosa Intestinal/virologia , RNA Viral/sangue , Carga Viral , Replicação ViralRESUMO
Recently, we developed a maternal-fetal macaque model using a highly pathogenic HIV-2 strain, HIV-2287, to study the time course of HIV transmission in utero. Most pregnant macaques (Macaca nemestrina) infected with HIV-2287 (10-103 infective doses) transmitted HIV to their fetuses, as verified by positive identification of virus-infected mononuclear cells and free viral RNA in fetal blood. To determine whether an antiretroviral drug combination therapy composed of two dideoxynucleosides, azidothymidine (15 mg/kg) and dideoxyinosine (15 mg/kg), and a protease inhibitor, indinavir (25 mg/kg), could completely inhibit mother-to-fetus HIV transmission, we administered these drugs orally through gastric catheters to five pregnant macaques infected with 10 infective doses of HIV-2287. Beginning 30 minutes after HIV inoculation, the dams were given the combination antiviral therapy three times daily until delivery by cesarean section. Drug treatment reduced the maternal virus load to a minimally detectable level but did not prevent primary HIV-2287 infection. All fetal and infant blood samples were virus negative by internally controlled RNA polymerase chain reaction (QC-RNA-PCR) and virus coculture assays. Fetal and infant CD4+ T-cell levels remained normal throughout the experiment. These findings strongly suggest that combination chemotherapy with azidothymidine, dideoxyinosine, and indinavir can suppress maternal viral load enough to prevent mother-to-fetus transmission of HIV.
Assuntos
Didanosina/uso terapêutico , Infecções por HIV/transmissão , HIV-2 , Indinavir/uso terapêutico , Transmissão Vertical de Doenças Infecciosas , Zidovudina/uso terapêutico , Animais , Didanosina/toxicidade , Quimioterapia Combinada , Feminino , Feto/virologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , Indinavir/toxicidade , Macaca nemestrina , Gravidez , Inibidores da Transcriptase Reversa/uso terapêutico , Subpopulações de Linfócitos T , Carga Viral , Zidovudina/toxicidadeRESUMO
Non-human primate models for acquired immunodeficiency syndrome (AIDS) are important for studies of prevention and intervention strategies. Ideally, such models would make use of human immunodeficiency virus type 1 (HIV-1) and animals that are readily available for research. HIV-1 was obtained from an infected macaque, and passaged sequentially in three groups of two Macaca nemestrina neonates each. Evidence for enhanced viral replication was first found in one of the group 2 animals, and in both group 3 animals. Observations that underlie this conclusion are sustained viral recovery from peripheral blood mononuclear cells (PBMCs), increased and accelerated production of antiviral antibodies, and the ability to detect plasma viral ribonucleic acid (RNA) months after infection. There was no evidence of CD4 depletion in any of the animals during the follow-up period. These data suggest that a useful non-human primate model for AIDS can be attained in pigtailed macaques (M. nemestrina).
Assuntos
Síndrome da Imunodeficiência Adquirida/fisiopatologia , HIV-1/fisiologia , Subpopulações de Linfócitos/imunologia , Replicação Viral , Síndrome da Imunodeficiência Adquirida/imunologia , Sequência de Aminoácidos , Animais , Contagem de Linfócito CD4 , Sequência Consenso , Modelos Animais de Doenças , Produtos do Gene env/genética , Genes env , Variação Genética , Anticorpos Anti-HIV/sangue , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , Macaca nemestrina , Dados de Sequência Molecular , RNA Viral/sangue , Carga ViralRESUMO
Lymphoid tissues are the primary target during the initial virus dissemination that occurs in HIV-1-infected individuals. Recent advances in antiretroviral therapy and techniques to monitor virus load in humans have demonstrated that the early stages of viral infection and host response are major determinants of the outcome of individual infections. Relatively little is known about immunopathogenic events occurring during the acute phase of HIV infection. We analyzed viral dissemination within lymphoid tissues by in situ hybridization and by combined immunohistochemistry/in situ hybridization during the acute infection phase (12 hours to 28 days) in pig-tailed macaques (Macaca nemestrina), challenged intravenously with a virulent strain of HIV-2, HIV-2(287). Two stages in viral dissemination were clearly evident within the first 28 days after HIV-2(287) infection. First, a massive increase in individual HIV-2-infected cells, mostly CD3+ T lymphocytes and a smaller percentage of macrophages and interdigitating dendritic cells, was identified within lymph nodes which peaked on the 10th day after HIV-2 infection. A shift of HIV-2 distribution was demonstrable between day 10 and day 14 after HIV-2 infection. Coincident with a marked reduction in individual HIV-2 RNA+ cells by day 14 postinfection, there was a dramatic increase in germinal center-associated HIV-2 RNA. High concentrations of HIV-2 RNA persisted in germinal centers in all animals by days 21 and 28 postinfection. Thus, HIV-2 appears to go through an initial, highly disseminated cellular phase followed by localization in the follicular dendritic cell network with relatively few infected cells. In this nonhuman primate model of HIV-associated immunopathogenesis, using a virus derived from a human pathogen, we identified a significant shift in the pattern of HIV-2 localization within a narrow time frame (day 10 to day 14). This shift in virus localization and behavior indicates that there may be a discrete but remarkably narrow window for therapeutic interventions that interrupt this stage in the natural course of HIV infection. Reproducibility and the accelerated time course of disease development make this model an excellent candidate for such intervention studies.
Assuntos
Centro Germinativo/virologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-2/isolamento & purificação , Linfonodos/virologia , Animais , Anticorpos Antivirais/análise , Células Dendríticas/virologia , Feminino , Centro Germinativo/patologia , HIV-2/genética , HIV-2/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Macaca nemestrina , Masculino , Mesentério , RNA Viral/metabolismo , Linfócitos T/virologia , Fatores de TempoRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection alters the expression of host cell genes at both the mRNA and protein levels. To obtain a more comprehensive view of the global effects of HIV infection of CD4-positive T-cells at the mRNA level, we performed cDNA microarray analysis on approximately 1500 cellular cDNAs at 2 and 3 days postinfection (p.i.) with HIV-1. Host cell gene expression changed little at 2 days p.i., but at 3 days p.i. 20 cellular genes were identified as differentially expressed. Genes involved in T-cell signaling, subcellular trafficking, and transcriptional regulation, as well as several uncharacterized genes, were among those whose mRNAs were differentially regulated. These results support the hypothesis that HIV-1 infection alters expression of a broad array of cellular genes and provides a framework for future functional studies on the differentially expressed mRNA products.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Regulação da Expressão Gênica , HIV-1/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linfócitos T CD4-Positivos/patologia , Linhagem Celular , DNA Complementar , Perfilação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
Thrombotic microangiopathy (TMA) has been increasingly reported in human immunodeficiency virus (HIV)-infected humans over the past decade. The pathogenesis is unknown. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques (Macaca nemestrina), infected intravenously with a virulent HIV-2 strain, HIV-2(287), in addition to that of four uninfected control macaques. Necropsies were performed between 12 hours and 28 days after infection. HIV-2 antigen was detectable in peripheral blood mononuclear cell (PBMC) cocultures in all animals after 10 days of HIV-2 infection; a rapid decline in CD4(+) PBMC (<350/microliter) was seen in five of six animals 21 days and 28 days after infection. No macaque developed features of clinical AIDS. Typical lesions of human HIV-associated nephropathy were undetectable. Six of the 27 HIV-2-infected macaques demonstrated both histological TMA lesions (thrombi in glomerular capillary loops and small arteries, mesangiolysis) and ultrastructural lesions (mesangiolysis, subendothelial lucency, platelet thrombi in glomerular capillary lumina). Extrarenal thrombi were detected in the gastrointestinal and adrenal microvasculature of macaques that had developed renal TMA. None of the control animals demonstrated features of renal TMA at necropsy. In a retrospective analysis of kidneys obtained from 39 additional macaques infected with HIV-2(287), seven cases demonstrated TMA. In situ hybridization showed no detectable HIV-2 RNA in kidney sections of 65/66 HIV-2-infected macaques, including all 13 TMA cases. Expression of the chemokine receptor CXCR4, the putative coreceptor for HIV-2(287), was absent in intrinsic renal cells in all HIV-2-infected macaques. The HIV-2-infected macaque may be a useful model of human HIV-associated TMA. Our data do not support a role of direct HIV-2 infection of intrinsic renal cells as an underlying mechanism.
Assuntos
Infecções por HIV/patologia , HIV-2 , Microcirculação/virologia , Trombose/patologia , Trombose/virologia , Animais , Análise Química do Sangue , Linfócitos T CD4-Positivos/metabolismo , Modelos Animais de Doenças , Feminino , Proteína gp120 do Envelope de HIV/análise , Proteína gp120 do Envelope de HIV/sangue , Humanos , Hibridização In Situ , Rim/anatomia & histologia , Rim/patologia , Rim/ultraestrutura , Linfonodos/patologia , Macaca nemestrina , Masculino , Microcirculação/patologia , Receptores CXCR4/análise , Receptores CXCR4/sangue , Fatores de TempoRESUMO
The early kinetics of antibody expression following transmucosal infection by SIV(Mne) were examined in several mucosal compartments in Macaca nemestrina. Five male-female pairs of macaques were inoculated intrarectally with SIV(Mne) E11S, a biological clone, and serially euthanized at 1, 2, 4, 8, and 12 weeks postinoculation. Plasma, tears, saliva, rectal secretions, and vaginal washes were collected serially and just prior to euthanasia. Both total and SIV-specific IgG and IgA levels were measured by immunoglobulin isotype-specific quantitative enzyme-linked immunosorbent assays (ELISAs), and were further examined by conventional and enhanced chemiluminescence (ECL) immunoblots. Virus coculture, polymerase chain reaction, and in situ hybridization assays revealed the systemic spread of virus as early as 1 week postinoculation in 8 of 10 animals. ECL immunoblots detected SIV-specific antibodies in mucosal samples collected 1 week postinoculation. The most dramatic increases in both total and SIV-specific IgA levels were detected in rectal secretion samples. In contrast, plasma and nonrectal mucosal samples from the same time points increased only slightly, suggesting that the most robust antibody response occurred at the portal of infection. Our results show that the SIV-infected macaque is an excellent model for studies designed to assess mucosal immune responses to primate lentivirus infections. Additional studies will assess the correlation between the antiviral protection afforded by candidate vaccines and mucosal antibody responses.
Assuntos
Anticorpos Antivirais/biossíntese , Imunidade nas Mucosas , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Animais , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Immunoblotting , Isotipos de Imunoglobulinas/imunologia , Macaca nemestrina , Masculino , Modelos Imunológicos , Infecções Oportunistas/imunologia , Reto , Síndrome de Imunodeficiência Adquirida dos Símios/virologiaRESUMO
The natural histories of disease progression induced by two closely related molecular clones of SIVmac were evaluated to determine the utility of these viruses for modeling fast and slow progression to AIDS in Macaca nemestrina. Viral and immune parameters were measured to determine differential progression. Survival time, viral load and CD4+ T cell decline all were indicative of distinct rates of progression, while early measurements of interferon-gamma (IFNgamma) producing cells did not indicate significant differences.
Assuntos
Contagem de Linfócito CD4 , Síndrome de Imunodeficiência Adquirida dos Símios/fisiopatologia , Vírus da Imunodeficiência Símia/classificação , Animais , Anticorpos Antivirais/sangue , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Citocinas/biossíntese , Progressão da Doença , Macaca nemestrina , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/isolamento & purificação , Vírus da Imunodeficiência Símia/fisiologia , Análise de Sobrevida , Fatores de TempoRESUMO
In an earlier study we found that pigtailed macaques (Macaca nemestrina) that were experimentally infected with human immunodeficiency virus type 1 (HIV-1) initially became viremic and seroconverted, but HIV-1 replication diminished markedly over time. In an attempt to develop a longer term pathogenic model, blood from HIV-1-infected macaques was serially transfused into three groups of naive macaques. Transfer was successful through two transfusions as shown by repeated virus isolations and confirmed by the development of cell-free plasma viremia and by seroconversion. Three to five weeks after transfusion, plasma levels of HIV-1 RNA from several macaques in the first two groups exceeded those of the initially inoculated macaques. However, animals in the third group had diminished RNA levels, were virus culture negative, and did not seroconvert. Sequence analyses of env-region clones from infected animals revealed only minimal changes over the course of the passages. These results confirm HIV-1 replication in M. nemestrina during the acute phase of infection. However, adaptation of HIV-1 to a macaque-pathogenic variant did not occur during serial passage, possibly because the animals were able to restrict HIV-1 replication below a level required for a pathogenic variant to emerge. Whether such containment is a function of the host's immune response or a virus cell incompatibility remains to be determined.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Macaca nemestrina/virologia , Adaptação Fisiológica , Animais , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/fisiopatologia , HIV-1/genética , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/virologia , Análise de Sequência de DNA , Inoculações Seriadas , Viremia , Replicação ViralRESUMO
OBJECTIVES: Receptive anal intercourse but not orogenital sex has been identified as a major risk factor for transmission of HIV-1. Recent studies using simian immunodeficiency virus (SIV) in rhesus macaques have demonstrated relatively efficient infection following oral administration, indicating that modes of transmission may vary between HIV-1 and SIV. Here, we investigate whether HIV-1 infection of macaques via the oral route is more efficient than via the rectal route. DESIGN: Eleven Macaca nemestrina neonates were exposed to HIV-1 via different routes (four oral, two intravenous, and five rectal). One animal was orally inoculated with a sham inoculum and two control animals were not exposed. METHODS: All animals were followed for virological signs of infection, and for pathogenesis associated with HIV-1 infection by general physical examinations, complete blood cell counts and lymphocyte subset analysis, and full necropsies. RESULTS: Three out of five rectally exposed macaques and both of the intravenously inoculated animals became infected with HIV-1, whereas none of the orally exposed animals showed evidence of HIV-1 infection. Clinical observations following exposure included failure to thrive in the orally inoculated animals and low CD4/CD8 ratios in the rectally exposed macaques. CONCLUSIONS: The finding that, contrary to what has been reported for SIV, transmission of HIV-1 via the oral route is not more efficient than via the rectal route, indicates important biological differences between HIV-1 and SIV, with direct implications for the spread of HIV and associated AIDS, and for development of anti-HIV-1 vaccines.
Assuntos
Infecções por HIV/transmissão , HIV-1/fisiologia , Animais , Animais Recém-Nascidos , Relação CD4-CD8 , Linfócitos T CD4-Positivos , Linhagem Celular , Modelos Animais de Doenças , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Subpopulações de Linfócitos , Macaca nemestrina , Mucosa Bucal , RetoRESUMO
Using PCR to monitor early (LTR/LTR (long terminal repeat)) and late (LTR/gag) products of reverse transcription and the formation of HIV-1 LTR circles (indicating nuclear import), we explored the relationship between T cell activation signals and early events in the life cycle of HIV-1 infection. The combination of TCR ligation with either CD28 cross-linking or exogenous IL-2 was required for HIV-1 LTR circle formation in resting CD4+ T cells. Ligation of the TCR or CD28 receptors or addition of IL-2 alone did not induce this process. However, cross-linking the TCR, or IL-2 alone, unlike CD28 ligation, could induce the completion of viral reverse transcription. In contrast, the initiation of HIV-1 reverse transcription could occur in resting CD4+ T cells without any stimulation. Cyclosporin A (CsA), an inhibitor of T cell activation, completely blocked HIV-1 DNA nuclear import in activated CD4+ T cells. The completion of HIV-1 reverse transcription was blocked by CsA in infected CD4+ T cells activated by TCR ligation and IL-2, but not in cells stimulated by TCR and CD28 ligation. The costimulation of CD3 and CD28 mAbs in the presence of IL-2 could not overcome the CsA inhibitory effect on nuclear import of viral DNA. Therefore, the factor(s) involved in a CsA-sensitive pathway plays a critical role in HIV-1 DNA transport from the cytoplasm into the nucleus. Production and movement of HIV-1 DNA in resting CD4+ T cells require two signals: one signal from the TCR, which normally regulates the G0 to G1 transition, induces completion of viral reverse transcription; the other signal through CD28 or an IL-2R-dependent process is sensitive to CsA treatment and regulates viral DNA entry into the nucleus.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Núcleo Celular/genética , Ciclosporina/farmacologia , DNA Viral/metabolismo , HIV-1/genética , Interfase/imunologia , Ativação Linfocitária/efeitos dos fármacos , Anticorpos Monoclonais/imunologia , Transporte Biológico/genética , Transporte Biológico/imunologia , Antígenos CD28/imunologia , Antígenos CD28/farmacologia , Complexo CD3/imunologia , Complexo CD3/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Interleucina-2/farmacologia , Interfase/genética , Ativação Linfocitária/genéticaRESUMO
The lack of a representative animal model that permits frequent in utero fetal blood sampling is a major limiting factor for the study of maternal-fetal HIV transmission. Therefore, we have developed a maternal-fetal virus infection model using chronically catheterized macaques to simultaneously study the time-course of viral infection in the mother and the response of the fetus to maternal HIV infection. Pregnant macaques were infected with 10(3) infectious units of HIV-2(287); every 3 days blood samples from both the mother and the fetus as well as amniotic fluid samples were collected. We found a varying degree of peak and time-to-peak virus load, virus-infected PBMCs, and free virus (determined by QC-RNA-PCR method) in maternal blood. Two of the three mothers with more than 10(8) copies of viral RNA/ml of plasma at peak viremia transmitted the virus to their fetuses at about 14 days post-infection. As observed with HIV-2(287) infected mothers, virus-infected fetuses also produced a rapid rate of CD4+ cell decline in utero.
Assuntos
Transmissão de Doença Infecciosa , Infecções por HIV/embriologia , Infecções por HIV/transmissão , HIV-2 , Linfócitos/imunologia , Complicações Infecciosas na Gravidez , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Cateteres de Demora , Desenvolvimento Embrionário e Fetal , Feminino , Idade Gestacional , Infecções por HIV/imunologia , HIV-2/isolamento & purificação , Linfócitos/virologia , Macaca mulatta , Reação em Cadeia da Polimerase , Gravidez , RNA Viral/sangue , Viremia/sangue , Viremia/transmissãoRESUMO
We previously demonstrated that cellular mRNAs are degraded in CD4 positive lymphocytes infected by the human immunodeficiency virus, HIV-1, but not in cells infected by the simian lentivirus, SIV. To begin to define the molecular mechanisms underlying this RNA degradation, we have established an in vitro RNA degradation assay utilizing extracts from both infected and uninfected cells. We found that in vitro transcribed, 32P-radiolabeled actin RNA was degraded in extracts prepared from CEM, CEMx174, and C8166 cells which were infected with HIV-1. Minimal actin RNA degradation was observed in extracts prepared from uninfected cells. Similarly little degradation was observed in cell-free extracts prepared from SIV-infected cells. To determine if viral RNA sequences could impart enhanced stability to cellular RNAs in our in vitro assay, we prepared radiolabeled RNAs that contained selected viral RNA determinants. One such RNA contained the HIV-1 specific TAR (transactivating region) sequence (nucleotides 1-111) appended to a reporter CAT RNA. Like the cellular actin RNA, these TARCAT RNAs were degraded in HIV-1-infected cell extracts, but not in extracts from uninfected cells or extracts prepared from SIV-infected cells. In contrast, an RNA containing only authentic HIV-1 sequences comprising TAR and gag sequences was more stable than actin RNA in HIV-1-infected extracts. These results, taken together, suggest that the in vitro assay reproduces events that occur in vivo and provide a starting point for identifying the factor responsible for cellular RNA degradation in HIV-1-infected cells.
Assuntos
HIV-1/metabolismo , RNA Mensageiro/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Actinas/genética , Animais , Sequência de Bases , Linhagem Celular , Primers do DNA , Humanos , Macaca , Dados de Sequência Molecular , RNA Viral/metabolismoRESUMO
We have examined the effects of interferon (IFN)-alpha/beta on HIV-1 and SIV replication in CD4+ T-cell lines. To enable us to examine these effects on a single cycle of virus replication, cells were synchronously infected with HIV-1 LAI or SIV mac251. Cell lines included MT4 cells which were responsive to IFN and, as controls, C8166 cells which failed to respond to interferon treatment. Similar to previous reports, we found that replication of both HIV-1 and SIV was markedly inhibited in responsive MT4 cell lines treated with IFN. No such decreases were observed in HIV-1-infected, IFN-treated C8166 cells. Levels of both intracellular and extracellular viral antigens decreased in both HIV-1- and SIV-infected MT4 cells treated with IFN. Whereas steady state levels of viral-specific RNAs dramatically declined in SIV-infected cells, no such decrease was observed in IFN-treated HIV-1-infected cells. In accordance with these data, the rate of viral protein synthesis did not significantly change in HIV-1-infected, IFN-treated MT-4 cells. Western blot analysis of extracts prepared from IFN-treated HIV-1-infected cells revealed a decreased accumulation of most HIV-1-specific glycoproteins and proteins with the exception of the pr55 gag precursor. Pulse-chase experiments confirmed the enhanced stability of pr55 in IFN-treated cells, but also unexpectedly demonstrated the accelerated and quantitative processing of the p26 precursor (p24 capsid [CA] plus p2) to the final processed p24 (CA) polypeptide. These data, taken together, suggest that IFN deregulated viral protein processing and caused reduced protein stability in HIV-1-infected cells while inhibiting an earlier stage of replication in SIV-infected cells.
Assuntos
HIV-1/efeitos dos fármacos , Interferon-alfa/farmacologia , Interferon beta/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/citologia , Linhagem Celular , Expressão Gênica , Genes Virais , Antígenos HIV/metabolismo , HIV-1/fisiologia , Humanos , Vírus da Imunodeficiência Símia/fisiologia , Proteínas Virais/metabolismoRESUMO
We previously reported that pigtailed macaques (Macaca nemestrina) became infected after intravenous inoculation with the LAI strain of human immunodeficiency virus type 1 (HIV-1), an isolate that replicates in both M. nemestrina lymphocytes and blood-derived monocyte/macrophages. In the current study we investigated the presence of HIV-1 and pathology in the postmortem brains of four of these macaques. Histopathological findings revealed focal lesions in white matter in the frontal and occipital lobes of one macaque, with myelin loss, nerve fibre loss, and gliosis within these lesions. Semi-quantitative, solution-based PCR revealed HIV-1 DNA in the brains of two of the other macaques. Using slide-based PCR-driven in situ hybridization, we studied these two macaques further and detected intranuclear, circular HIV-1 DNA in vascular endothelia and other non-neuronal brain cells. These findings indicate that M. nemestrina brain can be infected with HIV-1 in vivo and may provide a useful animal model for understanding early HIV-1 brain infection in humans.
Assuntos
Complexo AIDS Demência/patologia , Complexo AIDS Demência/virologia , Encéfalo/virologia , HIV-1/isolamento & purificação , Macaca nemestrina/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/patologia , Células Cultivadas , Sequência Consenso , DNA Circular/análise , DNA Viral/análise , Modelos Animais de Doenças , Proteína gp120 do Envelope de HIV/química , HIV-1/patogenicidade , Humanos , Hibridização In Situ/métodos , Dados de Sequência Molecular , Monócitos/virologia , Fragmentos de Peptídeos/química , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/análise , RNA Viral/análiseRESUMO
We investigated the role of blood dendritic cells (DC) in transmission of HIV-1 from infected to uninfected CD4+ T cells, and the accessory molecules involved. DC promoted transmission from infected to uninfected CD4+ cells, but blood DC themselves were not infectable. DC-mediated transmission was blocked by mAb to CD4 and MHC class II, but strongly increased by mAb to CD40 on DC or CD28 on T cells. The DC-dependent infection was inhibitable by anti-CD80 and a soluble fusion protein of the CD80 ligand, CTLA4; soluble CTLA4Ig also blocked infection augmented by crosslinking CD40. We also demonstrated that mAb to CD40 up-regulate the expression of CTLA4 ligands CD80 and B70/B7-2 (CD86) on DC. These data suggest that the dialog between CD40-CD40 ligand (CD40L) and CD28-CD80 counter-receptors on DC and T cells may be linked to HIV infection in vivo.