RESUMEN
Lentiviruses infect myeloid cells, leading to acute infection followed by persistent/latent infections not cleared by the host immune system. HIV and SIV are lentiviruses that infect CD4+ lymphocytes in addition to myeloid cells in blood and tissues. HIV infection of myeloid cells in brain, lung, and heart causes tissue-specific diseases that are mostly observed during severe immunosuppression, when the number of circulating CD4+ T cells declines to exceeding low levels. Antiretroviral therapy (ART) controls viral replication but does not successfully eliminate latent virus, which leads to viral rebound once ART is interrupted. HIV latency in CD4+ lymphocytes is the main focus of research and concern when HIV eradication efforts are considered. However, myeloid cells in tissues are long-lived and have not been routinely examined as a potential reservoir. Based on a quantitative viral outgrowth assay (QVOA) designed to evaluate latently infected CD4+ lymphocytes, a similar protocol was developed for the assessment of latently infected myeloid cells in blood and tissues. Using an SIV ART model, it was demonstrated that myeloid cells in blood and brain harbor latent SIV that can be reactivated and produce infectious virus in vitro, demonstrating that myeloid cells have the potential to be an additional latent reservoir of HIV that should be considered during HIV eradication strategies.
Asunto(s)
Sistema Nervioso Central/virología , Modelos Animales de Enfermedad , Macaca mulatta/virología , Macrófagos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Latencia del Virus , Animales , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , Humanos , Carga ViralRESUMEN
Enteropathy in HIV infection is not eliminated with combination antiretroviral therapy and is possibly linked to microbial translocation. We used a rapidly progressing SIV/pigtailed macaque model of HIV to examine enteropathy and microbial translocation. Histologic evidence of intestinal disease was observed in only half of infected macaques during late-stage infection (LSI). Combination antiretroviral therapy initiated during acute infection prevented intestinal disease. In the ileum and colon, enteropathy was associated with increased caspase-3 staining, decreased CD3+ T cells, and increased SIV-infected cells. CD3+ T cells were preserved in LSI animals without intestinal disease, and levels of CD3 staining in all LSI animals strongly correlated with the number of infected cells in the intestine and plasma viral load. Unexpectedly, there was little evidence of microbial translocation as measured by soluble CD14, soluble CD163, lipopolysaccharide binding protein, and microbial 16s ribosomal DNA. Loss of epithelial integrity indicated by loss of the tight junction protein claudin-3 was not observed during acute infection despite significantly fewer T cells. Claudin-3 was reduced in LSI animals with severe intestinal disease but did not correlate with increased microbial translocation. LSI animals that did not develop intestinal disease had increased T-cell intracytoplasmic antigen 1-positive cytotoxic T lymphocytes, suggesting a robust adaptive cytotoxic T-lymphocyte response may, in part, confer resilience to SIV-induced intestinal damage.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/patología , Enteropatía por VIH/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Enfermedad Aguda , Animales , Antígenos CD/metabolismo , Terapia Antirretroviral Altamente Activa , Caspasa 3/metabolismo , Claudina-3/metabolismo , Colon/enzimología , Colon/patología , Modelos Animales de Enfermedad , Quimioterapia Combinada , Células Epiteliales/metabolismo , Enteropatía por VIH/sangre , Enteropatía por VIH/virología , Íleon/enzimología , Íleon/patología , Inmunohistoquímica , Intestinos/patología , Macaca mulatta , Proteínas de Unión a Poli(A)/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/sangre , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/metabolismo , Carga ViralRESUMEN
Mounting evidence implicates antiretroviral (ARV) drugs as potential contributors to the persistence and evolution of clinical and pathological presentation of HIV-associated neurocognitive disorders in the post-ARV era. Based on their ability to induce endoplasmic reticulum (ER) stress in various cell types, we hypothesized that ARV-mediated ER stress in the central nervous system resulted in chronic dysregulation of the unfolded protein response and altered amyloid precursor protein (APP) processing. We used in vitro and in vivo models to show that HIV protease inhibitor (PI) class ARVs induced neuronal damage and ER stress, leading to PKR-like ER kinase-dependent phosphorylation of the eukaryotic translation initiation factor 2α and enhanced translation of ß-site APP cleaving enzyme-1 (BACE1). In addition, PIs induced ß-amyloid production, indicative of increased BACE1-mediated APP processing, in rodent neuroglial cultures and human APP-expressing Chinese hamster ovary cells. Inhibition of BACE1 activity protected against neuronal damage. Finally, ARVs administered to mice and SIV-infected macaques resulted in neuronal damage and BACE1 up-regulation in the central nervous system. These findings implicate a subset of PIs as potential mediators of neurodegeneration in HIV-associated neurocognitive disorders.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Inhibidores de la Proteasa del VIH/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/patología , Células Cultivadas , Macaca , Masculino , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Estabilidad Proteica/efectos de los fármacos , Ratas , Ritonavir/farmacología , Respuesta de Proteína Desplegada/efectos de los fármacos , eIF-2 Quinasa/metabolismoRESUMEN
Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs, to promote HIV cure. In addition to contributing to our understanding of the pathogenesis of HAND, the SIV/pigtailed macaque model also provides an excellent opportunity to test innovative approaches to eliminate the latent HIV reservoir in the brain.
Asunto(s)
Antivirales/farmacología , Sistema Nervioso Central/efectos de los fármacos , Disfunción Cognitiva/tratamiento farmacológico , Modelos Animales de Enfermedad , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Complejo SIDA Demencia/tratamiento farmacológico , Complejo SIDA Demencia/inmunología , Complejo SIDA Demencia/fisiopatología , Complejo SIDA Demencia/virología , Animales , Terapia Antirretroviral Altamente Activa , Sistema Nervioso Central/virología , Disfunción Cognitiva/inmunología , Disfunción Cognitiva/fisiopatología , Disfunción Cognitiva/virología , Humanos , Macaca nemestrina , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Virus de la Inmunodeficiencia de los Simios/fisiología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/virología , Carga Viral/efectos de los fármacos , Latencia del Virus/fisiologíaRESUMEN
UNLABELLED: Despite the success of combined antiretroviral therapy (ART), human immunodeficiency virus (HIV) infection remains a lifelong infection because of latent viral reservoirs in infected patients. The contribution of CD4(+) T cells to infection and disease progression has been extensively studied. However, during early HIV infection, macrophages in brain and other tissues are infected and contribute to tissue-specific diseases, such as encephalitis and dementia in brain and pneumonia in lung. The extent of infection of monocytes and macrophages has not been rigorously assessed with assays comparable to those used to study infection of CD4(+) T cells and to evaluate the number of CD4(+) T cells that harbor infectious viral genomes. To assess the contribution of productively infected monocytes and macrophages to HIV- and simian immunodeficiency virus (SIV)-infected cells in vivo, we developed a quantitative virus outgrowth assay (QVOA) based on similar assays used to quantitate CD4(+) T cell latent reservoirs in HIV- and SIV-infected individuals in whom the infection is suppressed by ART. Myeloid cells expressing CD11b were serially diluted and cocultured with susceptible cells to amplify virus. T cell receptor ß RNA was measured as a control to assess the potential contribution of CD4(+) T cells in the assay. Virus production in the supernatant was quantitated by quantitative reverse transcription-PCR. Productively infected myeloid cells were detected in blood, bronchoalveolar lavage fluid, lungs, spleen, and brain, demonstrating that these cells persist throughout SIV infection and have the potential to contribute to the viral reservoir during ART. IMPORTANCE: Infection of CD4(+) T cells and their role as latent reservoirs have been rigorously assessed; however, the frequency of productively infected monocytes and macrophages in vivo has not been similarly studied. Myeloid cells, unlike lymphocytes, are resistant to the cytopathic effects of HIV. Moreover, tissue-resident macrophages have the ability to self-renew and persist in the body for months to years. Thus, tissue macrophages, once infected, have the characteristics of a potentially stable viral reservoir. A better understanding of the number of productively infected macrophages is crucial to further evaluate the role of infected myeloid cells as a potential viral reservoir. In the study described here we compared the frequency of productively infected CD4(+) T cells and macrophages in an SIV-infected macaque model. We developed a critical assay that will allow us to quantitate myeloid cells containing viral genomes that lead to productive infection in SIV-infected macaques and assess the role of macrophages as potential reservoirs.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Genoma Viral , Macrófagos/virología , Monocitos/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Carga Viral , Animales , Antígeno CD11b/análisis , Modelos Animales de Enfermedad , Reservorios de Enfermedades/virología , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T , Infecciones por VIH/virología , Humanos , Macaca mulatta , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Replicación ViralRESUMEN
The effects of HIV infection on spleen and its cellular subsets have not been fully characterized, particularly for macrophages in which diverse populations exist. We used an accelerated SIV-infected macaque model to examine longitudinal effects on T-cell and macrophage populations and their susceptibilities to infection. Substantial lymphoid depletion occurred, characterized by follicular burn out and a loss of CD3 T lymphocytes, which was associated with cellular activation and transient dysregulations in CD4/CD8 ratios and memory effector populations. In contrast, the loss of CD68 and CD163(+)CD68(+) macrophages and increase in CD163 cells was irreversible, which began during acute infection and persisted until terminal disease. Mac387 macrophages and monocytes were transiently recruited into spleen, but were not sufficient to mitigate the changes in macrophage subsets. Type I interferon, M2 polarizing genes, and chemokine-chemokine receptor signaling were up-regulated in spleen and drove macrophage alterations. SIV-infected T cells were numerous within the white pulp during acute infection, but were rarely observed thereafter. CD68, CD163, and Mac387 macrophages were highly infected, which primarily occurred in the red pulp independent of T cells. Few macrophages underwent apoptosis, indicating that they are a long-lasting target for HIV/SIV. Our results identify macrophages as an important contributor to HIV/SIV infection in spleen and in promoting morphologic changes through the loss of specific macrophage subsets that mediate splenic organization.
Asunto(s)
Macrófagos/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Bazo/inmunología , Bazo/patología , Linfocitos T/inmunología , Animales , Inmunohistoquímica , Hibridación in Situ , Macaca nemestrina , Análisis de Secuencia por Matrices de Oligonucleótidos , Síndrome de Inmunodeficiencia Adquirida del Simio/patologíaRESUMEN
BACKGROUND: Sensitive assays are needed for detection of residual human immunodeficiency virus (HIV) in patients with undetectable plasma viral loads to determine whether eradication strategies are effective. The gold standard quantitative viral outgrowth assay (QVOA) underestimates the magnitude of the viral reservoir. We sought to determine whether xenograft of leukocytes from HIV type 1 (HIV)-infected patients with undetectable plasma viral loads into immunocompromised mice would result in viral amplification. METHODS: Peripheral blood mononuclear cells or purified CD4(+) T cells from HIV or simian immunodeficiency virus (SIV)-infected subjects with undetectable plasma viral loads were adoptively transferred into NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. The mice were monitored for viremia following depletion of human CD8(+) T cells to minimize antiviral activity. In some cases, humanized mice were also treated with activating anti-CD3 antibody. RESULTS: With this murine viral outgrowth assay (MVOA), we successfully amplified replication-competent HIV or SIV from all subjects tested, including 5 HIV-positive patients receiving suppressive antiretroviral therapy (ART) and 6 elite controllers or suppressors who were maintaining undetectable viral loads without ART, including an elite suppressor from whom we were unable to recover virus by QVOA. CONCLUSIONS: Our results suggest that the MVOA has the potential to serve as a powerful tool to identify residual HIV in patients with undetectable viral loads.
Asunto(s)
Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , Carga Viral , Animales , Terapia Antirretroviral Altamente Activa , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Modelos Animales de Enfermedad , Infecciones por VIH/tratamiento farmacológico , VIH-1/crecimiento & desarrollo , Humanos , Interleucina-2/sangre , Leucocitos Mononucleares/virología , Macaca , Masculino , Ratones , Virus de la Inmunodeficiencia de los Simios/crecimiento & desarrollo , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificación , Viremia/veterinariaRESUMEN
Immune pressure exerted by MHC class I-restricted cytotoxic T cells drives the development of viral escape mutations, thereby regulating HIV disease progression. Nonetheless, the relationship between host immunity and HIV central nervous system (CNS) disease remains poorly understood. The simian immunodeficiency virus (SIV) macaque model recapitulates key features of HIV infection including development of AIDS and CNS disease. To investigate cell-mediated immunity regulating SIV CNS disease progression, we compared the incidence of SIV encephalitis and the influence of MHC class I allele expression on the development of CNS disease in rhesus macaques (Macaca mulatta) versus pigtailed macaques (Macaca nemestrina). After inoculation with the immunosuppressive swarm SIV/DeltaB670 and the neurovirulent molecular clone SIV/17E-Fr, pigtailed macaques progressed more rapidly to AIDS, had higher plasma and cerebrospinal fluid (CSF) viral loads, and were more likely to progress to SIV-associated encephalitis (SIVE) compared to rhesus macaques. In addition, MHC class I alleles were neuroprotective in both species (Mamu-A*001 in rhesus macaques and Mane-A1*084:01:01 in pigtailed macaques); animals expressing these alleles were less likely to develop SIV encephalitis and correspondingly had lower viral replication in the brain. Species-specific differences in susceptibility to SIV disease demonstrated that cell mediated immune responses are critical to SIV CNS disease progression.
Asunto(s)
Susceptibilidad a Enfermedades/inmunología , Macaca mulatta/virología , Macaca nemestrina/virología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Complejo SIDA Demencia/inmunología , Animales , Progresión de la Enfermedad , Antígenos de Histocompatibilidad Clase I/inmunología , Macaca mulatta/inmunología , Macaca nemestrina/inmunología , Virus de la Inmunodeficiencia de los SimiosRESUMEN
Activation of the kynurenine pathway (KP) of tryptophan catabolism likely contributes to HIV-associated neurological disorders. However, KP activation in brain tissue during HIV infection has been understudied, and the effect of combination antiretroviral therapy (cART) on KP induction in the brain is unknown. To examine these questions, tryptophan, kynurenine, 3-hydroxykynurenine, quinolinic acid, and serotonin levels were measured longitudinally during SIV infection in the striatum and CSF from untreated and cART-treated pigtailed macaques. Messenger RNA (mRNA) levels of KP enzymes also were measured in the striatum. In untreated macaques, elevations in KP metabolites coincided with transcriptional induction of upstream enzymes in the KP. Striatal KP induction was also temporally associated-but did not directly correlate-with serotonin losses in the brain. CSF quinolinic acid/tryptophan ratios were found to be the earliest predictor of neurological disease in untreated SIV-infected macaques, outperforming other KP metabolites as well as the putative biomarkers interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Finally, cART did not restore KP metabolites to control levels in the striatum despite the control of the virus, though CSF metabolite levels were normalized in most animals. Overall, these results demonstrate that cerebral KP activation is only partially resolved with cART and that CSF QUIN/TRP ratios are an early, predictive biomarker of CNS disease.
Asunto(s)
Encéfalo/metabolismo , Quinurenina/metabolismo , Ácido Quinolínico/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/metabolismo , Triptófano/metabolismo , Animales , Antirretrovirales/farmacología , Encéfalo/virología , Ensayo de Inmunoadsorción Enzimática , Cromatografía de Gases y Espectrometría de Masas , Inmunohistoquímica , Macaca , Reacción en Cadena de la PolimerasaRESUMEN
The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.
Asunto(s)
Infecciones por Virus ADN/veterinaria , Ranavirus/clasificación , Tortugas/inmunología , Animales , Infecciones por Virus ADN/inmunología , Infecciones por Virus ADN/mortalidad , Infecciones por Virus ADN/virología , ADN Viral/sangre , ADN Viral/aislamiento & purificación , Tortugas/virologíaRESUMEN
We recently demonstrated direct evidence of increased monoamine oxidase (MAO) activity in the brain of a simian immunodeficiency virus (SIV) model of human immunodeficiency virus (HIV)-associated central nervous system (CNS) disease, consistent with previously reported dopamine deficits in both SIV and HIV infection. In this study, we explored potential mechanisms behind this elevated activity. MAO B messenger RNA was highest in macaques with the most severe SIV-associated CNS lesions and was positively correlated with levels of CD68 and GFAP transcripts in the striatum. MAO B messenger RNA also correlated with viral loads in the CNS of SIV-infected macaques and with oxidative stress. Furthermore, in humans, striatal MAO activity was elevated in individuals with HIV encephalitis, compared with activity in HIV-seronegative controls. These data suggest that the neuroinflammation and oxidative stress caused by SIV infection in the CNS may provide the impetus for increased transcription of MAO B and that MAO, and more broadly, oxidative stress, have significant potential as therapeutic targets in CNS disease due to HIV.
Asunto(s)
Complejo SIDA Demencia/enzimología , Encéfalo/enzimología , Monoaminooxidasa/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/enzimología , Adulto , Animales , Química Encefálica , Cuerpo Estriado/enzimología , Femenino , Perfilación de la Expresión Génica , Glutatión/análisis , Humanos , Macaca nemestrina/virología , Masculino , Persona de Mediana Edad , Estrés Oxidativo , Reacción en Cadena en Tiempo Real de la Polimerasa , Carga ViralRESUMEN
Elevated cerebrospinal fluid (CSF) levels of markers of oxidative stress, neuronal injury, and inflammation and decreased neurotransmitter levels have been reported in HIV-associated neurocognitive disorders (HAND). Minocycline may have a neuroprotective effect by inhibiting inducible nitric oxide synthase, which produces nitric oxide, a compound that induces oxygen free radical production. In A5235, "Phase II, Randomized, Placebo-Controlled, Double-Blind Study of Minocycline in the Treatment of HIV-Associated Cognitive Impairment," minocycline was not associated with cognitive improvement, but the effect on the above CSF measures was not examined previously. The objective of this study was to examine the effect of minocycline on markers of oxidative stress, neuronal injury, neurotransmitter levels, and inflammation from CSF in participants in A5235. One hundred seven HIV+ individuals received either minocycline 100 mg or placebo orally every 12 h for 24 weeks. Twenty-one HIV+ individuals received the optional lumbar punctures. Lipid and protein markers of oxidative stress (e.g., ceramides and protein carbonyls), glutamate, neurotransmitter precursors, kynurenine metabolites, neurofilament heavy chain, and inflammatory cytokines were measured in the CSF before and after treatment. The 24-week change in ceramides was larger in a beneficial direction in the minocycline group compared to the placebo group. The two groups did not differ in the 24-week changes for other markers.These results suggest that minocycline may decrease lipid markers of oxidative stress (ceramides) in individuals with HAND; however, an effect of minocycline on other CSF markers was not observed. A larger sample size is needed to further validate these results.
Asunto(s)
Complejo SIDA Demencia/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Minociclina/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Complejo SIDA Demencia/líquido cefalorraquídeo , Complejo SIDA Demencia/patología , Complejo SIDA Demencia/virología , Síndrome de Inmunodeficiencia Adquirida/líquido cefalorraquídeo , Síndrome de Inmunodeficiencia Adquirida/patología , Síndrome de Inmunodeficiencia Adquirida/virología , Administración Oral , Adulto , Ceramidas/líquido cefalorraquídeo , Citocinas/líquido cefalorraquídeo , Método Doble Ciego , Femenino , Ácido Glutámico/líquido cefalorraquídeo , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Quinurenina/líquido cefalorraquídeo , Masculino , Persona de Mediana Edad , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/virología , Óxido Nítrico Sintasa de Tipo II/líquido cefalorraquídeo , Estrés OxidativoRESUMEN
Effective combined antiretroviral therapy (cART) in HIV-infected patients has made HIV a treatable infection; however, debilitating HIV-associated neurocognitive disorders (HAND) can still affect approximately 50% of HIV-infected individuals even under cART. While cART has greatly reduced the prevalence of the most severe form of HAND, milder forms have increased in prevalence, leaving the total proportion of HIV-infected individuals suffering from HAND relatively unchanged. In this study, an in vitro drug screen identified fluconazole and paroxetine as protective compounds against HIV gp120 and Tat neurotoxicity. Using an accelerated, consistent SIV/macaque model of HIV-associated CNS disease, we tested the in vivo neuroprotective capabilities of combination fluconazole/paroxetine (FluPar) treatment. FluPar treatment protected macaques from SIV-induced neurodegeneration, as measured by neurofilament light chain in the CSF, APP accumulation in axons, and CaMKIIα in the frontal cortex, but did not significantly reduce markers of neuroinflammation or plasma or CNS viral loads. Since HIV and SIV neurodegeneration is often attributed to accompanying neuroinflammation, this study provides proof of concept that neuroprotection can be achieved even in the face of ongoing neuroinflammation and viral replication.
Asunto(s)
Fluconazol/farmacología , Neuronas/efectos de los fármacos , Nootrópicos/farmacología , Paroxetina/farmacología , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Complejo SIDA Demencia/tratamiento farmacológico , Complejo SIDA Demencia/fisiopatología , Complejo SIDA Demencia/virología , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Síndrome de Inmunodeficiencia Adquirida/virología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Humanos , Macaca nemestrina , Proteínas de Neurofilamentos/líquido cefalorraquídeo , Proteínas de Neurofilamentos/genética , Neuronas/patología , Neuronas/virología , Cultivo Primario de Células , Ratas , Síndrome de Inmunodeficiencia Adquirida del Simio/líquido cefalorraquídeo , Síndrome de Inmunodeficiencia Adquirida del Simio/fisiopatología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Virus de la Inmunodeficiencia de los Simios/fisiología , Carga Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
HIV-associated neurocognitive disorder (HAND), characterized by a wide spectrum of behavioral, cognitive, and motor dysfunctions, continues to affect approximately 50 % of HIV(+) patients despite the success of combination antiretroviral drug therapy (cART) in the periphery. Of note, potential toxicity of antiretroviral drugs in the central nervous system (CNS) remains remarkably underexplored and may contribute to the persistence of HAND in the cART era. Previous studies have shown antiretrovirals (ARVs) to be neurotoxic in the peripheral nervous system in vivo and in peripheral neurons in vitro. Alterations in lipid and protein metabolism, mitochondrial damage, and oxidative stress all play a role in peripheral ARV neurotoxicity. We hypothesized that ARVs also induce cellular stresses in the CNS, ultimately leading to neuronal damage and contributing to the changing clinical and pathological picture seen in HIV-positive patients in the cART era. In this report, we show that ARVs are neurotoxic in the CNS in both pigtail macaques and rats in vivo. Furthermore, in vitro, ARVs lead to accumulation of reactive oxygen species (ROS), and ultimately induction of neuronal damage and death. Whereas ARVs alone caused some activation of the endogenous antioxidant response in vitro, augmentation of this response by a fumaric acid ester, monomethyl fumarate (MMF), blocked ARV-induced ROS generation, and neuronal damage/death. These findings implicate oxidative stress as a contributor to the underlying mechanisms of ARV-induced neurotoxicity and will provide an access point for adjunctive therapies to complement ARV therapy and reduce neurotoxicity in this patient population.
Asunto(s)
Complejo SIDA Demencia/patología , Antirretrovirales/toxicidad , Encéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Animales , Western Blotting , Encéfalo/patología , Encéfalo/virología , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Técnica del Anticuerpo Fluorescente , Macaca , Masculino , Neuronas/patología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Simian immunodeficiency virus (SIV) infection in macaques is so far the best animal model for human immunodeficiency virus type 1 (HIV-1) studies, but suppressing viral replication in infected animals remains challenging. Using a novel single-round infectivity assay, we quantitated the antiviral activities of antiretroviral drugs against SIV. Our results emphasize the importance of the dose-response curve slope in determining the inhibitory potential of antiretroviral drugs and provide useful information for regimen selection in treating SIV-infected animals in models of therapy and virus eradication.
Asunto(s)
Fármacos Anti-VIH/farmacología , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Animales , Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Humanos , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Proteínas del Envoltorio Viral/genética , Replicación Viral/efectos de los fármacosRESUMEN
HIV infection of the CNS is an early event after primary infection, resulting in neurological complications in a significant number of individuals despite antiretroviral therapy (ART). The main cells infected with HIV within the CNS are macrophages/microglia and a small fraction of astrocytes. The role of these few infected astrocytes in the pathogenesis of neuroAIDS has not been examined extensively. Here, we demonstrate that few HIV-infected astrocytes (4.7 ± 2.8% in vitro and 8.2 ± 3.9% in vivo) compromise blood-brain barrier (BBB) integrity. This BBB disruption is due to endothelial apoptosis, misguided astrocyte end feet, and dysregulation of lipoxygenase/cyclooxygenase, BK(Ca) channels, and ATP receptor activation within astrocytes. All of these alterations in BBB integrity induced by a few HIV-infected astrocytes were gap junction dependent, as blocking these channels protected the BBB from HIV-infected astrocyte-mediated compromise. We also demonstrated apoptosis in vivo of BBB cells in contact with infected astrocytes using brain tissue sections from simian immunodeficiency virus-infected macaques as a model of neuroAIDS, suggesting an important role for these few infected astrocytes in the CNS damage seen with HIV infection. Our findings describe a novel mechanism of bystander BBB toxicity mediated by low numbers of HIV-infected astrocytes and amplified by gap junctions. This mechanism of toxicity contributes to understanding how CNS damage is spread even in the current ART era and how minimal or controlled HIV infection still results in cognitive impairment in a large population of infected individuals.
Asunto(s)
Astrocitos/virología , Barrera Hematoencefálica/virología , Encéfalo/virología , Uniones Comunicantes/virología , Animales , Astrocitos/patología , Barrera Hematoencefálica/patología , Encéfalo/patología , Recuento de Células , Células Cultivadas , Células Endoteliales/patología , Células Endoteliales/virología , Técnica del Anticuerpo Fluorescente , Uniones Comunicantes/patología , VIH-1 , Humanos , Etiquetado Corte-Fin in Situ , Macaca nemestrina , Síndrome de Inmunodeficiencia Adquirida del Simio/patología , Síndrome de Inmunodeficiencia Adquirida del Simio/virologíaRESUMEN
In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8(+) T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8(+) T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4(+) T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8(+) T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.
Asunto(s)
Antirretrovirales/administración & dosificación , Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/genética , Mutación Missense , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Sustitución de Aminoácidos/genética , Animales , Linfocitos T CD8-positivos/inmunología , Productos del Gen gag/genética , Productos del Gen gag/inmunología , Epítopos Inmunodominantes/inmunología , Macaca , Selección Genética , Síndrome de Inmunodeficiencia Adquirida del Simio/virologíaRESUMEN
Interferon-ß induction occurs during acute simian immunodeficiency virus (SIV) infection in the brain. We have examined expression and function of cytosolic RNA sensors, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5), in vivo in the brain of our consistent, accelerated SIV-macaque model and in vitro in SIV-infected macaque macrophages to identify the pathway of type I interferon (IFN) induction. MDA5 messenger RNA (mRNA) and protein were expressed at higher levels in the brain than RIG-I, with protein expression correlating with the severity of disease from 42 until 84 days post-inoculation. The siRNA experiments reveal that mRNA expression of IFN-inducible gene MxA is dependent on MDA5, but not RIG-I. Finally, we demonstrate that SIV infection leads to the production of double-stranded RNA in vivo, which may act as the MDA5 ligand. We have shown for the first time to our knowledge the functional role of MDA5 in the innate immune response to SIV infection.
Asunto(s)
Encefalitis Viral/inmunología , Inmunidad Innata , Interferón-alfa/inmunología , Interferón beta/inmunología , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Virus de la Inmunodeficiencia de los Simios/inmunología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Encefalitis Viral/metabolismo , Proteínas de Unión al GTP/metabolismo , Interferón-alfa/metabolismo , Interferón beta/metabolismo , Macaca nemestrina , Macrófagos/metabolismo , Modelos Animales , Proteínas de Resistencia a Mixovirus , ARN Helicasas/genética , ARN Helicasas/inmunología , Índice de Severidad de la Enfermedad , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
Of the 33 million people infected with the human immunodeficiency virus (HIV) worldwide, 40-60% of individuals will eventually develop neurocognitive sequelae that can be attributed to the presence of HIV-1 in the central nervous system (CNS) and its associated neuroinflammation despite antiretroviral therapy. PrP(C) (protease resistant protein, cellular isoform) is the nonpathological cellular isoform of the human prion protein that participates in many physiological processes that are disrupted during HIV-1 infection. However, its role in HIV-1 CNS disease is unknown. We demonstrate that PrP(C) is significantly increased in both the CNS of HIV-1-infected individuals with neurocognitive impairment and in SIV-infected macaques with encephalitis. PrP(C) is released into the cerebrospinal fluid, and its levels correlate with CNS compromise, suggesting it is a biomarker of HIV-associated neurocognitive impairment. We show that the chemokine (c-c Motif) Ligand-2 (CCL2) increases PrP(C) release from CNS cells, while HIV-1 infection alters PrP(C) release from peripheral blood mononuclear cells. Soluble PrP(C) mediates neuroinflammation by inducing astrocyte production of both CCL2 and interleukin 6. This report presents the first evidence that PrP(C) dysregulation occurs in cognitively impaired HIV-1-infected individuals and that PrP(C) participates in the pathogenesis of HIV-1-associated CNS disease.
Asunto(s)
Biomarcadores/metabolismo , Trastornos del Conocimiento/etiología , Infecciones por VIH/complicaciones , VIH-1/patogenicidad , Inflamación/etiología , Proteínas PrPC/metabolismo , Adulto , Animales , Astrocitos/citología , Astrocitos/metabolismo , Encefalopatías/etiología , Encefalopatías/metabolismo , Células Cultivadas , Quimiocina CCL2 , Trastornos del Conocimiento/metabolismo , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Infecciones por VIH/virología , Humanos , Técnicas para Inmunoenzimas , Inflamación/metabolismo , Macaca nemestrina , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/metabolismo , Neuronas/metabolismo , Neuronas/patología , Proteínas PrPC/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/complicaciones , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/patogenicidadRESUMEN
The CNS remains vulnerable to HIV-induced damage despite highly active antiretroviral therapy (HAART). Using a rigorous simian immunodeficiency virus (SIV) macaque model of HAART that combines three classes of antiretroviral drugs (a protease inhibitor, a reverse transcriptase inhibitor, and an integrase inhibitor), we examined immune responses and virus replication in the plasma and cerebrospinal fluid (CSF) following HAART initiation during acute infection (4 days postinoculation (p.i.)). HAART-treated macaques did not experience the level of acute CD4+ and CD8+ T cell and NK cell count suppression in the peripheral blood normally observed during acute infection. Initiation of HAART produced a rapid four-log decline in viral load in plasma and a slower two-log decline of viral RNA in the CSF over the subsequent 17 days of infection. Despite a dramatic reduction of viral RNA levels in the brain at 21 days p.i., viral DNA levels were not different between the two groups. Expression of most cytokine mRNA in brain of HAART-treated macaques did not significantly differ from untreated controls. Expression of the IFN responsive gene MxA was significantly reduced in the brain of HAART-treated macaques, suggesting control of hyperactive immune responses. Control of virus replication likely was enhanced by significant increases in CD4+ and CD8+ T cell trafficking in the brain of infected animals on HAART therapy and the concomitant increase in levels of IFNγ. Collectively, these data indicate preserved innate and adaptive immune activity in the brain following HAART initiation during acute SIV infection in this macaque model, suggesting profound benefits following acute treatment of SIV.