RESUMEN
Methods to detect intracellular kinase signaling intermediates by flow cytometry have been recently developed. Termed "phospho-flow," these methods employ fluorescence-conjugated monoclonal antibodies that recognize phosphorylated epitopes of intracellular kinases, and may be combined with surface phenotypic markers to observe changes in kinase pathways by cellular subset. Effector functions, like cytokine production, are processes intrinsically linked to intracellular signaling and kinase activity within each cell. Methodologies that would simultaneously detect changes to signaling pathways as well as effector responses at the single-cell level would allow for mapping of the functional consequences induced by signaling pathway modifications. However, there are challenges to developing such a combined protocol, relating to the different kinetics of rapid signaling events and the more prolonged time required to induce and observe cytokine responses. In this report, we describe the development of an assay that accommodates differences in protocol conditions and response kinetics, merging phospho-flow cytometry, and intracellular cytokine staining methods into a single experimental protocol. We examined intracellular ERK1/2 phosphorylation and IFN-γ production by CD4+ and CD8+ T cells upon polyclonal stimulation with PMA and ionomycin, while monitoring expression of the cytolytic molecule perforin and the T cell activation marker CD38. We present a method that allows observation of kinase phosphorylation and cytokine production within the same cell after stimuli, while maintaining a stable cellular phenotype. Monitoring of signaling and effector functions in distinct immune subsets provides a platform to investigate and relate intracellular kinase signaling activity to immune cell effector function and phenotype in disease states.
Asunto(s)
Citocinas/biosíntesis , Citometría de Flujo , Ionomicina/farmacología , Activación de Linfocitos/inmunología , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Anticuerpos Monoclonales/inmunología , Citocinas/inmunología , Citometría de Flujo/métodos , Humanos , Fosforilación , Fosfotransferasas/metabolismo , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Natural killer (NK) cells are innate lymphocytes that play an important role against viral infections and cancer. This effect is achieved through a complex mosaic of inhibitory and activating receptors expressed by NK cells that ultimately determine the magnitude of the NK-cell response. The T-cell immunoglobulin- and mucin domain-containing (Tim)-3 receptor was initially identified as a T-helper 1-specific type I membrane protein involved in regulating T-cell responses. Human NK cells transcribe the highest amounts of Tim-3 among lymphocytes. Tim-3 protein is expressed on essentially all mature CD56(dim)CD16(+) NK cells and is expressed heterogeneously in the immature CD56(bright)CD16(-) NK-cell subset in blood from healthy adults and in cord blood. Tim-3 expression was induced on CD56(bright)CD16(-) NK cells after stimulation with IL-15 or IL-12 and IL-18 in vitro, suggesting that Tim-3 is a maturation marker on NK cells. Whereas Tim-3 has been used to identify dysfunctional T cells, NK cells expressing high amounts of Tim-3 are fully responsive with respect to cytokine production and cytotoxicity. However, when Tim-3 was cross-linked with antibodies it suppressed NK cell-mediated cytotoxicity. These findings suggest that NK-cell responses may be negatively regulated when NK cells encounter target cells expressing cognate ligands of Tim-3.
Asunto(s)
Células Asesinas Naturales/inmunología , Subgrupos Linfocitarios/inmunología , Proteínas de la Membrana/inmunología , Linfocitos T Citotóxicos/inmunología , Biomarcadores/metabolismo , Antígeno CD56/inmunología , Antígeno CD56/metabolismo , Diferenciación Celular/inmunología , Línea Celular , Reactivos de Enlaces Cruzados/metabolismo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Inmunofenotipificación , Células Asesinas Naturales/citología , Ligandos , Subgrupos Linfocitarios/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Linfocitos T Citotóxicos/citología , Regulación hacia Arriba/inmunologíaRESUMEN
Chemokines and their receptors play an essential role within the immune system by dictating cellular migration. In vivo, receptor-ligand interactions rarely occur in isolation as cellular recruitment and migration are complex and highly coordinated processes often involving networks of multiple chemokines and multiple receptors. Simultaneous detection of multiple chemokine receptors on the single cell level is necessary to allow immunophenotyping studies that will help understand the intricacies of these networks. Chemokine receptors undergo a basal level of ongoing internalization, intracellular trafficking, and recycling back to the cell surface, even in the absence of the ligand. In the presence of ligand, receptor-ligand interactions enhance receptor internalization, reducing the cell surface receptor concentration, making precise determination of intrinsic levels challenging. Using multicolor flow cytometry, we sought to evaluate and optimize the simultaneous detection of cell surface expression levels of CCR2, CX3CR1, and CCR5 in primary human monocytes using a single antibody panel. We observed that staining for CCR2 alone or for CX3CR1 alone showed greater expression levels than when the cells were stained with the full panel of antibodies. Fluorescent-minus-one (FMO) controls revealed that ligation of the CCR5 monoclonal antibody to the receptor interfered with detection of CX3CR1 and CCR2. Sequential addition of antibodies during the staining procedure was sufficient to restore the detection levels, suggesting close proximity and possible functional interactions between CCR2/CCR5 and CX3CR1/CCR5 in monocytes. This study highlights the importance of optimizing staining procedures and using proper controls when simultaneously evaluating expression levels of multiple chemokine receptors by flow cytometry. Concurrent assessment of multiple receptors will provide insight and greater understanding of the complex interactions involved in cellular migration.
Asunto(s)
Citometría de Flujo/métodos , Monocitos/inmunología , Receptores CCR2/análisis , Receptores CCR5/análisis , Receptores de Quimiocina/análisis , Adulto , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Receptor 1 de Quimiocinas CX3C , Movimiento Celular , Femenino , Humanos , Masculino , Receptores CCR2/inmunología , Receptores CCR5/inmunología , Receptores de Quimiocina/inmunología , Coloración y EtiquetadoRESUMEN
Mitogen-activated protein kinase (MAPK) signaling pathways are dynamic and sensitive regulators of T cell function and differentiation. Altered MAPK signaling has been associated with the inflammatory and autoimmune diseases lupus and arthritis and with some pathogenic viral infections. HIV-1 infection is characterized by chronic immune inflammation, aberrantly heightened CD8(+) T cell activation levels, and altered T cell function. The relationship between MAPK pathway function, HIV-1-induced activation (CD38 and HLA-DR), and exhaustion (Tim-3) markers in circulating CD8(+) T cells remains unknown. Phosphorylation of the MAPK effector proteins ERK and p38 was examined by "phosflow" flow cytometry in 79 recently HIV-1-infected, antiretroviral-treatment-naïve adults and 21 risk-matched HIV-1-negative controls. We identified a subset of CD8(+) T cells refractory to phorbol 12-myristate 13-acetate plus ionomycin-induced ERK1/2 phosphorylation (referred to as p-ERK1/2-refractory cells) that was greatly expanded in HIV-1-infected adults. The CD8(+) p-ERK1/2-refractory cells were highly activated (CD38(+) HLA-DR(+)) but not exhausted (Tim-3 negative), tended to have low CD8 expression, and were enriched in intermediate and late transitional memory states of differentiation (CD45RA(-) CD28(-) CD27(+/-)). Targeting MAPK pathways to restore ERK1/2 signaling may normalize immune inflammation levels and restore CD8(+) T cell function during HIV-1 infection.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/patogenicidad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal , Adulto , Linfocitos T CD8-positivos/patología , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Infecciones por VIH/patología , Humanos , Activación de Linfocitos , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Carga ViralRESUMEN
The complexity of immunoregulation has focused attention on the CD4+ T "suppressor" regulatory cell (Treg), which helps maintain balance between immunity and tolerance. An immunoregulatory T-cell population that upon activation amplifies cellular immune responses was described in murine models more than 30 years ago; however, no study has yet identified a naturally occurring T "inducer" cell type. Here, we report that the ectoenzyme CD39/NTPDase1 (ecto-nucleoside triphosphate diphosphohydrolase 1) helps to delineate a novel population of human "inducer" CD4+ T cells (Tind) that significantly increases the proliferation and cytokine production of responder T cells in a dose-dependent manner. Furthermore, this unique Tind subset produces a distinct repertoire of cytokines in comparison to the other CD4+ T-cell subsets. We propose that this novel CD4+ T-cell population counterbalances the suppressive activity of suppressor Treg in peripheral blood and serves as a calibrator of immunoregulation.
Asunto(s)
Antígenos CD/inmunología , Apirasa/inmunología , Linfocitos T CD4-Positivos/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/citología , Proliferación Celular , Citocinas/biosíntesis , Citocinas/inmunología , Humanos , Subgrupos de Linfocitos T/citologíaRESUMEN
Chronic immune activation and progression to AIDS are observed after SIV infection in macaques but not in natural host primate species. To better understand this dichotomy, we compared acute pathogenic SIV infection in pigtailed macaques (PTs) to non-pathogenic infection in African green monkeys (AGMs). SIVagm-infected PTs, but not SIVagm-infected AGMs, rapidly developed systemic immune activation, marked and selective depletion of IL-17-secreting (Th17) cells, and loss of the balance between Th17 and T regulatory (Treg) cells in blood, lymphoid organs, and mucosal tissue. The loss of Th17 cells was found to be predictive of systemic and sustained T cell activation. Collectively, these data indicate that loss of the Th17 to Treg balance is related to SIV disease progression.
Asunto(s)
Interleucina-17/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Análisis de Varianza , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Chlorocebus aethiops , Colon/inmunología , Colon/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Interleucina-17/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Activación de Linfocitos , Macaca nemestrina , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Viral/análisis , ARN Viral/sangre , ARN Viral/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios , Estadísticas no Paramétricas , Linfocitos T Colaboradores-Inductores/virología , Linfocitos T Reguladores/virología , Carga ViralRESUMEN
Natural killer (NK) cells bridge the interface between innate and adaptive immunity and are implicated in the control of herpes simplex virus 2 (HSV-2) infection. In subjects infected with human immunodeficiency virus 1 (HIV-1), the critical impact of the innate immune response on disease progression has recently come into focus. Higher numbers of NK cells are associated with lower HIV-1 plasma viraemia. Individuals with the compound genotype of killer cell immunoglobulin-like receptor (KIR) 3DS1 and human leucocyte antigen (HLA)-Bw4-80I, or who have alleles of KIR3DL1 that encode proteins highly expressed on the NK cell surface, have a significant delay in disease progression. We studied the effect of HSV-2 co-infection in HIV-1-infected subjects, and show that HSV-2 co-infection results in a pan-lymphocytosis, with elevated absolute numbers of CD4(+) and CD8(+) T cells, and NK cells. The NK cells in HSV-2 co-infected subjects functioned more efficiently, with an increase in degranulation after in vitro stimulation. The number of NK cells expressing the activating receptors NKp30 and NKp46, and expressing KIR3DL1 or KIR3DS1, was inversely correlated with HIV-1 plasma viral load in subjects mono-infected with HIV-1, but not in subjects co-infected with HSV-2. This suggests that HSV-2 infection mediates changes within the NK cell population that may affect immunity in HIV-1 infection.
Asunto(s)
Seropositividad para VIH/inmunología , VIH-1/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 2/inmunología , Células Asesinas Naturales/metabolismo , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Recuento de Células , Células Cultivadas , Citotoxicidad Inmunológica , Progresión de la Enfermedad , Femenino , Seropositividad para VIH/complicaciones , Seropositividad para VIH/patología , Seropositividad para VIH/fisiopatología , VIH-1/patogenicidad , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Herpes Simple/complicaciones , Herpes Simple/patología , Herpes Simple/fisiopatología , Herpesvirus Humano 2/patogenicidad , Prueba de Histocompatibilidad , Humanos , Inmunidad Innata , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Células Asesinas Naturales/virología , Linfocitosis , Masculino , Persona de Mediana Edad , Receptores KIR3DL1/genética , Receptores KIR3DL1/metabolismo , Receptores KIR3DS1/genética , Receptores KIR3DS1/metabolismoRESUMEN
Little is known about the manipulation of IL-17 producing CD4+ T cells (T(H)17) on a per-cell basis in humans in vivo. Previous studies on the effects of IL-2 on IL-17 secretion in non-HIV models have shown divergent results. We hypothesized that IL-2 would mediate changes in IL-17 levels among recently HIV-1-infected adults receiving anti-retroviral therapy. We measured cytokine T cell responses to CD3/CD28, HIV-1 Gag, and CMV pp65 stimulation, and changes in multiple CD4+ T cell subsets. Those who received IL-2 showed a robust expansion of naive and total CD4+ T cell counts and T-reg counts. However, after IL-2 treatment, the frequency of T(H)17 cells declined, while counts of T(H)17 cells did not change due to an expansion of the CD4+ naïve T cell population (CD27+CD45RA+). Counts of HIV-1 Gag-specific T cells declined modestly, but CMV pp65 and CD3/CD28 stimulated populations did not change. Hence, in contrast with recent studies, our results suggest IL-2 is not a potent in vivo regulator of T(H)17 cell populations in HIV-1 disease. However, IL-2-mediated T-reg expansions may selectively reduce responses to certain antigen-specific populations, such as HIV-1 Gag.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/inmunología , Inmunoterapia , Interleucina-2/administración & dosificación , Células Th17/efectos de los fármacos , Adulto , Antirretrovirales/uso terapéutico , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Recuento de Células , Proliferación Celular/efectos de los fármacos , Quimioterapia Combinada , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Humanos , Inmunomodulación , Interleucina-17/biosíntesis , Interleucina-17/genética , Interleucina-2/uso terapéutico , Fosfoproteínas/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T , Células Th17/inmunología , Células Th17/patología , Células Th17/virología , Proteínas de la Matriz Viral/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/inmunologíaRESUMEN
A flurry of recent reports on the role of activating and inhibitory forms of the killer cell immunoglobulin-like receptors (KIR) in natural killer (NK) cell activity against human immunodeficiency virus type 1 (HIV-1) have yielded widely divergent results. The role of the activating NK receptor encoded by the KIR3DS1 allele and its putative ligands, members of the HLA class I Bw4Ile80 cluster, in early HIV-1 disease is controversial. We selected 60 treatment-naïve adults for study from the OPTIONS cohort of individuals with early HIV-1 infection in San Francisco. We performed NK cell functional assays measuring gamma interferon (IFN-gamma) and CD107a expression by NK cells in the unstimulated state and after stimulation by the major histocompatibility complex class I-deficient 721.221 B-lymphoblastoid cell line. In addition, we measured CD38 expression (a T-cell activation marker) on T and NK cells. Persons who have at least one copy of the KIR3DS1 gene had higher IFN-gamma and CD107a expression in the unstimulated state compared to those who do not possess this gene. After stimulation, both groups experienced a large induction of IFN-gamma and CD107a, with KIR3DS1 carriers achieving a greater amount of IFN-gamma expression. Differences in effector activity correlating with KIR3DS1 were not attributable to joint carriage of HLA Bw4Ile80 and KIR3DS1. We detected a partial but not complete dependence of KIR3DS1 on the members of B*58 supertype (B*57 and B*58) leading to higher NK cell function. Possessing KIR3DS1 was associated with lower expression of CD38 on both CD8(+) T and NK cells and with a loss or weakening of the known strong associations between CD8(+) T-cell expression of CD38 mean fluorescence intensity and the HIV-1 viral load. We observed that possessing KIR3DS1 was associated with higher NK cell effector functions in early HIV-1 disease, despite the absence of HLA Bw4Ile80, a putative ligand of KIR3DS1. Carriage of KIR3DS1 was associated with diminished CD8(+) T-cell activation, as determined by expression of CD38, and a disruption of the traditional relationship between viral load and activation in HIV-1 disease, which may lead to better clinical outcomes for these individuals.
Asunto(s)
Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/inmunología , Células Asesinas Naturales/inmunología , Receptores KIR3DS1/inmunología , ADP-Ribosil Ciclasa 1/biosíntesis , Adulto , Linfocitos T CD8-positivos/inmunología , Femenino , Citometría de Flujo , Genotipo , Infecciones por VIH/genética , Antígenos HLA-B/genética , Humanos , Interferón gamma/biosíntesis , Proteína 1 de la Membrana Asociada a los Lisosomas/biosíntesis , Masculino , Receptores KIR3DS1/genética , San Francisco , Linfocitos T/inmunologíaRESUMEN
APOBEC3G and APOBEC3F are human cytidine deaminases that serve as innate antiviral defense mechanisms primarily by introducing C-to-U changes in the minus strand DNA of retroviruses during replication (resulting in G-to-A mutations in the genomic sense strand sequence). The HIV-1 Vif protein counteracts this defense by promoting the proteolytic degradation of APOBEC3G and APOBEC3F in the host cell. In the absence of Vif expression, APOBEC3 is incorporated into HIV-1 virions and the viral genome undergoes extensive G-to-A mutation, or "hypermutation", typically rendering it non-viable within a single replicative cycle. Consequently, Vif is emerging as an attractive target for pharmacological intervention and therapeutic vaccination. Although a highly effective Vif inhibitor may result in mutational meltdown of the viral quasispecies, a partially effective Vif inhibitor may accelerate the evolution of drug resistance and immune escape due to the codon structure and recombinogenic nature of HIV-1. This hypothesis rests on two principal assumptions which are supported by experimental evidence: a) there is a dose response between intracellular APOBEC concentration and degree of viral hypermutation, and, b) HIV-1 can tolerate an elevated mutation rate, and a true error or extinction threshold is as yet undetermined. Rigorous testing of this hypothesis will have timely and critical implications for the therapeutic management of HIV/AIDS, and delve into the complexities underlying the induction of lethal mutagenesis in a viral pathogen.
Asunto(s)
Citidina Desaminasa/metabolismo , Citosina Desaminasa/metabolismo , VIH-1/genética , Mutación , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Desaminasa APOBEC-3G , Secuencia de Aminoácidos , Secuencia de Bases , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , Humanos , Datos de Secuencia Molecular , Mutación/efectos de los fármacos , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidoresRESUMEN
We sought to determine whether pretreatment low pol replication capacity (polRC) is associated with CD4 T-cell count gains during antiretroviral therapy (ART). Patients were recruited at north American and Australian sites. Viral polRC was measured before starting ART in all subjects. We examined 243 individuals for a median 260 days after initiating ART. Low baseline polRC was associated with greater CD4 T-cell gains independent of virological responses.
Asunto(s)
Fármacos Anti-VIH/uso terapéutico , Genes pol/fisiología , Infecciones por VIH/tratamiento farmacológico , VIH-1/fisiología , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Estudios de Seguimiento , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Pronóstico , ARN Viral/sangre , Replicación Viral/genéticaRESUMEN
Human endogenous retrovirus K (HERV-K) is distinctive among the retroviruses that comprise about 8% of the human genome in that multiple HERV-K proviruses encode full-length viral proteins, and many HERV-K proviruses formed during recent human evolution. HERV-K gag proteins are found in the cytoplasm of primary tumor cells of patients with seminoma. We identified HERV-K-specific T cells in patients with a past history of seminoma using the interferon-gamma ELISPOT assay and an MHC-HERV-K peptide-specific tetramer. A minority of apparently healthy subjects without evident germ cell tumors also made HERV-K-specific T cell responses. In summary, we detected T cell reactivity to HERV-K peptides in both past seminoma patients and a minority of apparently healthy controls.
Asunto(s)
Anticuerpos Antivirales/análisis , Retrovirus Endógenos/inmunología , Seminoma/inmunología , Linfocitos T/inmunología , Anticuerpos Antivirales/sangre , Retrovirus Endógenos/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Seminoma/metabolismo , Seminoma/virología , Linfocitos T/metabolismo , Integración ViralRESUMEN
The problem of relating genotype (as represented by amino acid sequence) to phenotypes is distinguished from standard regression problems by the nature of sequence data. Here we investigate an instance of such a problem where the phenotype of interest is HIV-1 replication capacity and contiguous segments of protease and reverse transcriptase sequence constitutes genotype. A variety of data analytic methods have been proposed in this context. Shortcomings of select techniques are contrasted with the advantages afforded by tree-structured methods. However, tree-structured methods, in turn, have been criticized on grounds of only enjoying modest predictive performance. A number of ensemble approaches (bagging, boosting, random forests) have recently emerged, devised to overcome this deficiency. We evaluate random forests as applied in this setting, and detail why prediction gains obtained in other situations are not realized. Other approaches including logic regression, support vector machines and neural networks are also applied. We interpret results in terms of HIV-1 reverse transcriptase structure and function.
RESUMEN
BACKGROUND: Sustained elevations in CD4 cell counts commonly occur despite incomplete viral suppression with protease inhibitor-based antiretroviral therapy. OBJECTIVES: To determine the incidence and risk factors associated with return of CD4 cell count to pre-therapy levels in patients experiencing virologic failure of protease inhibitor therapy. DESIGN: This is a clinic-based cohort study of HIV-infected adults who failed to maintain durable viral suppression on a protease inhibitor-based regimen. MAIN OUTCOME MEASURES: Virologic failure was defined as persistent plasma HIV RNA level > 500 copies/ml. Immunologic failure was defined as return of CD4 cell count to pre-therapy levels. RESULTS: A total of 291 patients experienced virologic failure on a protease inhibitor-based regimen and had a treatment-mediated CD4 cell increase above pre-therapy levels at the time of virologic failure. If patient data were censored at the time a successful salvage regimen was initiated, then the median time to immunologic failure after the onset of virologic failure was 3 years. If patient data were also censored at the time therapy was discontinued, then 36.8% of the cohort experienced immunologic failure after 3 years of continuous virologic failure. The change in viral load from a pre-treatment baseline, and not the absolute level of viremia achieved, was a strong and independent predictor of immunologic failure. Discontinuing therapy was associated with immunologic failure independent of viral load changes. CONCLUSION: Reduction in T CD4+ cell numbers may eventually occur during prolonged virologic failure of a protease inhibitor-based regimen and is predicted by the degree of virologic suppression below a pre-therapy 'set-point'.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/efectos de los fármacos , Viremia/tratamiento farmacológico , Adulto , Terapia Antirretroviral Altamente Activa , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/citología , Progresión de la Enfermedad , Infecciones por VIH/sangre , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/genética , Humanos , Indinavir/uso terapéutico , Nelfinavir/uso terapéutico , ARN Viral/sangre , Ritonavir/uso terapéutico , Saquinavir/uso terapéutico , Factores de Tiempo , Insuficiencia del Tratamiento , Viremia/sangre , Viremia/inmunología , Viremia/virologíaRESUMEN
OBJECTIVES: Primary, or transmitted, drug resistance is common among treatment naive patients recently infected with HIV-1, and impairs response to anti-retroviral therapy. We previously observed that patients with secondary resistance (developed in response to anti-retroviral treatment) who chose to stop an anti-retroviral regimen experience rapid overgrowth of drug resistant viruses by wild-type virus of higher pol replication capacity. We sought to determine if primary drug resistance would be lost at a rapid rate, and viral pol replication capacity would increase, in the absence of treatment. METHODS: We tracked drug resistance phenotype, genotype, viral pol replication capacity (single cycle recombinant assay incorporating a segment of the patient pol gene [pol RC]), plasma HIV-1 RNA, and CD4 T cell counts in the absence of treatment among patients in early HIV-1 infection. RESULTS: Six of 22 patients had evidence of primary drug resistance to at least one class of drug; three resistant to protease inhibitors, three resistant to non-nucleoside reverse transcriptase inhibitors, and four resistant to nucleoside reverse transcriptase inhibitors. All six patients maintained evidence of drug resistance for the period of observation. Among patients with baseline primary drug resistance pol RC did not increase over time. CONCLUSION: The selection environment of early infection is determined by immune pressure, and stochastic events, not viral pol replication capacity. In contrast to secondary resistant infections that are rapidly overgrown when therapy is stopped, primary drug resistance persists over time. Surveillance and clinical detection of primary resistance is feasible in the first year of infection.
Asunto(s)
Antirretrovirales/uso terapéutico , Farmacorresistencia Viral/inmunología , Infecciones por VIH/tratamiento farmacológico , VIH-1 , Replicación Viral/inmunología , Adulto , Antirretrovirales/inmunología , Recuento de Linfocito CD4 , Farmacorresistencia Viral/genética , Genes pol/genética , Genes pol/inmunología , Genotipo , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Inhibidores de la Proteasa del VIH/inmunología , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/efectos de los fármacos , VIH-1/inmunología , Humanos , Fenotipo , ARN Viral/sangre , Inhibidores de la Transcriptasa Inversa/inmunología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Replicación Viral/genéticaRESUMEN
OBJECTIVE: To determine whether treatment with growth hormone (GH) enhances thymopoiesis in individuals infected with HIV-1. METHODS: Five HIV-1-infected adults were treated with GH for 6-12 months in a prospective open-label study. Immunological analyses were performed before GH treatment and repeated at 3 month intervals after GH initiation. Thymic mass was analysed using computed tomography with quantitative density and volume analysis. Analysis of circulating lymphocytes, including naive and memory T cell subsets, was performed using multiparameter flow cytometry. RESULTS: GH treatment was associated with a marked increase in thymic mass in all GH recipients. Circulating naive CD4 T cells also increased significantly in all patients during GH therapy, suggesting an enhancement of thymopoiesis. CONCLUSION: GH has significant effects on the human immune system, including the reversal of thymic atrophy in HIV-1-infected adults. De-novo T cell production may thus be inducible in immunodeficient adults.
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Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , Hormona de Crecimiento Humana/uso terapéutico , Timo/efectos de los fármacos , Adulto , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Hormona de Crecimiento Humana/efectos adversos , Humanos , Leucopoyesis/efectos de los fármacos , Masculino , Persona de Mediana Edad , Timo/patologíaAsunto(s)
Infecciones por VIH/inmunología , VIH-1 , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/inmunología , Alelos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Progresión de la Enfermedad , Antígenos HLA-B/inmunología , Humanos , Receptores KIRRESUMEN
Galectin-9 (Gal-9) is a ß-galactosidase-binding lectin that promotes apoptosis, tissue inflammation, and T cell immune exhaustion, and alters HIV infection in part through engagement with the T cell immunoglobulin mucin domain-3 (Tim-3) receptor and protein disulfide isomerases (PDI). Gal-9 was initially thought to be an eosinophil attractant, but is now known to mediate multiple complex signaling events that affect T cells in both an immunosuppressive and inflammatory manner. To understand the kinetics of circulating Gal-9 levels during HIV infection we measured Gal-9 in plasma during HIV acquisition, in subjects with chronic HIV infection with differing virus control, and in uninfected individuals. During acute HIV infection, circulating Gal-9 was detected as early as 5 days after quantifiable HIV RNA and tracked plasma levels of interleukin (IL)-10, tumor necrosis factor (TNF)-α, and IL-1ß. In chronic HIV infection, Gal-9 levels positively correlated with plasma HIV RNA levels (r=0.29; p=0.023), and remained significantly elevated during suppressive antiretroviral therapy (median: 225.3 pg/ml) and in elite controllers (263.3 pg/ml) compared to age-matched HIV-uninfected controls (54 pg/ml). Our findings identify Gal-9 as a novel component of the first wave of the cytokine storm in acute HIV infection that is sustained at elevated levels in virally suppressed subjects and suggest that Gal-9:Tim-3 crosstalk remains active in elite controllers and antiretroviral (ARV)-suppressed subjects, potentially contributing to ongoing inflammation and persistent T cell dysfunction.
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Galectinas/inmunología , Infecciones por VIH/inmunología , Proteínas de la Membrana/metabolismo , ARN Viral/sangre , Antirretrovirales/uso terapéutico , Terapia Antirretroviral Altamente Activa , Galectinas/sangre , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Receptor 2 Celular del Virus de la Hepatitis A , Humanos , Tolerancia Inmunológica/inmunología , Interleucina-10/sangre , Interleucina-1beta/sangre , Proteína Disulfuro Isomerasas/metabolismo , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Coronary artery calcium (CAC) is a validated subclinical measure of atherosclerosis. Studies in the general population have linked blood inflammatory biomarkers including monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor (TNF)-α with the burden of CAC, but this relationship is often lost following correction for traditional cardiovascular risk factors. We assessed the relationship of various biomarkers to CAC, specifically in HIV-infected individuals on potent antiretroviral therapy (ART). Analyses utilized entry data from participants in the Hawaii Aging with HIV-Cardiovascular (HAHC-CVD) study. Computerized tomography examinations for CAC were obtained locally and analyzed by a central reading center in blinded fashion. Plasma biomarkers were assessed by multiplexing using Milliplex Human Cardiovascular Disease panels. Among a cohort of 130 subjects [88% male, median (IQR) age of 51 (46-57) years, CD4 count of 492 (341-635) cells/mm(3), 86.9% with HIV RNA ≤50 copies/ml], CAC was present in 46.9% of subjects. In univariate analyses higher levels of log-transformed MCP-1 and TNF-α were associated with the presence of CAC (p<0.05). In multivariate logistic regression models, MCP-1 and TNF-α remained significant after adjustment for traditional cardiovascular (CVD) risk factors. Similar results were found when analyses were assessed by Framingham risk score categories or when restricted to subjects with plasma HIV RNA ≤50 copies/ml. In contrast to findings in the general population, higher MCP-1 and TNF-α predict the presence of CAC independent of traditional CVD risk factors in HIV-infected subjects fully suppressed on ART, suggesting that HIV-mediated immune activation may play a role in CVD risk.
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Aterosclerosis/diagnóstico , Calcio/análisis , Quimiocina CCL2/sangre , Enfermedad de la Arteria Coronaria/diagnóstico , Infecciones por VIH/complicaciones , Plasma/química , Factor de Necrosis Tumoral alfa/sangre , Aterosclerosis/diagnóstico por imagen , Biomarcadores/sangre , Estudios de Cohortes , Enfermedad de la Arteria Coronaria/diagnóstico por imagen , Enfermedad de la Arteria Coronaria/patología , Femenino , Hawaii , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Tomografía Computarizada por Rayos XRESUMEN
OBJECTIVE: To assess the role of HIV and monocytes/macrophages in adipose tissue dysregulation. METHODS: Cross-sectional study in 5 groups: HIV seronegative, HIV+ antiretroviral therapy (ART)-naive, HIV+ nonlipoatrophic on zidovudine- and/or stavudine-containing ART, HIV+ lipoatrophic on similar ART, and HIV+ on abacavir- or tenofovir-containing ART. HIV DNA in circulating monocyte subsets was quantitated by real-time polymerase chain reaction. Biopsied subcutaneous fat was examined for macrophage content by CD68 staining. Isolated adipocytes and macrophages were cultured and the supernatant assayed for secretory products by Luminex multiplex cytokine technology. RESULTS: Sixty-nine subjects were enrolled. Lipoatrophic subjects had higher median HIV DNA levels (270.5 copies/10 cells) in circulating peripheral CD14CD16 co-expressing monocyte subsets compared with subjects who were ART-naive (25.0 copies), nonlipoatrophic (15.0 copies), or on abacavir/tenofovir (57.5 copies), P < 0.01. Group differences in adipocytes and adipose macrophage content were marginal. Although adipocyte secretory products were similar, HIV-infected subjects had higher adipose macrophage-derived interleukin (IL)-12p40, IL-6, IL-8, and monocyte inflammatory protein 1 alpha and lower eotaxin and interferon gamma levels than HIV seronegative subjects (P < 0.05). Within HIV-infected subjects, adipose macrophage secretory products were comparable between subjects naive with ART versus those on ART. CONCLUSIONS: Circulating HIV-infected and proinflammatory CD14CD16 monocyte subsets contribute to the pathogenesis of HIV-associated lipoatrophy. Among HIV-infected individuals, macrophages, rather than adipocytes, are the primary source of low-grade inflammation in subcutaneous adipose tissue. HIV infection modifies these macrophages to a more proinflammatory phenotype, and these changes are not substantially mitigated by the use of ART.