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1.
AIDS Res Hum Retroviruses ; 30(7): 654-64, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24786365

RESUMO

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.


Assuntos
Galectinas/imunologia , Infecções por HIV/imunologia , Proteínas de Membrana/metabolismo , RNA Viral/sangue , Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Galectinas/sangue , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Tolerância Imunológica/imunologia , Interleucina-10/sangue , Interleucina-1beta/sangue , Isomerases de Dissulfetos de Proteínas/metabolismo , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/sangue
2.
PLoS One ; 9(2): e90330, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24587328

RESUMO

BACKGROUND: Rates of insulin resistance are increased in HIV-infected patients on stable antiretroviral therapy (ART). Such increase may partially be due to HIV-induced immune dysregulation involving monocytes (MO) and its subsets. MATERIALS AND METHODS: Cross-sectional analysis of 141 HIV-infected subjects age ≥ 40 years on stable ART. Homeostatic model assessment-insulin resistance (HOMA-IR) and rates of metabolic syndrome were calculated. Subjects were classified by fasting glucose and oral glucose tolerance test (OGTT) into clinical diabetes categories. Multi-parametric flow cytometry was used to determine MO subset percentages: [classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)), non-classical (CD14(low/+)CD16(++)), and a recently identified fourth (CD14(low/+)CD16(-)) 'transitional' MO subset] and percentage of activated (CD38(+)HLA-DR(+)) CD8 T cells. Absolute levels of cells were calculated using clinical CBC and T cell subset data. Multiple plasma soluble biomarkers were assessed by Luminex technology. RESULTS: Median age 50 years, CD4 count (percent) 505 cells/µL (29%), and 89% male. Total MO (r=-0.23, p=0.006) and classical and non-classical MO subsets correlated negatively with CD4 percent. No correlations were seen with CD4 count as absolute values. Log-total MO and log-classical MO predicted HOMA-IR independently of HIV immuno-virologic and diabetes risk factors (ß=0.42, p=0.02 and ß=0.35, p=0.02, respectively) and were increased in subjects with metabolic syndrome (p=0.03 and p=0.05 respectively). Total and/or subset MO levels correlated with multiple soluble plasma biomarkers including CRP, IL-6, MMP-9, MPO, SAA, SAP and tPAI-1, with tPAI-1 independently predicting HOMA-IR (ß=0.74, p<0.001). CONCLUSIONS: MO levels increase with worsening HIV immune dysregulation as assessed by CD4 percent. CD4 percent may provide additional information about MO and metabolic risk in this population beyond absolute values. MO, and specifically classical MO, may contribute to insulin resistance and metabolic syndrome during chronic HIV infection. Multiple soluble plasma biomarkers including tPAI-1 increase with increase in MO. Levels of tPAI-1 independently predict the development of insulin resistance.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Resistência à Insulina/imunologia , Monócitos/imunologia , Fatores Etários , Biomarcadores/sangue , Biomarcadores/metabolismo , Glicemia , Estudos Transversais , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/imunologia , Diabetes Mellitus/metabolismo , Feminino , Humanos , Imunofenotipagem , Contagem de Linfócitos , Masculino , Síndrome Metabólica/imunologia , Síndrome Metabólica/metabolismo , Pessoa de Meia-Idade , Monócitos/metabolismo , Fatores de Risco , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
3.
Cytometry A ; 85(3): 268-76, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24464647

RESUMO

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.


Assuntos
Citocinas/biossíntese , Citometria de Fluxo , Ionomicina/farmacologia , Ativação Linfocitária/imunologia , Transdução de Sinais/efeitos dos fármacos , Subpopulações de Linfócitos T/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Anticorpos Monoclonais/imunologia , Citocinas/imunologia , Citometria de Fluxo/métodos , Humanos , Fosforilação , Fosfotransferases/metabolismo , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia
4.
Atherosclerosis ; 232(1): 52-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24401216

RESUMO

HIV infection causes systemic immune inflammation, and increases the risk for cardiovascular (CVD) disease even among those on virologically suppressive anti-retroviral treatment (ART). We performed a biostatistical analysis and screen of candidate cellular and plasma biomarkers for association with carotid artery intima-media thickness (CIMT), independent of traditional CVD risk factors such as age, gender, systolic blood pressure (SBP), lipid levels, smoking and diabetes. We conducted a multi-stage analysis based on a cross-sectional study of CVD risk in HIV-infected subjects age >45 years on ART for >6 months. The goal of this analysis was to identify candidate cellular and plasma biomarkers of CIMT in HIV-1 infected adults. We further sought to determine if these candidate biomarkers were independent of traditional CVD risk factors previously identified in HIV negative adults. High-resolution B-mode ultrasound images of the right common carotid common artery (CCA) were obtained. Plasma soluble inflammatory mediators, cytokines and chemokines were detected. Monocytes were defined by CD14/CD16 expression, and CD8+ T-cell activation by CD38/HLA-DR expression. Subjects were a median of 49.5 years old, 87% male, had a CIMT of 0.73 mm, FRS of 6%, a median viral load of 48 copies/mL, and CD4+ T cell count of 479 cells/µL. Soluble VCAM-1, and expansion of CD14dimCD16- monocytes each associated with higher CIMT independently of age and SBP. These factors are distinct components of a shared atherogenic process; 1) vascular endothelial molecular expression and 2) vascular monocytes that enter into the vascular endothelium and promote atherosclerotic plaque.


Assuntos
Doenças Cardiovasculares/metabolismo , Artérias Carótidas/patologia , Espessura Intima-Media Carotídea , Células Endoteliais/citologia , Infecções por HIV/complicações , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Adulto , Biomarcadores/metabolismo , Pressão Sanguínea , Estudos de Coortes , Estudos Transversais , Citocinas/metabolismo , Feminino , Proteínas Ligadas por GPI/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/citologia , Fenótipo , Receptores de IgG/metabolismo , Fatores de Risco , Molécula 1 de Adesão de Célula Vascular/metabolismo
5.
AIDS Res Hum Retroviruses ; 30(2): 142-6, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23984974

RESUMO

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.


Assuntos
Aterosclerose/diagnóstico , Cálcio/análise , Quimiocina CCL2/sangue , Doença da Artéria Coronariana/diagnóstico , Infecções por HIV/complicações , Plasma/química , Fator de Necrose Tumoral alfa/sangue , Aterosclerose/diagnóstico por imagem , Biomarcadores/sangue , Estudos de Coortes , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/patologia , Feminino , Havaí , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
6.
J Acquir Immune Defic Syndr ; 65(2): 151-9, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24091690

RESUMO

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.


Assuntos
Tecido Adiposo/imunologia , Tecido Adiposo/fisiopatologia , Infecções por HIV/complicações , Síndrome de Lipodistrofia Associada ao HIV/imunologia , Síndrome de Lipodistrofia Associada ao HIV/fisiopatologia , Macrófagos/imunologia , Monócitos/imunologia , Adulto , Estudos Transversais , Feminino , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade
7.
J Clin Virol ; 58(4): 635-40, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24239423

RESUMO

BACKGROUND: Dengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble ß-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties. OBJECTIVE: To investigate plasma Gal-9 levels as a biomarker for DENV infection. STUDY DESIGN: We enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness. RESULTS: During the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P<0.01). CONCLUSION: Gal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.


Assuntos
Dengue/sangue , Galectinas/sangue , Doença Aguda , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Citocinas/sangue , Dengue/epidemiologia , Dengue/imunologia , Dengue/fisiopatologia , Epidemias , Humanos , Filipinas/epidemiologia , Adulto Jovem
8.
PLoS One ; 8(10): e77412, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24143233

RESUMO

We recently observed that a large proportion of activated (CD38(+)HLA-DR(+)) CD8(+) T cells from recently HIV-1-infected adults are refractory to phosphorylation of ERK1/2 kinases (p-ERK1/2-refractory). Given that the ERK1/2 pathway mediates intracellular signaling critical for multiple T cell functions, including key effector functions, the loss of ERK1/2 responsiveness may have broad consequences for CD8(+) T cell function. In the current study, we hypothesized that the p-ERK1/2-refractory population, localized largely within the activated CD38(+)HLA-DR(+) CD8(+) T cell population, would display impairments in CD8(+) T cell effector functions, such as cytokine production and degranulation, compared to CD8(+) p-ERK1/2-responsive cells. We further hypothesized that the p-ERK1/2-refractory phenotype is persistent over time during untreated infection, and would correlate with poorer virologic control, in a manner independent of CD8(+) T cell activation level. We performed single-cell resolution, flow cytometric assays of phospho-kinase responses paired to intracellular cytokine staining in one assay to examine IFN-γ, perforin and CD107α responses in CD8(+) T cells by ERK1/2 signaling profile. On a per cell basis, p-ERK1/2-refractory cells, which fall predominantly within the activated CD8(+) T cell compartment, produced less IFN-γ in response to polyclonal or HIV-1 antigen-specific stimulation, and expressed lower levels of perforin and CD107α. The p-ERK1/2 refractory cell population displayed minimal overlap with the PD-1 and Tim-3 inhibitory exhaustion markers and predicted high viral load independent of activation, suggesting that ERK1/2 may be a unique marker and point of intervention for improving CD8(+) T cell function. Blunted effector functions, secondary to ERK1/2 signaling deficits concentrated within activated CD8(+) T cells, may contribute to immunodeficiency and underlie the predictive capacity of CD8(+) T cell activation on HIV-1 disease progression. (270/300).


Assuntos
Linfócitos T CD8-Positivos/citologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Adulto , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Interferon gama/biossíntese , Fenótipo , Fosfoproteínas/metabolismo , Especificidade da Espécie , Fatores de Tempo , Carga Viral/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/farmacologia
9.
PLoS One ; 8(9): e75500, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086545

RESUMO

Chronic infection by HIV increases the risk of cardiovascular disease (CVD) despite effective antiretroviral therapy (ART). The mechanisms linking HIV to CVD have yet to be fully elucidated. High plasma levels of the pro-inflammatory cytokine IL-6, which may be triggered by IL-1ß, is a biomarker of CVD risk in HIV-negative adults, and of all-cause mortality in HIV disease. Monocytes play a pivotal role in atherosclerosis, and may be major mediators of HIV-associated inflammation. We therefore hypothesized that monocytes from HIV-infected adults would display high inflammatory responses. Employing a 10-color flow cytometry intracellular cytokine staining assay, we directly assessed cytokine and chemokine responses of monocytes from the cryopreserved peripheral blood of 33 chronically HIV-1 infected subjects. Participants were 45 years or older, on virologically suppressive ART and at risk for CVD. This group was compared to 14 HIV-negative subjects matched for age and gender, with similar CVD risk. We simultaneously detected intracellular expression of IL-1ß, IL-6, IL-8 and TNF in blood monocytes in the basal state and after stimulation by triggers commonly found in the blood of treated, chronically HIV-infected subjects: lipopolysaccharide (LPS) and oxidized low-density lipoprotein (oxLDL). In the absence of stimulation, monocytes from treated HIV-infected subjects displayed a high frequency of cells producing IL-1ß (median 19.5%), compared to low levels in HIV-uninfected persons (0.9% p<0.0001). IL-8, which is induced by IL-1ß, was also highly expressed in the HIV-infected group in the absence of stimulation, 43.7% compared to 1.9% in HIV-uninfected subjects, p<0.0001. Strikingly, high basal expression of IL-1ß by monocytes predicted high IL-6 levels in the plasma, and high monocyte IL-6 responses in HIV-infected subjects. Hyper-inflammatory IL-1ß enriched monocytes may be a major source of IL-6 production and systemic inflammation in HIV-infected adults, and may contribute to the risk for all-cause mortality and cardiovascular disease in treated HIV infection.


Assuntos
Antirretrovirais/uso terapêutico , Doenças Cardiovasculares/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Monócitos/imunologia , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/imunologia , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Risco , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
10.
Cell Host Microbe ; 14(4): 411-21, 2013 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-24139399

RESUMO

Several human APOBEC3 deaminases can inhibit HIV-1 replication in vitro. HIV-1 Vif counteracts this restriction by targeting APOBEC3 for proteasomal degradation. Human APOBEC3H (A3H) is highly polymorphic, with natural variants differing considerably in anti-HIV-1 activity in vitro. To examine HIV-1 adaptation to variation in A3H activity in a natural infection context, we determined the A3H haplotypes and Vif sequences from 76 recently infected HIV-1 patients. We detected A3H-specific Vif changes suggesting viral adaptation. The patient-derived Vif sequences were used to engineer viruses that specifically differed in their ability to counteract A3H. Replication of these Vif-variant viruses in primary T cells naturally expressing active or inactive A3H haplotypes showed that endogenously expressed A3H restricts HIV-1 replication. Proviral DNA from A3H-restricted viruses showed high levels of G-to-A mutations in an A3H-specific GA dinucleotide context. Taken together, our data validate A3H expressed at endogenous levels as a bona fide HIV-1 restriction factor.


Assuntos
Adaptação Biológica , Aminoidrolases/antagonistas & inibidores , HIV-1/imunologia , HIV-1/fisiologia , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Aminoidrolases/imunologia , Células Cultivadas , Análise Mutacional de DNA , DNA Viral/química , DNA Viral/genética , Infecções por HIV/virologia , HIV-1/genética , HIV-1/isolamento & purificação , Haplótipos , Interações Hospedeiro-Patógeno , Humanos , Provírus/genética , Genética Reversa , Seleção Genética , Análise de Sequência de DNA , Linfócitos T/virologia , Replicação Viral
11.
Hawaii J Med Public Health ; 72(9 Suppl 4): 34-8, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24052917

RESUMO

UNLABELLED: Albuminuria (urinary excretion of more than 30 milligram of albumin per gram of creatinine) serves as an indicator of microvascular injury, which has been associated with atherosclerosis and cardiovascular disease in HIV-seronegative individuals. Albuminuria has been reported to be prevalent among HIV-seropositive individuals, however, the relationship between albuminuria and risk for cardiovascular disease in this population has not been well-studied. We examined the relationships between albuminuria and parameters of atherosclerosis including carotid intima-media thickness and traditional cardiovascular risk assessment among HIV-seropositive individuals receiving stable antiretroviral therapy. We utilized a cross-sectional baseline data from the Hawai'i Aging with HIV-Cardiovascular Study cohort. RESULTS: Data was available on 111 HIV-infected patients (median age of 52 (Q1,Q3: 46, 57), male 86%; diabetes 6%; hypertension 33%; dyslipidemia 50%; median CD4 count of 489 cells/mm(3) (341, 638); HIV RNA PCR < 48 copies/ml of 85%). Eighteen subjects (16.2%) had microalbuminuria, and two subjects (1.8%) had macroalbuminuria. Albuminuria was significantly associated with increased Framingham Risk Score (P=.002), insulin resistance by HOMA-IR (P=.02), diastolic blood pressure (P=.01), and carotid intima-media thickness (P =.04). The correlation between the amount of albuminuria and carotid intima-media thickness remained significant even after adjusting for age, gender, ethnicity, current smoking status, diabetes mellitus, diastolic blood pressure, fasting insulin level, CD4 count, and HIV-RNA viral load. CONCLUSION: Albuminuria is prevalent among HIV-infected patients receiving stable antiretroviral therapy. It is significantly related to previously defined markers of cardiovascular disease and metabolic syndrome among HIV-infected patients receiving stable antiretroviral therapy.


Assuntos
Albuminúria/epidemiologia , Doenças Cardiovasculares/epidemiologia , Espessura Intima-Media Carotídea , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Feminino , Havaí/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco
12.
Cytometry A ; 83(3): 280-6, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23426986

RESUMO

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.


Assuntos
Citometria de Fluxo/métodos , Monócitos/imunologia , Receptores CCR2/análise , Receptores CCR5/análise , Receptores de Quimiocinas/análise , Adulto , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Receptor 1 de Quimiocina CX3C , Movimento Celular , Feminino , Humanos , Masculino , Receptores CCR2/imunologia , Receptores CCR5/imunologia , Receptores de Quimiocinas/imunologia , Coloração e Rotulagem
13.
Blood ; 119(16): 3734-43, 2012 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-22383801

RESUMO

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.


Assuntos
Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Proteínas de Membrana/imunologia , Linfócitos T Citotóxicos/imunologia , Biomarcadores/metabolismo , Antígeno CD56/imunologia , Antígeno CD56/metabolismo , Diferenciação Celular/imunologia , Linhagem Celular , Reagentes de Ligações Cruzadas/metabolismo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Imunofenotipagem , Células Matadoras Naturais/citologia , Ligantes , Subpopulações de Linfócitos/metabolismo , Proteínas de Membrana/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Linfócitos T Citotóxicos/citologia , Regulação para Cima/imunologia
14.
J Virol ; 85(23): 12343-50, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21937661

RESUMO

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.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais , Adulto , Linfócitos T CD8-Positivos/patologia , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Infecções por HIV/patologia , Humanos , Ativação Linfocitária , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Carga Viral
15.
J Clin Immunol ; 30(5): 681-92, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20571894

RESUMO

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.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Infecções por HIV/imunologia , Imunoterapia , Interleucina-2/administração & dosagem , Células Th17/efeitos dos fármacos , Adulto , Antirretrovirais/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Contagem de Células , Proliferação de Células/efeitos dos fármacos , Quimioterapia Combinada , Infecções por HIV/tratamento farmacológico , HIV-1 , Humanos , Imunomodulação , Interleucina-17/biossíntese , Interleucina-17/genética , Interleucina-2/uso terapêutico , Fosfoproteínas/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Células Th17/imunologia , Células Th17/patologia , Células Th17/virologia , Proteínas da Matriz Viral/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia
16.
Sci Transl Med ; 2(32): 32ra36, 2010 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-20484731

RESUMO

The pathogenesis of human and simian immunodeficiency viruses is characterized by CD4(+) T cell depletion and chronic T cell activation, leading ultimately to AIDS. CD4(+) T helper (T(H)) cells provide protective immunity and immune regulation through different immune cell functional subsets, including T(H)1, T(H)2, T regulatory (T(reg)), and interleukin-17 (IL-17)-secreting T(H)17 cells. Because IL-17 can enhance host defenses against microbial agents, thus maintaining the integrity of the mucosal barrier, loss of T(H)17 cells may foster microbial translocation and sustained inflammation. Here, we study HIV-seropositive subjects and find that progressive disease is associated with the loss of T(H)17 cells and a reciprocal increase in the fraction of the immunosuppressive T(reg) cells both in peripheral blood and in rectosigmoid biopsies. The loss of T(H)17/T(reg) balance is associated with induction of indoleamine 2,3-dioxygenase 1 (IDO1) by myeloid antigen-presenting dendritic cells and with increased plasma concentration of microbial products. In vitro, the loss of T(H)17/T(reg) balance is mediated directly by the proximal tryptophan catabolite from IDO metabolism, 3-hydroxyanthranilic acid. We postulate that induction of IDO may represent a critical initiating event that results in inversion of the T(H)17/T(reg) balance and in the consequent maintenance of a chronic inflammatory state in progressive HIV disease.


Assuntos
Infecções por HIV/imunologia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Inflamação/imunologia , Interleucina-17/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Triptofano/metabolismo , Ácido 3-Hidroxiantranílico/metabolismo , Biópsia , Contagem de Linfócito CD4 , Relação CD4-CD8 , Células Cultivadas , Colo Sigmoide/enzimologia , Colo Sigmoide/imunologia , Colo Sigmoide/virologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Células Dendríticas/virologia , Progressão da Doença , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Infecções por HIV/virologia , Soropositividade para HIV , Humanos , Inflamação/enzimologia , Inflamação/virologia , Mediadores da Inflamação/imunologia , Interferon gama/imunologia , Cinurenina/metabolismo , Lipopolissacarídeos/imunologia , Linfonodos/enzimologia , Linfonodos/imunologia , Linfonodos/virologia , Estudos Prospectivos , Reto/enzimologia , Reto/imunologia , Reto/virologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/virologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/virologia , Fatores de Tempo , Regulação para Cima , Carga Viral
17.
Immunology ; 129(2): 186-96, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19824915

RESUMO

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.


Assuntos
Soropositividade para HIV/imunologia , HIV-1/imunologia , Herpes Simples/imunologia , Herpesvirus Humano 2/imunologia , Células Matadoras Naturais/metabolismo , Adulto , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Contagem de Células , Células Cultivadas , Citotoxicidade Imunológica , Progressão da Doença , Feminino , Soropositividade para HIV/complicações , Soropositividade para HIV/patologia , Soropositividade para HIV/fisiopatologia , HIV-1/patogenicidade , Antígenos HLA-B/genética , Antígenos HLA-B/metabolismo , Herpes Simples/complicações , Herpes Simples/patologia , Herpes Simples/fisiopatologia , Herpesvirus Humano 2/patogenicidade , Teste de Histocompatibilidade , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Células Matadoras Naturais/virologia , Linfocitose , Masculino , Pessoa de Meia-Idade , Receptores KIR3DL1/genética , Receptores KIR3DL1/metabolismo , Receptores KIR3DS1/genética , Receptores KIR3DS1/metabolismo
18.
Eur J Immunol ; 40(1): 134-41, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19877008

RESUMO

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.


Assuntos
Antígenos CD/imunologia , Apirase/imunologia , Linfócitos T CD4-Positivos/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/citologia , Proliferação de Células , Citocinas/biossíntese , Citocinas/imunologia , Humanos , Subpopulações de Linfócitos T/citologia
19.
PLoS Pathog ; 5(2): e1000295, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19214220

RESUMO

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.


Assuntos
Interleucina-17/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Análise de Variância , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Chlorocebus aethiops , Colo/imunologia , Colo/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Interleucina-17/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Ativação Linfocitária , Macaca nemestrina , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Viral/análise , RNA Viral/sangue , RNA Viral/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia , Estatísticas não Paramétricas , Linfócitos T Auxiliares-Indutores/virologia , Linfócitos T Reguladores/virologia , Carga Viral
20.
PLoS One ; 4(2): e4408, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19198651

RESUMO

BACKGROUND: The relationship of elevated T cell activation to altered T cell differentiation profiles, each defining features of HIV-1 infection, has not been extensively explored. We hypothesized that anti-retroviral suppression of T cell activation levels would lead to alterations in the T cell differentiation of total and HIV-1 specific CD8+ T cell responses among recently HIV-1 infected adults. METHODOLOGY/PRINCIPAL FINDINGS: We performed a longitudinal study simultaneously measuring T cell activation and maturation markers on both total and antigen-specific T cells in recently infected adults: prior to treatment; after the initiation of HAART; and after treatment was halted. Prior to treatment, HIV-1 Gag-specific CD8+ T cells were predominantly of a highly activated, intermediate memory (CD27+CD28-) phenotype, while CMV pp65-specific CD8+ T cells showed a late memory (CD27-CD28-), low activation phenotype. Participants with the highest fraction of late memory (CD27-CD28-) HIV-1-specific CD8+ T cells had higher CD4+ T cell counts (rho = +0.74, p = 0.004). In turn, those with the highest fraction of intermediate memory (CD27+ CD28-) HIV-1 specific CD8+ T cells had high total CD8+ T cell activation (rho = +0.68, p = 0.01), indicating poorer long-term clinical outcomes. The HIV-1 specific T cell differentiation profile was not readily altered by suppression of T cell activation following HAART treatment. CONCLUSIONS/SIGNIFICANCE: A more differentiated, less activated HIV-1 specific CD8+ T cell response may be clinically protective. Anti-retroviral treatment initiated two to four months after infection lowered T cell activation but had no effect on the differentiation profile of the HIV-1-specific response. Intervention during the first month of acute infection may be required to shift the differentiation phenotype of HIV-1 specific responses to a more clinically favorable profile.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , HIV-1/imunologia , Ativação Linfocitária/imunologia , Replicação Viral , Adulto , Linfócitos T CD8-Positivos/citologia , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Humanos , Fenótipo
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