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1.
Nat Commun ; 10(1): 1649, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967541

RESUMO

The recently developed droplet-based single-cell transcriptome sequencing (scRNA-seq) technology makes it feasible to perform a population-scale scRNA-seq study, in which the transcriptome is measured for tens of thousands of single cells from multiple individuals. Despite the advances of many clustering methods, there are few tailored methods for population-scale scRNA-seq studies. Here, we develop a Bayesian mixture model for single-cell sequencing (BAMM-SC) method to cluster scRNA-seq data from multiple individuals simultaneously. BAMM-SC takes raw count data as input and accounts for data heterogeneity and batch effect among multiple individuals in a unified Bayesian hierarchical model framework. Results from extensive simulation studies and applications of BAMM-SC to in-house experimental scRNA-seq datasets using blood, lung and skin cells from humans or mice demonstrate that BAMM-SC outperformed existing clustering methods with considerable improved clustering accuracy, particularly in the presence of heterogeneity among individuals.


Assuntos
Biologia Computacional/métodos , Análise de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Teorema de Bayes , Biópsia , Análise por Conglomerados , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica/métodos , Voluntários Saudáveis , Humanos , Leucócitos Mononucleares , Pulmão/citologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Pele/citologia , Pele/patologia , Software , Transcriptoma/genética
2.
Virology ; 521: 51-57, 2018 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-29879542

RESUMO

The relationships between HIV-1 DNA copy number, proviral transcriptional activity, and residual plasma viremia in individuals off and on ART are not well defined. To address this, we performed a cross-sectional study of 12 viremic donors and 23 ART-treated virologically suppressed (plasma HIV-1 RNA<20 copies/ml) donors. We report a strong association between HIV-1 DNA copy number and HIV-1 transcriptional activity in blood that persists on suppressive ART, but not between transcriptional activity and the levels of persistent viremia on ART. The latter finding contrasts with that in viremic donors and suggests that most HIV transcription in donors on suppressive ART does not result in virion production. This uncoupling of proviral transcription and viremia warrants closer investigation.

3.
AIDS ; 32(6): 699-708, 2018 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-29334544

RESUMO

OBJECTIVE: To define the relationships between molecular measures of viral persistence in blood (i.e., plasma viremia, cellular HIV-1 DNA, and mRNA) and expressed or inducible virus from resting CD4 T cells of individuals on suppressive antiretroviral therapy. DESIGN: We compared molecular measurements of HIV-1 in plasma and in uncultured peripheral blood mononuclear cells (PBMCs) to the levels of virions produced by either unstimulated or phorbol myristate acetate and ionomycin (PMA/iono)-stimulated PBMC or resting CD4 T cells from 21 donors on suppressive antiretroviral therapy. RESULTS: We found that unstimulated virion release from cultured resting CD4 T cells was positively correlated with the levels of plasma viremia in vivo (Spearman rho = 0.67, P = 0.0017). We also found that levels of both cellular HIV-1 DNA and unspliced HIV-1 mRNA per million uncultured PBMC were positively correlated with the levels of inducible virion release from both PMA/iono-stimulated PBMC (total HIV-1 DNA: rho = 0.64, P = 0.0017; unspliced HIV-1 RNA: rho = 0.77, P < 0.001) and PMA/iono-stimulated resting CD4 T cells (total HIV-1 DNA: rho = 0.75, P < 0.001; unspliced HIV-1 RNA: rho = 0.75, P < 0.001). CONCLUSION: These results show for the first time that there are strong associations between in-vivo measures of HIV-1 persistence and ex-vivo measures of spontaneous and inducible virus production from cultured PBMC and resting CD4 T cells. Findings from this study provide insight into the biology of HIV-1 persistence and suggest methods to guide the evaluation of clinical strategies to reduce the size of the viral reservoir.

4.
Proc Natl Acad Sci U S A ; 114(18): E3659-E3668, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28416661

RESUMO

Little is known about the fraction of human immunodeficiency virus type 1 (HIV-1) proviruses that express unspliced viral RNA in vivo or about the levels of HIV RNA expression within single infected cells. We developed a sensitive cell-associated HIV RNA and DNA single-genome sequencing (CARD-SGS) method to investigate fractional proviral expression of HIV RNA (1.3-kb fragment of p6, protease, and reverse transcriptase) and the levels of HIV RNA in single HIV-infected cells from blood samples obtained from individuals with viremia or individuals on long-term suppressive antiretroviral therapy (ART). Spiking experiments show that the CARD-SGS method can detect a single cell expressing HIV RNA. Applying CARD-SGS to blood mononuclear cells in six samples from four HIV-infected donors (one with viremia and not on ART and three with viremia suppressed on ART) revealed that an average of 7% of proviruses (range: 2-18%) expressed HIV RNA. Levels of expression varied from one to 62 HIV RNA molecules per cell (median of 1). CARD-SGS also revealed the frequent expression of identical HIV RNA sequences across multiple single cells and across multiple time points in donors on suppressive ART consistent with constitutive expression of HIV RNA in infected cell clones. Defective proviruses were found to express HIV RNA at levels similar to those proviruses that had no obvious defects. CARD-SGS is a useful tool to characterize fractional proviral expression in single infected cells that persist despite ART and to assess the impact of experimental interventions on proviral populations and their expression.


Assuntos
Antirretrovirais/administração & dosagem , Regulação Viral da Expressão Gênica/efeitos dos fármacos , HIV-1/metabolismo , Leucócitos Mononucleares/metabolismo , Provírus/metabolismo , RNA Viral/biossíntese , Transcrição Genética/efeitos dos fármacos , Feminino , Humanos , Leucócitos Mononucleares/virologia , Masculino
5.
Curr Opin Virol ; 18: 14-9, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26985878

RESUMO

Strategies to achieve a cure for HIV-1 infection can be broadly classified into three categories: eradication cure (elimination of all viral reservoirs), functional cure (immune control without reservoir eradication), or a hybrid cure (reservoir reduction with improved immune control). The many HIV-1 cure strategies being investigated include modification of host cells to resist HIV-1, engineered T cells to eliminate HIV-infected cells, broadly HIV-1 neutralizing monoclonal antibodies, and therapeutic vaccination, but the 'kick and kill' strategy to expose latent HIV-1 with latency reversing agents (LRAs) and kill the exposed cells through immune effector functions is currently the most actively pursued. It is unknown, however, whether LRAs can deplete viral reservoirs in vivo or whether current LRAs are sufficiently safe for clinical use.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos
6.
J Clin Microbiol ; 54(4): 902-11, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26763968

RESUMO

Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4(+)T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10copies/10(6)PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10copies/10(6)PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r= 0.77 to 1;P= 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4(+)T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription.


Assuntos
DNA Viral/análise , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Leucócitos Mononucleares/virologia , RNA Viral/análise , Carga Viral/métodos , Adulto , DNA Viral/genética , Feminino , HIV-1/genética , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
7.
J Virol ; 90(3): 1673-6, 2016 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-26559835

RESUMO

Quantifying induced virion production from single proviruses is important for assessing the effects of HIV-1 latency reversal agents. Limiting dilution ex vivo cultures of resting CD4(+) T cells from 14 HIV-positive volunteers revealed that virion production after T-cell activation from individual proviruses varies by 10,000-fold to 100,000-fold. High-producing proviruses were associated with increases in cell-associated HIV-1 DNA levels, suggesting that reactivated proviruses proliferate. Single-cell analyses are needed to investigate differences in proviral expansion and virus production following latency reversal.


Assuntos
Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Ativação Linfocitária , Provírus/fisiologia , Vírion/isolamento & purificação , Ativação Viral , Replicação Viral , Células Cultivadas , DNA Viral/análise , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos
8.
AIDS ; 29(16): 2121-9, 2015 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-26544577

RESUMO

BACKGROUND: Combination antiretroviral therapy (ART) suppresses HIV-1 replication, but does not restore CD4 T-cell counts in all individuals. To investigate the effects of maraviroc on HIV-1 persistence and the relations between virologic and immunologic parameters in individuals with incomplete CD4 T-cell recovery, we performed a prospective, open-label pilot trial in which maraviroc was added to a suppressive ART regimen for 24 weeks. DESIGN: A5256 was a single-arm trial in which individuals on suppressive ART with incomplete CD4 T-cell recovery added maraviroc for 24 weeks. METHODS: We quantified low-level, residual viremia in plasma and total HIV-1 DNA and 2-long terminal repeat (2-LTR) circles in peripheral blood mononuclear cells before and after maraviroc intensification. We also evaluated markers of CD4 and CD8 T-cell immune activation (%CD38HLA-DR) and apoptosis (%caspase3/Bcl-2). RESULTS: No effect of maraviroc was found on the probability of detectable plasma viremia (≥1 copy/ml; n = 31, exact McNemar P = 1.0) or detectable 2-LTR circles (n = 28, P = 0.25) or on total HIV-1 DNA (n = 28, 90% confidence interval -0.1, +0.3 log10 copies/10 CD4 T-cells). Premaraviroc HIV-1 DNA levels were inversely related to premaraviroc %CD38HLA-DR CD4 T-cells (Spearman = -0.52, P = 0.004), and lower premaraviroc HIV-1 DNA levels were associated with larger decreases in %CD38HLA-DR CD4 T-cells during maraviroc intensification (Spearman = 0.44, P = 0.018). CONCLUSION: In individuals on suppressive ART with incomplete CD4 T-cell recovery, maraviroc intensification did not affect measures of HIV-1 persistence but did decrease persistent CD4 T-cell immune activation especially in individuals with low preintensification levels of HIV-1 DNA.


Assuntos
Antirretrovirais/uso terapêutico , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/imunologia , Cicloexanos/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/isolamento & purificação , Triazóis/uso terapêutico , Carga Viral , Contagem de Linfócito CD4 , Linfócitos T CD8-Positivos/imunologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Masculino , Maraviroc , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento
9.
J Virol ; 89(11): 6155-60, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25833043

RESUMO

Simian immunodeficiency virus SIVsab infection is completely controlled in rhesus macaques (RMs) through functional immune responses. We report that in SIVsab-infected RMs, (i) viral replication is controlled to <0 to 3 copies/ml, (ii) about one-third of the virus strains in reservoirs are replication incompetent, and (iii) rebounding virus after CD8(+) cell depletion is replication competent and genetically similar to the original virus stock, suggesting early reservoir seeding. This model permits assessment of strategies aimed at depleting the reservoir without multidrug antiretroviral therapy.


Assuntos
Tolerância Imunológica , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Replicação Viral , Animais , Linfócitos T CD8-Positivos/imunologia , Macaca mulatta , Masculino , Vírus da Imunodeficiência Símia/genética
10.
PLoS Biol ; 12(12): e1002030, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25549104

RESUMO

We have developed and tested two linked but separable structured inquiry exercises using a set of Drosophila melanogaster GAL4 enhancer trap strains for an upper-level undergraduate laboratory methods course at Bucknell University. In the first, students learn to perform inverse PCR to identify the genomic location of the GAL4 insertion, using FlyBase to identify flanking sequences and the primary literature to synthesize current knowledge regarding the nearest gene. In the second, we cross each GAL4 strain to a UAS-CD8-GFP reporter strain, and students perform whole mount CNS dissection, immunohistochemistry, confocal imaging, and analysis of developmental expression patterns. We have found these exercises to be very effective in teaching the uses and limitations of PCR and antibody-based techniques as well as critical reading of the primary literature and scientific writing. Students appreciate the opportunity to apply what they learn by generating novel data of use to the wider research community.


Assuntos
Currículo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Laboratórios , Aprendizagem , Fatores de Transcrição/genética , Universidades , Animais , Sequência de Bases , Encéfalo/metabolismo , Regulação da Expressão Gênica , Genes de Insetos , Dados de Sequência Molecular , Corpos Pedunculados/metabolismo , Reação em Cadeia da Polimerase
11.
J Clin Microbiol ; 52(11): 3944-51, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25187636

RESUMO

A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.


Assuntos
Antirretrovirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Viremia/virologia , Primers do DNA/genética , Humanos , Sondas de Oligonucleotídeos/genética , RNA Viral/análise , RNA Viral/sangue , RNA Viral/genética , Sensibilidade e Especificidade
12.
Proc Natl Acad Sci U S A ; 111(19): 7078-83, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24706775

RESUMO

Reversal of proviral latency is being pursued as a curative strategy for HIV-1 infection. Recent clinical studies of in vivo administration of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) show increases in unspliced cellular HIV-1 RNA levels in resting CD4(+) T cells. A critical unknown, however, is the proportion of latent proviruses that can be transcriptionally reactivated by SAHA or T-cell activation. In this study, we quantified the fraction of HIV-1 proviruses in resting CD4(+) T cells from patients on suppressive antiretroviral therapy that were reactivated ex vivo with SAHA or antibodies to CD3/CD28. At concentrations of SAHA achieved clinically, only 0.079% of proviruses in resting CD4(+) T cells were reactivated to produce virions, compared with 1.5% of proviruses in cells treated with anti-CD3/CD28 antibodies after correcting for spontaneous virion production in the medium control. A significant positive correlation (ρ = 0.67, P < 0.001) was found between levels of virions in the supernatant and unspliced cellular HIV-1 RNA following anti-CD3/CD28 treatment, but not following SAHA treatment (ρ = 0.21, P = 0.99). These results reveal that the majority of HIV-1 proviruses are not reactivated by current therapeutic approaches and that more effective means of reversing proviral latency will likely be required to deplete HIV-1 reservoirs.


Assuntos
Antirretrovirais/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/crescimento & desenvolvimento , Ácidos Hidroxâmicos/uso terapêutico , Latência Viral/efeitos dos fármacos , Adulto , Idoso , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Feminino , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Provírus/genética , Provírus/crescimento & desenvolvimento , RNA Viral/genética , Viremia/tratamento farmacológico , Viremia/imunologia , Viremia/virologia , Latência Viral/imunologia , Vorinostat
13.
PLoS One ; 9(3): e92118, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24638072

RESUMO

The first cure of HIV-1 infection was achieved through complex, multimodal therapy including myeloablative chemotherapy, total body irradiation, anti-thymocyte globulin, and allogeneic stem cell transplantation with a CCR5 delta32 homozygous donor. The contributions of each component of this therapy to HIV-1 eradication are unclear. To assess the impact of cytotoxic chemotherapy alone on HIV-1 persistence, we longitudinally evaluated low-level plasma viremia and HIV-1 DNA in PBMC from patients in the ACTG A5001/ALLRT cohort on suppressive antiretroviral therapy (ART) who underwent chemotherapy for HIV-1 related lymphoma without interrupting ART. Plasma HIV-1 RNA, total HIV-1 DNA and 2-LTR circles (2-LTRs) in PBMC were measured using sensitive qPCR assays. In the 9 patients who received moderately intensive chemotherapy for HIV-1 related lymphoma with uninterrupted ART, low-level plasma HIV-1 RNA did not change significantly with chemotherapy: median HIV-1 RNA was 1 copy/mL (interquartile range: 1.0 to 20) pre-chemotherapy versus 4 copies/mL (interquartile range: 1.0 to 7.0) post-chemotherapy. HIV-1 DNA levels also did not change significantly, with median pre-chemotherapy HIV-1 DNA of 355 copies/106 CD4+ cells versus 228 copies/106 CD4+ cells post-chemotherapy. 2-LTRs were detectable in 2 of 9 patients pre-chemotherapy and in 3 of 9 patients post-chemotherapy. In summary, moderately intensive chemotherapy for HIV-1 related lymphoma in the context of continuous ART did not have a prolonged impact on HIV-1 persistence. Clinical trials registration unique identifier: NCT00001137.


Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , Infecções por HIV/patologia , Linfoma Relacionado a AIDS/tratamento farmacológico , Linfoma Relacionado a AIDS/virologia , Viremia/tratamento farmacológico , Adulto , Idoso , Estudos de Coortes , DNA Viral/sangue , Infecções por HIV/sangue , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Linfoma Relacionado a AIDS/sangue , Linfoma Relacionado a AIDS/patologia , Masculino , Pessoa de Meia-Idade , Viremia/complicações , Viremia/patologia
14.
J Infect Dis ; 208(6): 884-91, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23801609

RESUMO

OBJECTIVE: The goal of this study was to define viral kinetics after initiation of raltegravir (RAL)-based antiretroviral therapy (ART). METHODS: ART-naive patients received RAL, tenofovir disoproxil fumarate, and emtricitabine for 72 weeks. Human immunodeficiency virus type 1 (HIV-1) RNA were measured by ultrasensitive and single-copy assays, and first (d1)-, second (d2)-, and, third (d3)-phase decay rates were estimated by mixed-effects models. Decay data were compared to historical estimates for efavirenz (EFV)- and ritonavir/lopinavir (LPV/r)-based regimens. RESULTS: Bi- and tri-exponential models for ultrasensitive assay (n = 38) and single-copy assay (n = 8) data, respectively, provided the best fits over 8 and 72 weeks. The median d1 with ultrasensitive data was 0.563/day (interquartile range [IQR], 0.501-0.610/day), significantly slower than d1 for EFV-based regimens [P < .001]). The median duration of d1 was 15.1 days, transitioning to d2 at an HIV-1 RNA of 91 copies/mL, indicating a longer duration of d1 and a d2 transition at lower viremia levels than with EFV. Median patient-specific decay estimates with the single-copy assay were 0.607/day (IQR, 0.582-0.653) for d1, 0.070/day (IQR, 0.042-0.079) for d2, and 0.0016/day (IQR, 0.0005-0.0022) for d3; the median d1 duration was 16.1 days, transitioning to d2 at 69 copies/mL. d3 transition occurred at 110 days, at 2.6 copies/mL, similar to values for LPV/r-based regimens. CONCLUSIONS: Models using single-copy assay data revealed 3 phases of decay with RAL-containing ART, with a longer duration of first-phase decay consistent with RAL-mediated blockade of productive infection from preintegration complexes.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Pirrolidinonas/uso terapêutico , Estabilidade de RNA/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/uso terapêutico , Adulto , Benzoxazinas/uso terapêutico , Feminino , Infecções por HIV/sangue , Infecções por HIV/virologia , HIV-1/genética , Humanos , Lopinavir/uso terapêutico , Masculino , Pessoa de Meia-Idade , Organofosfonatos/uso terapêutico , Projetos Piloto , Estudos Prospectivos , RNA Viral/metabolismo , Raltegravir Potássico , Ritonavir/uso terapêutico , Tenofovir , Carga Viral
15.
Clin Vaccine Immunol ; 20(8): 1320-8, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23803902

RESUMO

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), ß-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.


Assuntos
Peptídeos Catiônicos Antimicrobianos/biossíntese , Quimiocina CXCL13/biossíntese , Mucosa Intestinal/imunologia , Celulas de Paneth/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Perfilação da Expressão Gênica , Intestino Delgado/imunologia , Linfonodos/imunologia , Macaca mulatta , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , alfa-Defensinas/biossíntese , beta-Defensinas/biossíntese
16.
J Acquir Immune Defic Syndr ; 63(4): 438-41, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23493152

RESUMO

A cure of HIV-1 has been achieved in one individual through allogeneic stem cell transplantation with a CCR5[INCREMENT]32 homozygous donor. Whether myeloablation and autologous stem cell transplantation for lymphoma in patients on suppressive antiretroviral therapy can eliminate HIV-1 reservoirs is unknown. Low-level plasma viremia and total HIV-1 DNA and 2-LTR circles in blood mononuclear cells were quantified after autologous transplantation in 10 patients on suppressive antiretroviral therapy using quantitative polymerase chain reaction assays capable of single-copy nucleic acid detection. Plasma viremia was detectable in 9 patients, whereas HIV-1 DNA was detectable in all 10 patients, indicating that HIV-1 had not been eliminated.


Assuntos
DNA Viral/sangue , Repetição Terminal Longa de HIV , HIV-1 , Transplante de Células-Tronco Hematopoéticas , Linfoma Relacionado a AIDS/sangue , RNA Viral/sangue , Carga Viral , Adulto , Antirretrovirais/uso terapêutico , Estudos Transversais , Humanos , Leucócitos Mononucleares , Linfoma Relacionado a AIDS/terapia , Linfoma Relacionado a AIDS/virologia , Masculino , Pessoa de Meia-Idade , Agonistas Mieloablativos/uso terapêutico , Transplante Autólogo , Adulto Jovem
17.
Neural Dev ; 7: 31, 2012 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-22967828

RESUMO

BACKGROUND: In holometabolous insects such as Drosophila melanogaster, neuroblasts produce an initial population of diverse neurons during embryogenesis and a much larger set of adult-specific neurons during larval life. In the ventral CNS, many of these secondary neuronal lineages differ significantly from one body segment to another, suggesting a role for anteroposterior patterning genes. RESULTS: Here we systematically characterize the expression pattern and function of the Hox gene Ultrabithorax (Ubx) in all 25 postembryonic lineages. We find that Ubx is expressed in a segment-, lineage-, and hemilineage-specific manner in the thoracic and anterior abdominal segments. When Ubx is removed from neuroblasts via mitotic recombination, neurons in these segments exhibit the morphologies and survival patterns of their anterior thoracic counterparts. Conversely, when Ubx is ectopically expressed in anterior thoracic segments, neurons exhibit complementary posterior transformation phenotypes. CONCLUSION: Our findings demonstrate that Ubx plays a critical role in conferring segment-appropriate morphology and survival on individual neurons in the adult-specific ventral CNS. Moreover, while always conferring spatial identity in some sense, Ubx has been co-opted during evolution for distinct and even opposite functions in different neuronal hemilineages.


Assuntos
Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Homeodomínio/metabolismo , Sistema Nervoso/crescimento & desenvolvimento , Sistema Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Morte Celular/genética , Drosophila , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Larva , Fatores de Transcrição/genética
18.
Curr HIV/AIDS Rep ; 9(1): 91-100, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22215419

RESUMO

Quantification of plasma HIV-1 RNA below the limit of FDA-approved assays by a single copy quantitative PCR assays (SCA) has provided significant insights into HIV-1 persistence despite potent antiretroviral therapy as well as a means to assess the impact of therapeutic strategies, such as treatment intensification, on residual viremia. In this review, we discuss insights gained from plasma HIV-1 RNA SCA and highlight the need for additional assays to characterize better the cellular and tissue reservoirs of HIV-1. Accurate, reproducible, and sensitive assays to quantify HIV-1 reservoirs, before and after therapeutic interventions, are essential tools in the quest for a cure of HIV-1 infection.


Assuntos
Reservatórios de Doenças/virologia , Infecções por HIV/virologia , HIV-1/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Viral/sangue , HIV-1/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Viremia/virologia
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