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1.
Nature ; 578(7793): 160-165, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31969707

RESUMO

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.

3.
Nat Immunol ; 20(7): 902-914, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209404

RESUMO

Lupus nephritis is a potentially fatal autoimmune disease for which the current treatment is ineffective and often toxic. To develop mechanistic hypotheses of disease, we analyzed kidney samples from patients with lupus nephritis and from healthy control subjects using single-cell RNA sequencing. Our analysis revealed 21 subsets of leukocytes active in disease, including multiple populations of myeloid cells, T cells, natural killer cells and B cells that demonstrated both pro-inflammatory responses and inflammation-resolving responses. We found evidence of local activation of B cells correlated with an age-associated B-cell signature and evidence of progressive stages of monocyte differentiation within the kidney. A clear interferon response was observed in most cells. Two chemokine receptors, CXCR4 and CX3CR1, were broadly expressed, implying a potentially central role in cell trafficking. Gene expression of immune cells in urine and kidney was highly correlated, which would suggest that urine might serve as a surrogate for kidney biopsies.


Assuntos
Rim/imunologia , Nefrite Lúpica/imunologia , Biomarcadores , Biópsia , Análise por Conglomerados , Biologia Computacional/métodos , Células Epiteliais/metabolismo , Citometria de Fluxo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Interferons/metabolismo , Rim/metabolismo , Rim/patologia , Leucócitos/imunologia , Leucócitos/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Anotação de Sequência Molecular , Células Mieloides/imunologia , Células Mieloides/metabolismo , Análise de Célula Única , Transcriptoma
4.
Sci Adv ; 5(1): eaau9223, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30746468

RESUMO

Specialized immune cell subsets are involved in autoimmune disease, cancer immunity, and infectious disease through a diverse range of functions mediated by overlapping pathways and signals. However, subset-specific responses may not be detectable in analyses of whole blood samples, and no efficient approach for profiling cell subsets at high throughput from small samples is available. We present a low-input microfluidic system for sorting immune cells into subsets and profiling their gene expression. We validate the system's technical performance against standard subset isolation and library construction protocols and demonstrate the importance of subset-specific profiling through in vitro stimulation experiments. We show the ability of this integrated platform to identify subset-specific disease signatures by profiling four immune cell subsets in blood from patients with systemic lupus erythematosus (SLE) and matched control subjects. The platform has the potential to make multiplexed subset-specific analysis routine in many research laboratories and clinical settings.

5.
Cancer Immunol Immunother ; 68(3): 421-432, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30564891

RESUMO

Targeting immune checkpoint pathways, such as programmed death ligand-1 (PD-L1, also known as CD274 or B7-H1) or its receptor programmed cell death-1 (PD-1) has shown improved survival for patients with numerous types of cancers, not limited to lung cancer, melanoma, renal cell carcinoma, and Hodgkin lymphoma. PD-L1 is a co-inhibitory molecule whose expression on the surface of tumor cells is associated with worse prognosis in many tumors. Here we describe a splice variant (secPD-L1) that does not splice into the transmembrane domain, but instead produces a secreted form of PD-L1 that has a unique 18 amino acid tail containing a cysteine that allows it to homodimerize and more effectively inhibit lymphocyte function than monomeric soluble PD-L1. We show that recombinant secPD-L1 can dimerize and inhibit T-cell proliferation and IFN-gamma production in vitro. The secPD-L1 variant is expressed by malignant cells in vitro that also express high levels of full-length PD-L1. Transcriptomic analysis of gene expression across The Cancer Genome Atlas found the strongest association of secPD-L1 with full-length PD-L1, but also with subsets of immunologic genes, such as in myeloid-derived suppressor cells. Moreover, the splice variant is also expressed in normal tissues and within normal peripheral blood cells it is preferentially expressed in activated myeloid cells. This is the first report of a form of secreted PD-L1 that homodimerizes and is functionally active. SecPD-L1 may function as a paracrine negative immune regulator within the tumor, since secPD-L1 does not require a cell-to-cell interaction to mediate its inhibitory effect.


Assuntos
Antígeno B7-H1/genética , Imunossupressores/farmacologia , Multimerização Proteica , Processamento de RNA , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/química , Antígeno B7-H1/farmacologia , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Células Supressoras Mieloides/fisiologia , Placenta/metabolismo , Gravidez , Microambiente Tumoral
6.
Cell Rep ; 25(1): 107-117.e3, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30282021

RESUMO

A detailed understanding of the mechanisms that establish or maintain the latent reservoir of HIV will guide approaches to eliminate persistent infection. We used a cell line and primary cell models of HIV latency to investigate viral RNA (vRNA) expression and the role of the host transcriptome using single-cell approaches. Single-cell vRNA quantitation identified distinct populations of cells expressing various levels of vRNA, including completely silent populations. Strikingly, single-cell RNA-seq of latently infected primary cells demonstrated that HIV downregulation occurred in diverse transcriptomic environments but was significantly associated with expression of a specific set of cellular genes. In particular, latency was more frequent in cells expressing a transcriptional signature that included markers of naive and central memory T cells. These data reveal that expression of HIV proviruses within the latent reservoir are influenced by the host cell transcriptional program. Therapeutic modulation of these programs may reverse or enforce HIV latency.


Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/fisiologia , Linfócitos T CD4-Positivos/virologia , Regulação para Baixo , Células HEK293 , HIV/genética , Infecções por HIV/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Células Jurkat , Provírus/genética , Provírus/fisiologia , RNA Viral/biossíntese , RNA Viral/genética , Análise de Célula Única , Transcrição Genética , Latência Viral
7.
Arthritis Res Ther ; 20(1): 139, 2018 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-29996944

RESUMO

BACKGROUND: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. METHODS: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. RESULTS: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 µg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. CONCLUSIONS: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.


Assuntos
Artrite Reumatoide/patologia , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/métodos , Membrana Sinovial/patologia , Criopreservação , Humanos
9.
Chaos ; 26(6): 063110, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27368775

RESUMO

Dynamical systems are frequently used to model biological systems. When these models are fit to data, it is necessary to ascertain the uncertainty in the model fit. Here, we present prediction deviation, a metric of uncertainty that determines the extent to which observed data have constrained the model's predictions. This is accomplished by solving an optimization problem that searches for a pair of models that each provides a good fit for the observed data, yet has maximally different predictions. We develop a method for estimating a priori the impact that additional experiments would have on the prediction deviation, allowing the experimenter to design a set of experiments that would most reduce uncertainty. We use prediction deviation to assess uncertainty in a model of interferon-alpha inhibition of viral infection, and to select a sequence of experiments that reduces this uncertainty. Finally, we prove a theoretical result which shows that prediction deviation provides bounds on the trajectories of the underlying true model. These results show that prediction deviation is a meaningful metric of uncertainty that can be used for optimal experimental design.


Assuntos
Aprendizagem , Modelos Teóricos , Incerteza , Infecções por HIV , Humanos
10.
Retrovirology ; 13(1): 45, 2016 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-27363431

RESUMO

Humans encode seven APOBEC3 proteins (A-H), with A3G, 3F and 3H as the major factors restricting HIV-1 replication. HIV-1, however, encodes Vif, which counteracts A3 proteins by chaperoning them to the proteasome where they are degraded. Vif polymorphisms found in HIV-1s isolated from infected patients have varying anti-A3G potency when assayed in vitro, but there are few studies demonstrating this in in vivo models. Here, we created Friend murine leukemia viruses encoding vif alleles that were previously shown to differentially neutralize A3G in cell culture or that were originally found in primary HIV-1 isolates. Infection of transgenic mice expressing different levels of human A3G showed that these naturally occurring Vif variants differed in their ability to counteract A3G during in vivo infection, although the effects on viral replication were not identical to those seen in cultured cells. We also found that the polymorphic Vifs that attenuated viral replication reverted to wild type only in A3G transgenic mice. Finally, we found that the level of A3G-mediated deamination was inversely correlated with the level of viral replication. This animal model should be useful for studying the functional significance of naturally occurring vif polymorphisms, as well as viral evolution in the presence of A3G.


Assuntos
Desaminase APOBEC-3G/metabolismo , Infecções por HIV/virologia , HIV-1/genética , Polimorfismo Genético , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Desaminase APOBEC-3G/genética , Alelos , Animais , Modelos Animais de Doenças , Vírus da Leucemia Murina de Friend/genética , Vírus da Leucemia Murina de Friend/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação , Replicação Viral
11.
PLoS One ; 11(3): e0152316, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27010978

RESUMO

Type 1 interferons such as interferon-alpha (IFNα) inhibit replication of Human immunodeficiency virus (HIV-1) by upregulating the expression of genes that interfere with specific steps in the viral life cycle. This pathway thus represents a potential target for immune-based therapies that can alter the dynamics of host-virus interactions to benefit the host. To obtain a deeper mechanistic understanding of how IFNα impacts spreading HIV-1 infection, we modeled the interaction of HIV-1 with CD4 T cells and IFNα as a dynamical system. This model was then tested using experimental data from a cell culture model of spreading HIV-1 infection. We found that a model in which IFNα induces reversible cellular states that block both early and late stages of HIV-1 infection, combined with a saturating rate of conversion to these states, was able to successfully fit the experimental dataset. Sensitivity analysis showed that the potency of inhibition by IFNα was particularly dependent on specific network parameters and rate constants. This model will be useful for designing new therapies targeting the IFNα network in HIV-1-infected individuals, as well as potentially serving as a template for understanding the interaction of IFNα with other viruses.


Assuntos
Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Interferon-alfa/química , Replicação Viral/fisiologia , Algoritmos , Antivirais/química , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Simulação por Computador , Humanos , Imunidade Inata , Concentração Inibidora 50 , Modelos Estatísticos , Reprodutibilidade dos Testes , Fatores de Tempo , Replicação Viral/efeitos dos fármacos
12.
Science ; 351(6277): 1048-54, 2016 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-26912368

RESUMO

Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.


Assuntos
Anticorpos/imunologia , Afinidade de Anticorpos/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Imagem Molecular/métodos , Animais , Anticorpos/genética , Afinidade de Anticorpos/genética , HIV-1/imunologia , Humanos , Camundongos , Microscopia de Fluorescência por Excitação Multifotônica , Orthomyxoviridae/imunologia , Análise de Sequência de DNA , Análise de Célula Única
13.
J Virol ; 89(1): 155-64, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25320301

RESUMO

UNLABELLED: Interleukin-1 beta (IL-1ß) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1ß secretion through the DNA-sensing molecule IFI16, the effect of IL-1ß on the course of infection is unknown. To test whether IL-1ß secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1ß) that encodes the mature form of IL-1ß. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1ß exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1ß-infected mice correlated with a large expansion of Gr-1(+) CD11b(+) myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1ß infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1ß was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1ß secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1ß represents a new model of proinflammatory retroviral infection. IMPORTANCE: Interleukin-1 beta (IL-1ß) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1ß on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1ß) that encodes IL-1ß and promotes abundant IL-1ß secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1ß as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.


Assuntos
Interleucina-1beta/metabolismo , Vírus da Leucemia Murina/imunologia , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/patologia , Infecções por Retroviridae/patologia , Infecções Tumorais por Vírus/patologia , Animais , Interações Hospedeiro-Patógeno , Interleucina-1beta/genética , Vírus da Leucemia Murina/genética , Vírus da Leucemia Murina/fisiologia , Leucemia Experimental/virologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Infecções por Retroviridae/virologia , Esplenomegalia/patologia , Infecções Tumorais por Vírus/virologia , Carga Viral , Virulência , Replicação Viral
14.
PLoS Pathog ; 10(5): e1004145, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24851906

RESUMO

The apolipoprotein B editing complex 3 (A3) cytidine deaminases are among the most highly evolutionarily selected retroviral restriction factors, both in terms of gene copy number and sequence diversity. Primate genomes encode seven A3 genes, and while A3F and 3G are widely recognized as important in the restriction of HIV, the role of the other genes, particularly A3A, is not as clear. Indeed, since human cells can express multiple A3 genes, and because of the lack of an experimentally tractable model, it is difficult to dissect the individual contribution of each gene to virus restriction in vivo. To overcome this problem, we generated human A3A and A3G transgenic mice on a mouse A3 knockout background. Using these mice, we demonstrate that both A3A and A3G restrict infection by murine retroviruses but by different mechanisms: A3G was packaged into virions and caused extensive deamination of the retrovirus genomes while A3A was not packaged and instead restricted infection when expressed in target cells. Additionally, we show that a murine leukemia virus engineered to express HIV Vif overcame the A3G-mediated restriction, thereby creating a novel model for studying the interaction between these proteins. We have thus developed an in vivo system for understanding how human A3 proteins use different modes of restriction, as well as a means for testing therapies that disrupt HIV Vif-A3G interactions.


Assuntos
Citidina Desaminase/fisiologia , Proteínas/fisiologia , Infecções por Retroviridae/genética , Infecções por Retroviridae/virologia , Carga Viral/genética , Desaminase APOBEC-3G , Animais , Células Cultivadas , HIV-1/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Retroviridae/fisiologia , Montagem de Vírus/genética , Internalização do Vírus , Replicação Viral/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética
15.
J Virol ; 87(13): 7357-66, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23616654

RESUMO

Early events during retroviral infection play a critical role in determining the course of infection and pathogenesis, but the mechanisms that regulate this phase of infection are poorly understood. Toll-like receptor 7 (TLR7) is required for promoting germinal center reactions and virus-specific neutralizing antibodies at later time points postinfection, but TLR7's role in early acute infection has not been determined. By infecting TLR7-deficient mice with a retroviral pathogen, Friend virus (FV), I determined that TLR7 potently inhibits retroviral replication during the first 5 days of infection and is required for rapid secretion of virus-specific IgM and interleukin-10 (IL-10) in response to infection. Although the IgM response was nonneutralizing, plasmas from wild-type mice but not TLR7-deficient mice inhibited FV replication when passively transferred to infected mice, suggesting an indirect mechanism of antibody function. Interestingly, IL-10 was secreted primarily by CD4 T cells, and IL-10-deficient mice also exhibited accelerated early virus spread, demonstrating that this response inhibits acute infection. Surprisingly, TLR7-deficient mice exhibited normal or elevated secretion of proinflammatory cytokines during acute infection, revealing the existence of a TLR7-independent retrovirus-sensing pathway that drives inflammatory cytokine secretion. Together, these results establish a previously unappreciated role for lymphocytes in mediating rapid TLR7-dependent inhibition of early retroviral infection through nonneutralizing IgM and IL-10.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Vírus da Leucemia Murina de Friend/imunologia , Glicoproteínas de Membrana/imunologia , Infecções por Retroviridae/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Citocinas/imunologia , Citometria de Fluxo , Imunoglobulina M/imunologia , Interleucina-10/imunologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptor 7 Toll-Like/genética , Replicação Viral/imunologia
16.
Immunology ; 136(4): 370-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22444240

RESUMO

The discovery of host-encoded gene products that sense molecular patterns in infectious microbes, and the demonstration of their role in triggering innate and adaptive immune responses, has been a key milestone in our understanding of immunology. Twenty-three years after Janeway first outlined the fundamental concepts of the 'pattern recognition' model, and 15 years since the identification of Toll-like receptors (TLRs) as pattern recognition receptors (PRRs), new insights continue to be revealed, and questions remain. For example, innate immune responses to microbes that are mediated by PRRs have historically been viewed as the domain of innate immune cell populations such as dendritic cells and macrophages. New evidence, however, has pointed to the role of B-cell-intrinsic TLR activation in shaping antibody responses. These studies have revealed that TLRs regulate a complex transcriptional network that controls multiple steps in the development of antigen-specific antibodies. This review covers these recent developments regarding the role of TLRs in B-cell gene expression and function in vitro and in vivo, and highlights the remaining challenges in the field, with particular emphasis on the role of TLRs in antibody responses to viral infection. A more complete understanding of how TLRs regulate antibody responses will lead to improved vaccine design.


Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Receptores Toll-Like/imunologia , Animais , Humanos , Imunidade Inata , Ativação Linfocitária , Camundongos , Transdução de Sinais , Receptores Toll-Like/metabolismo , Vírus/imunologia
17.
Biotechnol Lett ; 34(5): 853-6, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22207147

RESUMO

Heparin covalently attached to a water-insoluble resin suspended in HIV-infected aqueous buffer or whole blood captures the virus; subsequent physical separation of the immobilized heparin reduced the viral titers by over 80 and 50%, respectively. The detoxification concept has been validated by both circulating an HIV-1 solution through a column packed with the heparin-sepharose beads and successively mixing an HIV-1 solution with fresh beads.


Assuntos
Sangue/virologia , Descontaminação/métodos , HIV-1/isolamento & purificação , Heparina/metabolismo , Humanos , Ligação Proteica , Carga Viral
18.
PLoS Pathog ; 7(10): e1002293, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21998589

RESUMO

The development of vaccines that can enhance immunity to viral pathogens is an important goal. However, the innate molecular pathways that regulate the strength and quality of the immune response remain largely uncharacterized. To define the role of Toll-like receptor (TLR) signaling in control of a model retroviral pathogen, Friend virus (FV), I generated mice in which the TLR signaling adapter Myd88 was selectively deleted in dendritic cell (DC) or in B cell lineages. Deletion of Myd88 in DCs had little effect on immune control of FV, while B cell specific deletion of Myd88 caused a dramatic increase in viral infectious centers and a significantly reduced antibody response, indicating that B cell-intrinsic TLR signaling plays a crucial role, while TLR signaling in DCs is less important. I then identified the single-stranded RNA sensing protein TLR7 as being required for antibody-mediated control of FV by analyzing mice deficient in TLR7. Remarkably, B cells in infected TLR7-deficient mice upregulated CD69 and CD86 early in infection, but failed to develop into germinal center B cells. CD4 T cell responses were also attenuated in the absence of TLR7, but CD8 responses were TLR7 independent, suggesting the existence of additional pathways for detection of retroviral particles. Together these results demonstrate that the vertebrate immune system detects retroviruses in vivo via TLR7 and that this pathway regulates a key checkpoint controlling development of germinal center B cells.


Assuntos
Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Glicoproteínas de Membrana/metabolismo , Infecções por Retroviridae/imunologia , Receptor 7 Toll-Like/metabolismo , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Vírus da Leucemia Murina de Friend/metabolismo , Vírus da Leucemia Murina de Friend/patogenicidade , Centro Germinativo/virologia , Interferon gama/genética , Interferon gama/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Receptor 7 Toll-Like/genética , Regulação para Cima
19.
Virology ; 387(2): 313-21, 2009 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-19304304

RESUMO

Cytidine deamination is the primary mechanism by which APOBEC3G restricts HIV-1; however, several studies have reported that APOBEC3G also inhibits virus replication via a mechanism that is independent of deamination. Using active site APOBEC3G mutants, we have re-evaluated the biological relevance of deaminase-independent APOBEC3G-mediated restriction of HIV-1. APOBEC3G proteins with Glu-->Ala mutations in AS1, AS2 or AS1 and AS2 were stably expressed at physiological levels in CEM-SS T cells and 293T cells and the ability of the cells to support Deltavif HIV-1 replication was then tested. The AS2 and AS1/AS2 mutants were packaged efficiently into virions but in single-cycle or multi-cycle HIV-1 replication assays, were found to lack antiviral activity. The AS1 mutant, which retained deaminase activity, maintained near wild-type antiviral function. To determine the potency of APOBEC3G antiviral activity, cell lines were established that that expressed low levels of wild-type APOBEC3G and generated virions that contained as few as 1-2 APOBEC3G molecules. Even at very low copy number, APOBEC3G caused a significant reduction in infectivity, suggesting that a single molecule of packaged APOBEC3G inactivates the virus. The high potency of APOBEC3G is consistent with a catalytic mechanism of restriction in which a single molecule can induce a string of mutations but difficult to reconcile with a deaminase-independent, non-catalytic mechanism. Analysis of the reverse transcript sequences showed that the G-->A mutations were clustered, likely reflecting the action of single APOBEC3G molecules acting processively. We conclude that cytidine deamination is the mechanism by which APOBEC3G restricts HIV-1.


Assuntos
Citidina Desaminase/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , Replicação Viral , Desaminase APOBEC-3G , Motivos de Aminoácidos , Linhagem Celular , Citidina Desaminase/genética , Infecções por HIV/enzimologia , Humanos , Imunidade Inata , Mutação Puntual , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo
20.
PLoS Pathog ; 5(2): e1000298, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19214214

RESUMO

Although retroviruses have been extensively studied for many years, basic questions about how retroviral infections are detected by the immune system and which innate pathways are required for the generation of immune responses remain unanswered. Defining these pathways and how they contribute to the anti-retroviral immune responses would assist in the development of more effective vaccines for retroviral pathogens such as HIV. We have investigated the roles played by CD11c(+) dendritic cells (DCs) and by Toll-like receptor (TLR) signaling pathways in the generation of an anti-retroviral immune response against a mouse retroviral pathogen, Friend murine leukemia virus (F-MLV). Specific deletion of DCs during F-MLV infection caused a significant increase in viral titers at 14 days post-infection, indicating the importance of DCs in immune control of the infection. Similarly, Myd88 knockout mice failed to control F-MLV, and sustained high viral titers (10(7) foci/spleen) for several months after infection. Strikingly, both DC-depleted mice and Myd88 knockout mice exhibited only a partial reduction of CD8(+) T cell responses, while the IgG antibody response to F-MLV was completely lost. Furthermore, passive transfer of immune serum from wild-type mice to Myd88 knockout mice rescued control of F-MLV. These results identify TLR signaling and CD11c(+) DCs as playing critical roles in the humoral response to retroviruses.


Assuntos
Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vírus da Leucemia Murina de Friend/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Infecções por Retroviridae/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Vírus da Leucemia Murina de Friend/fisiologia , Imunização Passiva , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Infecções por Retroviridae/virologia , Transdução de Sinais , Estatísticas não Paramétricas , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia , Replicação Viral
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