Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.621
Filtrar
1.
Clin Chim Acta ; 508: 110-114, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32405080

RESUMO

BACKGROUND: We observe changes of the main lymphocyte subsets (CD16+CD56、CD19、CD3、CD4、and CD8) in COVID-19-infected patients and explore whether the changes are associated with disease severity. METHODS: One-hundred and fifty-four cases of COVID-19-infected patients were selected and divided into 3 groups (moderate group, severe group and critical group). The flow cytometry assay was performed to examine the numbers of lymphocyte subsets. RESULTS: CD3+, CD4+ and CD8 + T lymphocyte subsets were decreased in COVID-19-infected patients. Compared with the moderate group and the sever group, CD3+, CD4+ and CD8+ T cells in the critical group decreased greatly (P < 0.001, P = 0.005 or P = 0.001). CONCLUSIONS: Reduced CD3+, CD4+, CD8+ T lymphocyte counts may reflect the severity of the COVID-19. Monitoring T cell changes has important implications for the diagnosis and treatment of severe patients who may become critically ill.


Assuntos
Betacoronavirus/patogenicidade , Doenças Cardiovasculares/diagnóstico , Infecções por Coronavirus/diagnóstico , Diabetes Mellitus/diagnóstico , Pneumopatias/diagnóstico , Pneumonia Viral/diagnóstico , Subpopulações de Linfócitos T/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Complexo CD3/genética , Complexo CD3/imunologia , Antígenos CD4/genética , Antígenos CD4/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/mortalidade , Doenças Cardiovasculares/fisiopatologia , Estudos de Coortes , Comorbidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/fisiopatologia , Diabetes Mellitus/imunologia , Diabetes Mellitus/mortalidade , Diabetes Mellitus/fisiopatologia , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Pneumopatias/imunologia , Pneumopatias/mortalidade , Pneumopatias/fisiopatologia , Masculino , Pessoa de Meia-Idade , Pandemias , Seleção de Pacientes , Pneumonia Viral/imunologia , Pneumonia Viral/mortalidade , Pneumonia Viral/fisiopatologia , Prognóstico , Índice de Gravidade de Doença , Análise de Sobrevida , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia
2.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376625

RESUMO

Downregulation of BST-2/tetherin and CD4 by HIV-1 viral protein U (Vpu) promotes viral egress and allows infected cells to evade host immunity. Little is known however about the natural variability in these Vpu functions among the genetically diverse viral subtypes that contribute to the HIV-1 pandemic. We collected Vpu isolates from 332 treatment-naive individuals living with chronic HIV-1 infection in Uganda, Rwanda, South Africa, and Canada. Together, these Vpu isolates represent four major HIV-1 group M subtypes (A [n = 63], B [n = 84], C [n = 94], and D [n = 59]) plus intersubtype recombinants and uncommon strains (n = 32). The ability of each Vpu clone to downregulate endogenous CD4 and tetherin was quantified using flow cytometry following transfection into an immortalized T-cell line and compared to that of a reference Vpu clone derived from HIV-1 subtype B NL4.3. Overall, the median CD4 downregulation function of natural Vpu isolates was similar to that of NL4.3 (1.01 [interquartile range {IQR}, 0.86 to 1.18]), while the median tetherin downregulation function was moderately lower than that of NL4.3 (0.90 [0.79 to 0.97]). Both Vpu functions varied significantly among HIV-1 subtypes (Kruskal-Wallis P < 0.0001). Specifically, subtype C clones exhibited the lowest CD4 and tetherin downregulation activities, while subtype D and B clones were most functional for both activities. We also identified Vpu polymorphisms associated with CD4 or tetherin downregulation function and validated six of these using site-directed mutagenesis. Our results highlight the marked extent to which Vpu function varies among global HIV-1 strains, raising the possibility that natural variation in this accessory protein may contribute to viral pathogenesis and/or spread.IMPORTANCE The HIV-1 accessory protein Vpu enhances viral spread by downregulating CD4 and BST-2/tetherin on the surface of infected cells. Natural variability in these Vpu functions may contribute to HIV-1 pathogenesis, but this has not been investigated among the diverse viral subtypes that contribute to the HIV-1 pandemic. In this study, we found that Vpu function differs significantly among HIV-1 subtypes A, B, C, and D. On average, subtype C clones displayed the lowest ability to downregulate both CD4 and tetherin, while subtype B and D clones were more functional. We also identified Vpu polymorphisms that associate with functional differences among HIV-1 isolates and subtypes. Our study suggests that genetic diversity in Vpu may play an important role in the differential pathogenesis and/or spread of HIV-1.


Assuntos
Antígenos CD/biossíntese , Antígenos CD4/biossíntese , Regulação para Baixo , Infecções por HIV , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Antígenos CD/genética , Antígenos CD4/genética , Linhagem Celular Transformada , Doença Crônica , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/genética , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Proteínas Virais Reguladoras e Acessórias/genética
3.
Nat Commun ; 11(1): 948, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075963

RESUMO

Eliciting protective titers of HIV-1 broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development, but current vaccine strategies have yet to induce bnAbs in humans. Many bnAbs isolated from HIV-1-infected individuals are encoded by immunoglobulin gene rearrangments with infrequent naive B cell precursors and with unusual genetic features that may be subject to host regulatory control. Here, we administer antibodies targeting immune cell regulatory receptors CTLA-4, PD-1 or OX40 along with HIV envelope (Env) vaccines to rhesus macaques and bnAb immunoglobulin knock-in (KI) mice expressing diverse precursors of CD4 binding site HIV-1 bnAbs. CTLA-4 blockade augments HIV-1 Env antibody responses in macaques, and in a bnAb-precursor mouse model, CTLA-4 blocking or OX40 agonist antibodies increase germinal center B and T follicular helper cells and plasma neutralizing antibodies. Thus, modulation of CTLA-4 or OX40 immune checkpoints during vaccination can promote germinal center activity and enhance HIV-1 Env antibody responses.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Fatores Imunológicos/imunologia , Vacinação/métodos , Vacinas contra a AIDS/administração & dosagem , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Bloqueadores/imunologia , Anticorpos Neutralizantes/sangue , Linfócitos B/imunologia , Antígenos CD4/genética , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Humanos , Fatores Imunológicos/administração & dosagem , Ativação Linfocitária , Macaca mulatta/imunologia , Camundongos , Camundongos Transgênicos , Receptores OX40/agonistas , Receptores OX40/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Transcriptoma , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
4.
PLoS One ; 15(1): e0227889, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31971988

RESUMO

BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially life-threatening disease, and until today there is no other treatment available than surgical intervention. Dipeptidyl peptidase-4 (DPP4)-inhibitors, used clinically to treat type 2 diabetes, have in murine models been shown to attenuate aneurysm formation and decrease aortic wall matrix degradation, inflammation and apoptosis. Our aim was to investigate if DPP4 is present, active and differentially expressed in human AAA. METHODS AND RESULTS: DPP4 gene expression was elevated in both media and adventitia of AAA tissue compared with control tissue, as measured by microarrays and qPCR, with consistent findings in external data. The plasma activity of DPP4 was however lower in male patients with AAA compared with age- and gender-matched controls, independently of comorbidity or medication. Immunohistochemical double staining revealed co-localization of DPP4 with cells positive for CD68, CD4 and -8, CD20, and SMA. Gene set enrichment analysis demonstrated that expression of DPP4 in AAA tissue correlated with expression of biological processes related to B- and T-cells, extracellular matrix turnover, peptidase activity, oxidative stress and angiogenesis whereas it correlated negatively with muscle-/actin-related processes. CONCLUSION: DPP4 is upregulated in both media and adventitia of human AAA and correlates with aneurysm pathophysiological processes. These results support previous murine mechanistic studies and implicate DPP4 as a target in AAA disease.


Assuntos
Aorta/metabolismo , Aneurisma da Aorta Abdominal/genética , Vasos Sanguíneos/metabolismo , Dipeptidil Peptidase 4/genética , Actinas/genética , Adulto , Idoso , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Aorta/patologia , Aneurisma da Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/patologia , Apoptose/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Vasos Sanguíneos/patologia , Antígenos CD4/genética , Antígenos CD8/genética , Dipeptidil Peptidase 4/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia , Linfócitos T/metabolismo
5.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31852789

RESUMO

The HIV-1 Env protein is exposed at the surface of virions and infected cells. Env fluctuates between different closed and open structural states and these conformations influence both viral infectivity and sensitivity to antibody binding and neutralization. We established a flow virometry assay to visualize Env proteins at the surface of human immunodeficiency virus type 1 (HIV-1) virions. The assay is performed on ultracentrifuged fluorescent viral particles that are stained with a panel of broadly neutralizing antibodies (bNAbs) and nonneutralizing antibodies (nnAbs) that probe different epitopes of Env. We used this assay to compare Env at the surface of producer cells and viral particles and to analyze the effect of Nef, CD4, and SERINC5 on Env accessibility to antibodies. We studied the laboratory-adapted strain NL4-3 and two transmitted/founder viruses, THRO and CH058. We confirm that antibody accessibility varies between viral strains and show that Nef, CD4, and SERINC5 additively impact Env conformations. We further demonstrate that the Env accessibility profile on virions is globally similar to that observed on HIV-1-infected cells, with some noticeable differences. For instance, nnAbs bind to virions more efficiently than to producer cells, likely reflecting changes in Env conformational states on mature viral particles. This test complements other techniques and provides a convenient and simple tool for quantifying and probing the structure of Env at the virion surface and to analyze the impact of viral and cellular proteins on these parameters.IMPORTANCE HIV-1 Env conformation is one of the key parameters determining viral infectivity. The flow virometry-based assay developed in this study allows for the characterization of proteins incorporated in HIV-1 particles. We studied the conformation of HIV-1 Env and the impact that the viral protein Nef and the cellular proteins CD4 and SERINC5 have on Env accessibility to antibodies. Our assay permitted us to highlight some noticeable differences in the conformation of Env between producer cells and viral particles. It contributes to a better understanding of the actual composition of HIV-1 particles.


Assuntos
Antígenos CD4/metabolismo , Citometria de Fluxo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Proteínas de Membrana/metabolismo , Vírion/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Antígenos CD4/genética , Linhagem Celular , Epitopos/genética , Epitopos/metabolismo , Anticorpos Anti-HIV/química , Infecções por HIV/genética , HIV-1/genética , Humanos , Proteínas de Membrana/genética , Conformação Proteica , Vírion/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
6.
Elife ; 82019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31644426

RESUMO

Numerous challenges have impeded HIV-1 vaccine development. Among these is the lack of a convenient small animal model in which to study antibody elicitation and efficacy. We describe a chimeric Rhabdo-Immunodeficiency virus (RhIV) murine model that recapitulates key features of HIV-1 entry, tropism and antibody sensitivity. RhIVs are based on vesicular stomatitis viruses (VSV), but viral entry is mediated by HIV-1 Env proteins from diverse HIV-1 strains. RhIV infection of transgenic mice expressing human CD4 and CCR5, exclusively on mouse CD4+ cells, at levels mimicking those on human CD4+ T-cells, resulted in acute, resolving viremia and CD4+ T-cell depletion. RhIV infection elicited protective immunity, and antibodies to HIV-1 Env that were primarily non-neutralizing and had modest protective efficacy following passive transfer. The RhIV model enables the convenient in vivo study of HIV-1 Env-receptor interactions, antiviral activity of antibodies and humoral responses against HIV-1 Env, in a genetically manipulatable host.


Assuntos
Anticorpos Antivirais/biossíntese , Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Vírus Reordenados/genética , Vesiculovirus/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Animais , Especificidade de Anticorpos , Antígenos CD4/genética , Antígenos CD4/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/virologia , Modelos Animais de Doenças , Efeito Fundador , Expressão Gênica , Infecções por HIV/genética , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Humanos , Camundongos , Camundongos Transgênicos , Vírus Reordenados/imunologia , Receptores CCR5/genética , Receptores CCR5/imunologia , Vesiculovirus/imunologia , Tropismo Viral/genética , Tropismo Viral/imunologia , Internalização do Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia
7.
J Immunol ; 203(5): 1208-1217, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31315887

RESUMO

The CD4Cre transgenic model has been widely used for T cell-specific gene manipulation. We report unexpected highly efficient Cre-mediated recombination in alveolar macrophages (AMFs), bronchial epithelial cells (BECs), and alveolar epithelial cells (AECs) in this strain of mice. Different from CD4 T cells, AMFs, AECs, and BECs do not express detectable Cre protein, suggesting that Cre protein is either very transiently expressed in these cells or only expressed in their precursors. Mice carrying a conditional constitutively active KRas (caKRas) allele and the CD4Cre transgene contain not only hyperactivated T cells but also develop severe AMF accumulation, AEC and BEC hyperplasia, and adenomas in the lung, leading to early lethality correlated with caKRas expression in these cells. We propose that caKRas-CD4Cre mice represent, to our knowledge, a novel model of proliferative pneumonitis involving macrophages and epithelial cells and that the CD4Cre model may offer unique usefulness for studying gene functions simultaneously in multilineages in the lung. Our observations, additionally, suggest that caution in data interpretation is warranted when using the CD4Cre transgenic model for T cell-specific gene manipulation, particularly when lung pathophysiological status is being examined.


Assuntos
Células Epiteliais Alveolares/metabolismo , Antígenos CD4/genética , Integrases/genética , Macrófagos Alveolares/metabolismo , Pneumonia/etiologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Recombinação Genética , Transgenes
8.
PLoS Biol ; 17(6): e3000304, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31181085

RESUMO

Individuals chronically infected with HIV-1 harbor complex viral populations within their bloodstreams. Recently, it has come to light that when these people infect others, the new infection is typically established by only one or a small number of virions from within this complex viral swarm. An important goal is to characterize the biological properties of HIV-1 virions that seed and exist early in new human infections because these are potentially the only viruses against which a prophylactic HIV-1 vaccine would need to elicit protection. This includes understanding how the Envelope (Env) protein of these virions interacts with the T-cell receptor CD4, which supports attachment and entry of HIV-1 into target cells. We examined early HIV-1 isolates for their ability to infect cells via the CD4 receptor of 15 different primate species. Primates were the original source of HIV-1 and now serve as valuable animal models for studying HIV-1. We find that most primary isolates of HIV-1 from the blood, including early isolates, are highly selective and enter cells through some primate CD4 receptor orthologs but not others. This phenotype is remarkably consistent, regardless of route of transmission, viral subtype, or time of isolation post infection. We show that the weak CD4 binding affinity of blood-derived HIV-1 isolates is what makes them sensitive to the small sequence differences in CD4 from one primate species to the next. To substantiate this, we engineered an early HIV-1 Env to have high, medium, or low binding affinity to CD4, and we show that it loses the ability to enter cells via the CD4 receptor of many primate species as the binding affinity gets weaker. Based on the phenotype of selective use of primate CD4, we find that weak CD4 binding appears to be a nearly universal property of HIV-1 circulating in the bloodstream. Therefore, weak binding to CD4 must be a selected and important property in the biology of HIV-1 in the body. We identify six primate species that encode CD4 receptors that fully support the entry of early HIV-1 isolates despite their low binding affinity for CD4. These findings will help inform long-standing efforts to model HIV-1 transmission and early disease in primates.


Assuntos
Antígenos CD4/imunologia , Infecções por HIV/imunologia , HIV-1/genética , Animais , Aotidae , Antígenos CD4/genética , Linhagem Celular , Modelos Animais de Doenças , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/genética , Soropositividade para HIV/genética , Soropositividade para HIV/imunologia , HIV-1/imunologia , Humanos , Macaca mulatta , Primatas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
9.
PLoS Pathog ; 15(6): e1007819, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194843

RESUMO

Recently identified broadly neutralizing antibodies (bnAbs) show great potential for clinical interventions against HIV-1 infection. However, resistant strains may impose substantial challenges. Here, we report on the identification and characterization of a panel of HIV-1 strains with broad and potent resistance against a large number of bnAbs, particularly those targeting the CD4-binding site (CD4bs). Site-directed mutagenesis revealed that several key epitope mutations facilitate resistance and are located in the inner domain, loop D, and ß23/loop V5/ß24 of HIV-1 gp120. The resistance is largely correlated with binding affinity of antibodies to the envelope trimers expressed on the cell surface. Our results therefore demonstrate the existence of broadly resistant HIV-1 strains against CD4bs neutralizing antibodies. Treatment strategies based on the CD4bs bnAbs must overcome such resistance to achieve optimal clinical outcomes.


Assuntos
Anticorpos Neutralizantes/imunologia , Antígenos CD4/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Anticorpos Neutralizantes/genética , Antígenos CD4/genética , Linhagem Celular , Epitopos/genética , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos
10.
mBio ; 10(3)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31186327

RESUMO

It has recently emerged that HIV-1 Nef counteracts the antiviral host proteins SERINC3 and SERINC5. In particular, SERINC5 inhibits the infectivity of progeny virions when incorporated. SERINC3 and SERINC5 are also counteracted by the unrelated murine leukemia virus glycosylated Gag (glycoGag) protein, which possesses a potent Nef-like activity on HIV-1 infectivity. We now report that a minimal glycoGag termed glycoMA can fully substitute for Nef in promoting HIV-1 replication in Jurkat T lymphoid cells, indicating that Nef enhances replication in these cells mainly by counteracting SERINCs. In contrast, the SERINC antagonist glycoMA was unable to substitute for Nef in MOLT-3 T lymphoid cells, in which HIV-1 replication was highly dependent on Nef, and remained so even in the absence of SERINC3 and SERINC5. As in MOLT-3 cells, glycoMA was unable to substitute for Nef in stimulating HIV-1 replication in primary human cells. Although the ability of Nef mutants to promote HIV-1 replication in MOLT-3 cells correlated with the ability to engage endocytic machinery and to downregulate CD4, Nef nevertheless rescued virus replication under conditions where CD4 downregulation did not occur. Taken together, our observations raise the possibility that Nef triggers the endocytosis of a novel antiviral factor that is active against both laboratory-adapted and primary HIV-1 strains.IMPORTANCE The Nef protein of HIV-1 and the unrelated glycoGag protein of a murine leukemia virus similarly prevent the uptake of antiviral host proteins called SERINC3 and SERINC5 into HIV-1 particles, which enhances their infectiousness. We now show that although both SERINC antagonists can in principle similarly enhance HIV-1 replication, glycoGag is unable to substitute for Nef in primary human cells and in a T cell line called MOLT-3. In MOLT-3 cells, Nef remained crucial for HIV-1 replication even in the absence of SERINC3 and SERINC5. The pronounced effect of Nef on HIV-1 spreading in MOLT-3 cells correlated with the ability of Nef to engage cellular endocytic machinery and to downregulate the HIV-1 receptor CD4 but nevertheless persisted in the absence of CD4 downregulation. Collectively, our results provide evidence for a potent novel restriction activity that affects even relatively SERINC-resistant HIV-1 isolates and is counteracted by Nef.


Assuntos
HIV-1/genética , HIV-1/fisiologia , Glicoproteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Replicação Viral/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Antígenos CD4/genética , Linhagem Celular , Endocitose , Glicosilação , Interações Hospedeiro-Patógeno , Humanos , Células Jurkat , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
11.
Nanoscale ; 11(29): 13750-13757, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31140518

RESUMO

Recent studies on metal-insulator-metal-based plasmonic antennas have shown that emitters could couple with higher-order gap-plasmon modes in sub-10-nm gaps to overcome quenching. However, these gaps are often physically inaccessible for functionalization and are not scalably manufacturable. Here, using a simple biomimetic batch-fabrication, a plasmonic metasurface is created consisting of closely-coupled nanodisks and nanoholes in a metal-insulator-metal arrangement. The quadrupolar mode of this system exhibits strong broadband resonance in the visible-near-infrared regime with minimal absorptive losses and effectively supresses quenching, making it highly suitable for broadband plasmon-enhanced fluorescence. Functionalizing the accessible insulator nanogap, analytes are selectively immobilized onto the plasmonic hotspot enabling highly-localized detection. Sensing the streptavidin-biotin complex, a 91-, 288-, 403- and 501-fold fluorescence enhancement is observed for Alexa Fluor 555, 647, 750 and 790, respectively. Finally, the detection of single-stranded DNA (gag, CD4 and CCR5) analogues of genes studied in the pathogenesis of HIV-1 between 10 pM-10 µM concentrations and then CD4 mRNA in the lysate of transiently-transfected cells with a 5.4-fold increase in fluorescence intensity relative to an untransfected control is demonstrated. This outcome promises the use of biomimetic Au metasurfaces as platforms for robust detection of low-abundance nucleic acids.


Assuntos
Materiais Biomiméticos/química , Ouro/química , Microscopia de Fluorescência , Nanoestruturas/química , Ácidos Nucleicos/análise , Proteínas de Bactérias/química , Biotina/análogos & derivados , Biotina/química , Antígenos CD4/genética , Corantes Fluorescentes/química , Ácidos Nucleicos/química , Polímeros/química , RNA Mensageiro/análise , Dióxido de Silício/química , Propriedades de Superfície
12.
Genome Med ; 11(1): 26, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039804

RESUMO

BACKGROUND: Genome wide association studies have identified > 200 susceptibility loci accounting for much of the heritability of multiple sclerosis (MS). Epstein-Barr virus (EBV), a memory B cell tropic virus, has been identified as necessary but not sufficient for development of MS. The molecular and immunological basis for this has not been established. Infected B cell proliferation is driven by signalling through the EBV produced cell surface protein LMP1, a homologue of the MS risk gene CD40. METHODS: We have investigated transcriptomes of B cells and EBV-infected B cells at Latency III (LCLs) and identified MS risk genes with altered expression on infection and with expression levels associated with the MS risk genotype (LCLeQTLs). The association of LCLeQTL genomic burden with EBV phenotypes in vitro and in vivo was examined. The risk genotype effect on LCL proliferation with CD40 stimulation was assessed. RESULTS: These LCLeQTL MS risk SNP:gene pairs (47 identified) were over-represented in genes dysregulated between B and LCLs (p < 1.53 × 10-4), and as target loci of the EBV transcription factor EBNA2 (p < 3.17 × 10-16). Overall genetic burden of LCLeQTLs was associated with some EBV phenotypes but not others. Stimulation of the CD40 pathway by CD40L reduced LCL proliferation (p < 0.001), dependent on CD40 and TRAF3 MS risk genotypes. Both CD40 and TRAF3 risk SNPs are in binding sites for the EBV transcription factor EBNA2, with expression of each correlated with EBNA2 expression dependent on genotype. CONCLUSIONS: These data indicate targeting EBV may be of therapeutic benefit in MS.


Assuntos
Linfócitos B/metabolismo , Antígenos CD4/genética , Herpesvirus Humano 4/fisiologia , Esclerose Múltipla/genética , Polimorfismo de Nucleotídeo Único , Fator 3 Associado a Receptor de TNF/genética , Linfócitos B/virologia , Células Cultivadas , Endonucleases/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Locos de Características Quantitativas , Transcriptoma , Latência Viral , Replicação Viral
13.
Gene ; 707: 22-29, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31026568

RESUMO

Reinforcing the immunity of pregnant sows can not only improve their own health condition but also increase the survival rate and healthy status of their piglets. This study aims to find single-nucleotide polymorphism (SNP) and molecular markers that are associated with the immune traits of pregnant sows. SLA-DOB and CD4 were selected as candidate genes, and blood samples were randomly collected from pregnant Landrace sows and used to detect T-lymphocyte subsets, interferon alpha, interleukin 6, Toll-like receptor 3, serum antibody immunoglobulin G, and porcine reproductive and respiratory syndrome virus-specific antibody. Then, association analyses were conducted for the polymorphic sites of candidate genes with immune traits. We found 12 mutations in the two genes and conducted an association study with eight of them. Our results indicated that among the eight mutations, SNP1, SNP2, and SNP3 of the SLA-DOB gene and Ins9, SNP10, and SNP11 in the CD4 gene are newly discovered mutations. Except for SNP1, SNP3, and SNP11, the other five SNPs are associated with at least one immune trait tested. Especially, SNP2 and Ins9 are significantly associated with at least one of the T-lymphocyte subgroups and at least one antibody. These novel mutations have potential important effects on the polymorphic loci of the above immune traits in pregnant sows. The results suggest that the SLA-DOB and CD4 genes and their genetic mutations can be considered as important candidate genes and mutations for the immunity of pregnant sows.


Assuntos
Antígenos CD4/genética , Estudos de Associação Genética/veterinária , Antígenos de Histocompatibilidade Classe I/genética , Imunidade , Mutação , Animais , Feminino , Haplótipos , Imunoglobulina G/sangue , Interleucina-6/sangue , Família Multigênica , Mutagênese Insercional , Polimorfismo de Nucleotídeo Único , Gravidez , Locos de Características Quantitativas , Suínos , Linfócitos T/imunologia , Receptor 3 Toll-Like/sangue , Fator de Necrose Tumoral alfa/sangue
14.
PLoS One ; 14(4): e0215993, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31013333

RESUMO

Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype-phenotype linkages.


Assuntos
Nanotecnologia , Peptídeos/química , Ligação Proteica/genética , Anticorpos de Domínio Único/química , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos/genética , Antígenos/imunologia , Antígenos CD4/genética , Antígenos CD4/imunologia , Genótipo , Humanos , Peptídeos/genética , Peptídeos/imunologia , Fenótipo , Pichia/química , Pichia/genética , Ligação Proteica/imunologia , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Ressonância de Plasmônio de Superfície
15.
Genes Dev ; 33(11-12): 669-683, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30975723

RESUMO

The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.


Assuntos
Antígenos CD4/genética , Fator 1 de Modelagem da Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Animais , Antígenos CD4/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Feminino , Regulação da Expressão Gênica , Inativação Gênica , Chaperonas de Histonas/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Masculino , Camundongos , Domínios Proteicos
16.
Proc Natl Acad Sci U S A ; 116(8): 3229-3238, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718403

RESUMO

Human and simian immunodeficiency viruses (HIV/SIVs) use CD4 as the primary receptor to enter target cells. Here, we show that the chimpanzee CD4 is highly polymorphic, with nine coding variants present in wild populations, and that this diversity interferes with SIV envelope (Env)-CD4 interactions. Testing the replication fitness of SIVcpz strains in CD4+ T cells from captive chimpanzees, we found that certain viruses were unable to infect cells from certain hosts. These differences were recapitulated in CD4 transfection assays, which revealed a strong association between CD4 genotypes and SIVcpz infection phenotypes. The most striking differences were observed for three substitutions (Q25R, Q40R, and P68T), with P68T generating a second N-linked glycosylation site (N66) in addition to an invariant N32 encoded by all chimpanzee CD4 alleles. In silico modeling and site-directed mutagenesis identified charged residues at the CD4-Env interface and clashes between CD4- and Env-encoded glycans as mechanisms of inhibition. CD4 polymorphisms also reduced Env-mediated cell entry of monkey SIVs, which was dependent on at least one D1 domain glycan. CD4 allele frequencies varied among wild chimpanzees, with high diversity in all but the western subspecies, which appeared to have undergone a selective sweep. One allele was associated with lower SIVcpz prevalence rates in the wild. These results indicate that substitutions in the D1 domain of the chimpanzee CD4 can prevent SIV cell entry. Although some SIVcpz strains have adapted to utilize these variants, CD4 diversity is maintained, protecting chimpanzees against infection with SIVcpz and other SIVs to which they are exposed.


Assuntos
Antígenos CD4/genética , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Vírus da Imunodeficiência Símia/genética , Proteínas do Envelope Viral/genética , Animais , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Evolução Molecular , Variação Genética/imunologia , HIV/genética , HIV/patogenicidade , Humanos , Pan troglodytes/genética , Pan troglodytes/imunologia , Polissacarídeos/genética , Polissacarídeos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/patogenicidade , Proteínas do Envelope Viral/imunologia
17.
J Virol ; 93(8)2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30728264

RESUMO

Lactobacillus bacteria are potential delivery vehicles for biopharmaceutical molecules because they are well-recognized as safe microorganisms that naturally inhabit the human body. The goal of this study was to employ these lactobacilli to combat human immunodeficiency virus type 1 (HIV-1) infection and transmission. By using a chromosomal integration method, we engineered Lactobacillus acidophilus ATCC 4356 to display human CD4, the HIV-1 receptor, on the cell surface. Since human CD4 can bind to any infectious HIV-1 particles, the engineered lactobacilli can potentially capture HIV-1 of different subtypes and prevent infection. Our data demonstrate that the CD4-carrying bacteria are able to adsorb HIV-1 particles and reduce infection significantly in vitro and also block intrarectal HIV-1 infection in a humanized mouse model in preliminary tests in vivo Our results support the potential of this approach to decrease the efficiency of HIV-1 sexual transmission.IMPORTANCE In the absence of an effective vaccine, alternative approaches to block HIV-1 infection and transmission with commensal bacteria expressing antiviral proteins are being considered. This report provides a proof-of-concept by using Lactobacillus bacteria stably expressing the HIV-1 receptor CD4 to capture and neutralize HIV-1 in vitro and in a humanized mouse model. The stable expression of antiviral proteins, such as CD4, following genomic integration of the corresponding genes into this Lactobacillus strain may contribute to the prevention of HIV-1 sexual transmission.


Assuntos
Antígenos CD4/metabolismo , Infecções por HIV/prevenção & controle , HIV-1/metabolismo , Lactobacillus acidophilus/metabolismo , Animais , Antígenos CD4/genética , Linhagem Celular , Feminino , Infecções por HIV/genética , Infecções por HIV/metabolismo , HIV-1/genética , Humanos , Lactobacillus acidophilus/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
J Biol Chem ; 294(15): 5736-5746, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30696772

RESUMO

HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion-inhibitor T20 (Fuzeon, enfuvirtide) and the C-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of entry inhibitors supposedly target the fusion peptide (FP) and are termed anchor inhibitors. These include the VIRIP peptide and VIRIP derivatives such as VIR165, VIR353, and VIR576. Here, we investigated the mechanism of inhibition by VIR165. We show that substitutions within the FP modulate sensitivity to VIR165, consistent with the FP being the drug target. Our results also revealed that VIR165 acts during an intermediate post-CD4-binding entry step that is overlapping but not identical to the step inhibited by fusion inhibitors such as T20. We found that some but not all resistance mutations to heptad repeat 2 (HR2)-targeting fusion inhibitors can provide cross-resistance to VIR165. In contrast, resistance mutations in the HR1-binding site for the fusion inhibitors did not cause cross-resistance to VIR165. However, Env with mutations located outside this binding site and thought to affect fusion kinetics, exhibited decreased sensitivity to VIR165. Although we found a strong correlation between Env stability and resistance to HR2-based fusion inhibitors, such correlation was not observed for Env stability and VIR165 resistance. We conclude that VIRIP analogs target the FP during an intermediate, post-CD4-binding entry step that overlaps with but is distinct from the step(s) inhibited by HR2-based fusion inhibitors.


Assuntos
Farmacorresistência Viral , HIV-1/fisiologia , Mutação , Fragmentos de Peptídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , alfa 1-Antitripsina/farmacologia , Produtos do Gene env do Vírus da Imunodeficiência Humana , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linhagem Celular , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Enfuvirtida/química , Enfuvirtida/farmacologia , Humanos , Maraviroc/química , Maraviroc/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , alfa 1-Antitripsina/química , alfa 1-Antitripsina/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo
19.
Plast Reconstr Surg ; 143(3): 518e-526e, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30601329

RESUMO

BACKGROUND: CD4 T cells have been implicated in the pathology of lymphedema. Interestingly, however, there have been case reports of lymphedema development in patients with low levels of CD4 T cells because of immunosuppression. In this study, the authors sought to delineate the effect of relative CD4 T-cell deficiency on the development of lymphedema in a mouse model. METHODS: A mouse model of relative CD4 T-cell deficiency was created through lethal total body irradiation of wild-type mice that then underwent bone marrow transplantation with progenitors harvested from CD4 knockout mice (wild-type/CD4 knockout). Irradiated CD4 knockout mice reconstituted with wild-type mouse-derived progenitors (CD4 knockout/wild-type), and unirradiated CD4 knockout and wild-type mice were used as controls. All mice underwent tail skin and lymphatic excision to induce lymphedema, and analysis was performed 6 weeks later. RESULTS: Wild-type/CD4 knockout chimeras were not protected from developing lymphedema. Despite a global deficit in CD4 T cells, these mice had swelling, fibrosis, inflammation, and impaired lymphatic transport function indistinguishable from that in wild-type and CD4 knockout/wild-type mice. In contrast, unirradiated CD4 knockout mice had no features of lymphedema after lymphatic injury. CONCLUSIONS: Relatively small numbers of bone marrow and peripheral CD4 T cells are sufficient to induce the development of lymphedema. These findings suggest that lymphatic injury results in expansion of CD4 T-cell populations in lymphedematous tissues.


Assuntos
Antígenos CD4/deficiência , Linfócitos T CD4-Positivos/imunologia , Linfedema/imunologia , Animais , Transplante de Medula Óssea , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/efeitos da radiação , Modelos Animais de Doenças , Feminino , Humanos , Vasos Linfáticos/patologia , Vasos Linfáticos/cirurgia , Linfedema/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimeras de Transplante , Irradiação Corporal Total
20.
Methods Mol Biol ; 1899: 43-54, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649764

RESUMO

Regulatory T cells (Tregs) are a population of lymphocytes that exerts suppressive effects upon the immune system. In human peripheral blood, the major population of T lymphocytes with suppressive capacity are defined by expression of the T cell co-receptor CD4 and the interleukin-2 receptor α-chain (CD25), combined with minimal expression of the interleukin-7 receptor α subunit (CD127). We begin by outlining the method for isolating peripheral blood mononuclear cells (PBMCs) from human blood by centrifugation of whole blood overlayed on a hydrophilic polysaccharide, with an additional erythrocyte lysis step. The protocol that follows utilizes Fluorescence-Activated Cell Sorting (FACS) for the isolation of this CD4+CD25+CD127lo population of regulatory T cells, with high yield and purity, from immunostained PBMCs. Prior to FACS isolation, this protocol exploits magnetic immunoselection for pre-enrichment of CD25+ PBMC, which reduces the duration of the subsequent FACS isolation.


Assuntos
Separação Celular/métodos , Citometria de Fluxo/métodos , Linfócitos T Reguladores/citologia , Antígenos CD4/genética , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-7/genética , Leucócitos Mononucleares/citologia , Contagem de Linfócitos , Linfócitos T Reguladores/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA