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1.
Phys Rev Lett ; 125(12): 128101, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-33016741

RESUMO

The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.


Assuntos
HIV-1/patogenicidade , Antígenos CD4/metabolismo , Linhagem Celular , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Humanos , Imagem Óptica , Imagem com Lapso de Tempo , Vírion/metabolismo , Vírion/patogenicidade
2.
J Cell Mol Med ; 24(19): 11603-11606, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32864865

RESUMO

A novel pneumonia-associated respiratory syndrome named coronavirus disease-2019 (COVID-19), which was caused by SARS-CoV-2,broke out in Wuhan, China, in the end of 2019. Unfortunately, there is no specific antiviral agent or vaccine available to treat SARS-CoV-2 infections. The information regarding the immunological characteristics in COVID-19 patients remains limited. Here, we collected the blood samples from 18 healthy donors (HD) and 38 COVID-19 patients to analyze changes on γδ T cell population. In comparison with HD, the γδ T cell percentage decreased, while the activation marker CD25 expression increased in response to SARS-CoV-2 infection. Interestingly, the CD4 expression was upregulated in γδ T cells reflecting the occurrence of a specific effector cell population, which may serve as a biomarker for the assessment of SARS-CoV-2 infection.


Assuntos
Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Betacoronavirus/fisiologia , Biomarcadores , Antígenos CD4/metabolismo , China , Citometria de Fluxo , Humanos , Imunidade Inata , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Pandemias , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo
4.
Nat Struct Mol Biol ; 27(9): 822-828, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32719457

RESUMO

The HIV-1 Nef protein suppresses multiple immune surveillance mechanisms to promote viral pathogenesis and is an attractive target for the development of novel therapeutics. A key function of Nef is to remove the CD4 receptor from the cell surface by hijacking clathrin- and adaptor protein complex 2 (AP2)-dependent endocytosis. However, exactly how Nef does this has been elusive. Here, we describe the underlying mechanism as revealed by a 3.0-Å crystal structure of a fusion protein comprising Nef and the cytoplasmic domain of CD4 bound to the tetrameric AP2 complex. An intricate combination of conformational changes occurs in both Nef and AP2 to enable CD4 binding and downregulation. A pocket on Nef previously identified as crucial for recruiting class I MHC is also responsible for recruiting CD4, revealing a potential approach to inhibit two of Nef's activities and sensitize the virus to immune clearance.


Assuntos
Antígenos CD4/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/metabolismo , Antígenos CD4/química , Cristalografia por Raios X , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química
5.
Proc Natl Acad Sci U S A ; 117(31): 18719-18728, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32690692

RESUMO

CD4-based decoy approaches against HIV-1 are attractive options for long-term viral control, but initial designs, including soluble CD4 (sCD4) and CD4-Ig, were ineffective. To evaluate a therapeutic that more accurately mimics HIV-1 target cells compared with monomeric sCD4 and dimeric CD4-Ig, we generated virus-like nanoparticles that present clusters of membrane-associated CD4 (CD4-VLPs) to permit high-avidity binding of trimeric HIV-1 envelope spikes. In neutralization assays, CD4-VLPs were >12,000-fold more potent than sCD4 and CD4-Ig and >100-fold more potent than the broadly neutralizing antibody (bNAb) 3BNC117, with >12,000-fold improvements against strains poorly neutralized by 3BNC117. CD4-VLPs also neutralized patient-derived viral isolates that were resistant to 3BNC117 and other bNAbs. Intraperitoneal injections of CD4-CCR5-VLP produced only subneutralizing plasma concentrations in HIV-1-infected humanized mice but elicited CD4-binding site mutations that reduced viral fitness. All mutant viruses showed reduced sensitivity to sCD4 and CD4-Ig but remained sensitive to neutralization by CD4-VLPs in vitro. In vitro evolution studies demonstrated that CD4-VLPs effectively controlled HIV-1 replication at neutralizing concentrations, and viral escape was not observed. Moreover, CD4-VLPs potently neutralized viral swarms that were completely resistant to CD4-Ig, suggesting that escape pathways that confer resistance against conventional CD4-based inhibitors are ineffective against CD4-VLPs. These findings suggest that therapeutics that mimic HIV-1 target cells could prevent viral escape by exposing a universal vulnerability of HIV-1: the requirement to bind CD4 on a target cell. We propose that therapeutic and delivery strategies that ensure durable bioavailability need to be developed to translate this concept into a clinically feasible functional cure therapy.


Assuntos
Antígenos CD4 , HIV-1 , Nanopartículas , Vírion , Fármacos Anti-HIV , Antígenos CD4/química , Antígenos CD4/metabolismo , Linhagem Celular , HIV-1/química , HIV-1/genética , HIV-1/metabolismo , Humanos , Mimetismo Molecular , Nanomedicina/métodos , Nanopartículas/química , Nanopartículas/metabolismo , Vírion/química , Vírion/metabolismo
6.
PLoS One ; 15(5): e0233037, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401825

RESUMO

Tumor-infiltrating lymphocyte (TIL) levels have prognostic and predictive values in treatment-naïve breast cancers. However, there have been controversies regarding TIL subset changes and their clinical implications in post-treatment breast cancers. This study aimed to explore change and prognostic significance of TIL subset infiltration after primary systemic therapy (PST) in breast cancer. One-hundred-fifty-five patients who had residual disease after anthracycline- or anthracycline plus taxane-based PST were included. The quantities of intratumoral and stromal TIL subsets (CD8+, CD4+, and FOXP3+ TILs) in pre- and post-PST breast cancer samples, as well as changes between them, were analyzed along with their correlations with clinicopathologic features and outcome of patients. As a whole, intratumoral CD8+ and CD4+ TILs increased after PST while stromal TILs decreased. Both intratumoral and stromal FOXP3+ TILs decreased after PST. The chemo-sensitive group [residual cancer burden (RCB) class I and II] showed the same pattern of change in intratumoral CD8+ TILs as the whole group, whereas the chemo-resistant group (RCB class III) showed no significant change in intratumoral CD8+ TIL infiltration after PST. Survival analyses for each TIL subset as well as their ratios revealed that high levels of intratumoral, stromal, and total CD8+ TIL infiltration after PST were independent predictors of longer patient survival. In subgroup analyses, CD8+ TIL infiltration after PST revealed prognostic significance in the chemo-resistant group but not in the chemo-sensitive group. In conclusion, infiltration of CD8+, CD4+, and FOXP3+ TIL changed after PST in the intratumoral and stromal compartments. Especially, increase of intratumoral CD8+ TILs was associated with chemo-responsiveness. Moreover, CD8+ TIL status in residual tumors after PST may be used as a potential prognostic marker in breast cancer patients who receive PST and provide additional prognostic information to chemo-resistant group.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Adulto , Idoso , Antraciclinas/uso terapêutico , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasia Residual , Prognóstico , Análise de Sobrevida , Taxoides/uso terapêutico , Resultado do Tratamento
7.
Sci Rep ; 10(1): 6598, 2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32313087

RESUMO

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype. Recent studies have shown that MHC class II (MHCII) expression and tumor infiltrating lymphocytes are important prognostic factors in patients with TNBC, although the relative importance of lymphocyte subsets and associated protein expression is incompletely understood. NanoString Digital Spatial Profiling (DSP) allows for spatially resolved, highly multiplexed quantification of proteins in clinical samples. In this study, we sought to determine if DSP could be used to characterize expression of MHCII and other immune related proteins in tumor epithelial versus stromal compartments of patient-derived TNBCs (N = 10) using a panel of 39 markers. We confirmed that a subset of TNBCs have elevated expression of HLA-DR in tumor epithelial cells; HLA-DR expression was also significantly higher in the tumors of patients with long-term disease-free survival when compared to patients that relapsed. HLA-DR expression in the epithelial compartment was correlated with high expression of CD4 and ICOS in the stromal compartment of the same tumors. We also identified candidate protein biomarkers with significant differential expression between patients that relapsed versus those that did not. In conclusion, DSP is a powerful method that allows for quantification of proteins in the immune microenvironment of TNBCs.


Assuntos
Proteínas de Neoplasias/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Microambiente Tumoral/imunologia , Antígenos CD4/metabolismo , Linhagem Celular Tumoral , Antígenos HLA-DR/imunologia , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Linfócitos/patologia , Prognóstico , Recidiva , Células Estromais/patologia , Neoplasias de Mama Triplo Negativas/patologia
8.
Nat Chem Biol ; 16(5): 529-537, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32152540

RESUMO

Combination antiretroviral therapy has transformed HIV-1 infection, once a fatal illness, into a manageable chronic condition. Drug resistance, severe side effects and treatment noncompliance bring challenges to combination antiretroviral therapy implementation in clinical settings and indicate the need for additional molecular targets. Here, we have identified several small-molecule fusion inhibitors, guided by a neutralizing antibody, against an extensively studied vaccine target-the membrane proximal external region (MPER) of the HIV-1 envelope spike. These compounds specifically inhibit the HIV-1 envelope-mediated membrane fusion by blocking CD4-induced conformational changes. An NMR structure of one compound complexed with a trimeric MPER construct reveals that the compound partially inserts into a hydrophobic pocket formed exclusively by the MPER residues, thereby stabilizing its prefusion conformation. These results suggest that the MPER is a potential therapeutic target for developing fusion inhibitors and that strategies employing an antibody-guided search for novel therapeutics may be applied to other human diseases.


Assuntos
Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Proteína gp41 do Envelope de HIV/química , Proteína gp41 do Envelope de HIV/metabolismo , Internalização do Vírus/efeitos dos fármacos , Sítios de Ligação , Antígenos CD4/metabolismo , Membrana Celular/metabolismo , Dequalínio/química , Dequalínio/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência , Células HEK293 , Proteína gp41 do Envelope de HIV/genética , HIV-1/patogenicidade , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutação , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície
9.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32161177

RESUMO

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41)3] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system.IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine.


Assuntos
Glicoproteínas/química , Glicoproteínas/efeitos dos fármacos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/efeitos dos fármacos , HIV-1/imunologia , Piperazinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Células A549 , Anticorpos Neutralizantes/imunologia , Antígenos CD4/efeitos dos fármacos , Antígenos CD4/metabolismo , Glicoproteínas/genética , Células HEK293 , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica
10.
BMC Cancer ; 20(1): 179, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32131780

RESUMO

BACKGROUND: The objective of this systematic review and meta-analysis was to determine the prognostic value of total tumor-infiltrating lymphocytes (TILs) and subtypes of TILs (CD4+, CD8+, and FOXP3+) in triple-negative breast cancer (TNBC). METHODS: A systematic search of the MEDLINE, EMBASE, and Web of Science databases was conducted to identified eligible articles published before August 2019. Study screening, data extraction, and risk of bias assessment were performed by two independent reviewers. Risk of bias on the study level was assessed using the ROBINS I tool and Quality in Prognosis Studies (QUIPS) tool. We performed a meta-analysis to obtain a pooled estimate of the prognostic role of TILs using Review Manager 5.3. RESULTS: In total, 37 studies were included in the final analysis. Compared to TNBC patients with low TIL levels, TNBC patients with high TIL levels showed a higher rate of pathological complete response (pCR) to treatment (odds ratio [OR] 2.14, 95% confidence interval [CI] 1.43-3.19). With each 10% increase in percentage of TILs, patients with TNBC had an increased pCR (OR 1.09, 95% CI 1.02-1.16). Compared to TNBC patients with low TIL levels, patients with high TIL levels had better overall survival (OS; hazard ratio [HR] 0.58, 95% CI 0.48-0.71) and disease-free survival (DFS; HR 0.66, 95% CI 0.57-0.76). Additionally, with a continuous increase in TIL levels, patients with TNBC had improved OS (HR 0.90, 95% CI 0.87-0.93) and DFS (HR 0.92, 95% CI 0.90-0.95). A high CD4+ TIL level was associated with better OS (HR 0.49, 95% CI 0.32-0.76) and DFS (HR 0.54, 95% CI 0.36-0.80). A high CD8+ TIL level was associated better DFS only (HR 0.55, 95% CI 0.38-0.81), as no statistical association was found with OS (HR 0.70, 95% CI 0.46-1.06). A high FOXP3+ TIL level also was associated with only DFS (HR 0.50, 95% CI 0.33-0.75) and not OS (HR 1.28, 95% CI 0.24-6.88). CONCLUSIONS: TNBC with a high level of TILs showed better short-term and long-term prognoses. High levels of specific phenotypes of TILs (CD4+, CD8+, and FOXP3+) were predictive of a positive long-term prognosis for TNBC.


Assuntos
Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias de Mama Triplo Negativas/imunologia , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Feminino , Fatores de Transcrição Forkhead/metabolismo , Humanos , Prognóstico , Neoplasias de Mama Triplo Negativas/patologia
11.
Surgery ; 167(6): 991-998, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32113580

RESUMO

BACKGROUND: Long-term outcomes after face transplantation are rarely reported in the scientific literature. Here we present outcome data of a partial face allograft recipient 10 years after transplantation. METHODS: Medical records were reviewed for functional and psychosocial outcomes as well as complications. Histopathologic analyses of autopsy tissues and characterization of skin immune cells were performed. RESULTS: The patient retained long-term motor and sensory function, though with a noticeable drop in sensory function after year 5. Social reintegration of the patient was marked by reconnection with his family and participation in public social activities. Immunosuppressive therapy consisted of tacrolimus (target levels 6-8 ng/mL after the first year), mycophenolate, and prednisone, while steroids were completely weaned between years 1 and 7. One acute cellular rejection episode of grade II or higher occurred on average per year and led to chronic skin changes (papillary dermal sclerosis with superficial hyalinization, epidermal thinning with loss of rete ridges, perieccrine fibrosis), but the allograft vessels, muscles, adipose tissue, and bone were spared. Allograft skin was characterized by increased number of CD4+ TNF-α/IL17A producing T-cells as compared with native skin. Long-term kidney function was maintained at 60 mL/min estimated glomerular filtration rate. Unfortunately, the preexisting hepatitis C virus infection with liver cirrhosis was resistant to 3 treatments with new direct-acting antivirals and eventually hepatocellular carcinoma developed, causing the patient's death 10 years after transplantation. CONCLUSION: This report suggests that face transplants can maintain their function for at least 10 years. Chronic skin changes can occur independently of allograft vasculopathy.


Assuntos
Transplante de Face , Pele/patologia , Transplantados , Aloenxertos , Antígenos CD4/metabolismo , Carcinoma Hepatocelular/virologia , Seguimentos , Taxa de Filtração Glomerular , Hepatite C Crônica/complicações , Humanos , Imunossupressores/uso terapêutico , Interleucina-17/metabolismo , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Pele/metabolismo , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
Phys Chem Chem Phys ; 22(10): 5548-5560, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32119016

RESUMO

The entry of human immunodeficiency virus type I (HIV-1) into host cells is initiated by binding to the cell-surface receptor CD4, which induces a conformational transition of the envelope (Env) glycoprotein gp120 from the closed, unliganded state to the open, CD4-bound state. Despite many available structures in these two states, detailed aspects on the dynamics and thermodynamics of gp120 remain elusive. Here, we performed microsecond-scale (µs-scale) multiple-replica molecular dynamics (MD) simulations to explore the differences in the conformational dynamics, protein motions, and thermodynamics between the unliganded and CD4-bound/complexed forms of gp120. Comparative analyses of MD trajectories reveal that CD4 binding promotes the structural deviations/changes and conformational flexibility, loosens the structural packing, and complicates the molecular motions of gp120. Comparison of the constructed free energy landscapes (FELs) reveals that the CD4-complexed gp120 has more conformational substates, larger conformational entropy, and lower thermostability than the unliganded form. Therefore, the unliganded conformation represents a structurally and energetically stable "ground state" for the full-length gp120. The observed great increase in the mobility of V1/V2 and V3 along with their more versatile movement directions in the CD4-bound gp120 compared to the unliganded form suggests that their orientations with respect to each other and to the structural core determine the differences in the conformational dynamics and thermodynamics between the two gp120 forms. The results presented here provide a basis by which to better understand the functional and immunological properties of gp120 and, furthermore, to deploy appropriate strategies for the development of anti-HIV-1 drugs or vaccines.


Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Simulação de Dinâmica Molecular , Termodinâmica , Ligantes , Ligação Proteica , Conformação Proteica
13.
Proc Natl Acad Sci U S A ; 117(13): 7382-7391, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32179688

RESUMO

Nef is an accessory protein of primate lentiviruses that is essential for efficient replication and pathogenesis of HIV-1. A conserved feature of Nef proteins from different lentiviral lineages is the ability to modulate host protein trafficking and down-regulate a number of cell surface receptors to enhance replication and promote immune evasion. Notably, the inability of Nef to down-regulate CD3 from infected T cells distinguishes HIV-1 Nef and its direct simian precursors from other primate lentiviruses. Why HIV-1 does not employ this potential immune evasion strategy is not fully understood. Using chimeric HIV-1 constructs expressing lentiviral Nef proteins that differ in their ability to down-modulate CD3, we show that retaining CD3 on the surface of infected primary T cells results in increased viral replication and cell-to-cell spread. We identified increased expression of envelope (Env) trimers at the cell surface and increased Env incorporation into virions as the determinants for the Nef- and CD3-dependent enhancement of viral infectivity. Importantly, this was independent of Nef-mediated antagonism of the host restriction factor SERINC5. CD3 retention on the surface of infected primary T cells also correlated with increased T cell signaling, activation, and cell death during cell-to-cell spread. Taken together, our results show that loss of an otherwise conserved function of Nef has a positive effect on HIV-1 replication, allowing for more efficient replication while potentially contributing to HIV-1 pathogenesis by triggering T cell activation and cell death during viral spread.


Assuntos
HIV-1/metabolismo , HIV-1/patogenicidade , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Complexo CD3/metabolismo , Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos , Regulação para Baixo , Soropositividade para HIV , HIV-1/genética , Humanos , Evasão da Resposta Imune , Ativação Linfocitária , Proteínas de Membrana/metabolismo , Cultura Primária de Células , Vírus da Imunodeficiência Símia/patogenicidade , Linfócitos T/imunologia , Proteínas Virais Reguladoras e Acessórias/metabolismo , Vírion/metabolismo , Replicação Viral , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
14.
J Immunol ; 204(6): 1543-1561, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-32066595

RESUMO

Elicitation of broadly neutralizing Ab (bNAb) responses toward the conserved HIV-1 envelope (Env) CD4 binding site (CD4bs) by vaccination is an important goal for vaccine development and yet to be achieved. The outcome of previous immunogenicity studies suggests that the limited accessibility of the CD4bs and the presence of predominant nonneutralizing determinants (nND) on Env may impede the elicitation of bNAbs and their precursors by vaccination. In this study, we designed a panel of novel immunogens that 1) preferentially expose the CD4bs by selective elimination of glycosylation sites flanking the CD4bs, and 2) minimize the nND immune response by engineering fusion proteins consisting of gp120 Core and one or two CD4-induced (CD4i) mAbs for masking nND epitopes, referred to as gp120-CD4i fusion proteins. As expected, the fusion proteins possess improved antigenicity with retained affinity for VRC01-class, CD4bs-directed bNAbs and dampened affinity for nonneutralizing Abs. We immunized C57BL/6 mice with these fusion proteins and found that overall the fusion proteins elicit more focused CD4bs Ab response than prototypical gp120 Core by serological analysis. Consistently, we found that mice immunized with selected gp120-CD4i fusion proteins have higher frequencies of germinal center-activated B cells and CD4bs-directed memory B cells than those inoculated with parental immunogens. We isolated three mAbs from mice immunized with selected gp120-CD4i fusion proteins and found that their footprints on Env are similar to VRC01-class bNAbs. Thus, using gp120-CD4i fusion proteins with selective glycan deletion as immunogens could focus Ab response toward CD4bs epitope.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Imunogenicidade da Vacina , Camundongos , Modelos Animais , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
15.
Immunology ; 160(1): 52-63, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32052861

RESUMO

As a pineal gland hormone, melatonin acts through its receptors to modulate the immune system. The immune system is composed of primary and secondary organs, and immune organs are adapted to the presence of the fetal alloantigen during pregnancy. However, it is unclear whether melatonin affects maternal immune organs during early pregnancy in sheep. In this study, the ovine thymus, lymph node, spleen and liver were sampled at day 16 of the oestrous cycle, and at days 13, 16 and 25 of pregnancy. The expression of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2) and cluster of differentiation 4 (CD4) was detected by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry experiments. Our results showed that during early pregnancy there was an upregulation of MT1 mRNA and protein in the thymus, lymph node and liver, and there was a downregulation in the spleen. The expression of MT2 mRNA and protein was increased in the thymus but decreased in the spleen and liver, and there was no significant change in the lymph node during early pregnancy. CD4 protein was upregulated in the thymus, lymph node and liver, but there were no significant changes in the spleen during early pregnancy. In conclusion, early pregnancy induces tissue-specific expression of MT1, MT2 and CD4, which may be due to the different functions of the thymus, lymph node, spleen and liver. Further, melatonin is involved in immune regulation of the maternal thymus, lymph node, spleen and liver during early pregnancy in sheep.


Assuntos
Antígenos CD4/metabolismo , Histocompatibilidade Materno-Fetal , Melatonina/metabolismo , Prenhez/imunologia , Receptores de Melatonina/metabolismo , Ovinos/imunologia , Animais , Feminino , Perfilação da Expressão Gênica , Tolerância Imunológica , Fígado/imunologia , Fígado/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Timo/imunologia , Timo/metabolismo , Regulação para Cima/imunologia
16.
Fish Shellfish Immunol ; 99: 239-242, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32058099

RESUMO

The co-receptor CD4 plays an important role in distinguishing between helper T-cell (Th) and cytotoxic T lymphocyte (CTL). In the present study, we investigated the molecular features of CD4-2 cDNA to facilitate understanding of their roles in cobia (Rachycentron canadum). Two CD4-2 molecules have been identified and exhibited 16.10% amino acids identity with each other. The cDNA of CD4-2A consists of a 993 bp ORF encoding 330 aa with long intracytoplasmic tail containing conserved protein tyrosine kinase p56Lck binding (C-X-C) motif, a transmembrane region, and two extracellular Ig-like (Ig-like) domains are predicted. Comparatively, the cDNA of cobia CD4-2B consists of a 990 bp ORF encoding 329 aa without a transmembrane domain as well as C-X-C motif, and three Ig-like domains are present. Homology comparison showed that the CD4-2A aa sequence of cobia showed high similarity and similar structural features to CD4-2 from other species, while the deduced CD4-2B protein shares higher structural similarity to CD4-1 group. Phylogenetic analysis indicated that cobia CD4-2A was closer with CD4-2 molecules in other fish species, distant from the clade formed by fish CD4-1 and mammalian CD4 sequences. However, cobia CD4-2B grouped with other known teleost CD4-1 sequences. The expression pattern of CD4-2A and CD4-2B mRNA during the embryonic development followed the trend of an initial increase after fertilized, providing evidence of maternal transfer of CD4-2 homologues to the developing cobia embryos and larvae. All of these results are useful for better understanding of cell-mediated immunity of cobia.


Assuntos
Antígenos CD4/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Perciformes/metabolismo , Animais , Antígenos CD4/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Perciformes/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-32073894

RESUMO

The adaptive immune response could play a major role in the resolution of lung injury. Although regulatory T cells (Tregs) have been implicated in promoting the resolution of lung injury, therapeutic strategies to enhance Treg quantity and activity at the site of injury need further exploration. In the current study, Akt inhibition using triciribine (TCBN), given 48 h after lipopolysaccharide (LPS) administration, increased Tregs-promoted resolution of acute lung injury (ALI). TCBN treatment enhanced the resolution of LPS-induced ALI on day 7 by reducing pulmonary edema and neutrophil activity associated with an increased number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs, specifically in the injured lungs and not in the spleen. Treatment of EL-4 T-lymphocytes with two Akt inhibitors (TCBN and MK-2206) for 72 h resulted in increased FoxP3 expression in vitro. On the other end, Treg-specific PTEN knockout (PTENTreg KO) mice that have a higher Akt activity in its Tregs exhibited a significant impairment in ALI resolution, increased edema, and neutrophil activity associated with a reduced number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs as compared with the control group. In conclusion, our study identifies a potential target for the treatment of late-stage ALI by promoting resolution through effector Treg-mediated suppression of inflammation.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T Reguladores/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Transferência Adotiva/métodos , Animais , Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Transcrição Forkhead/metabolismo , Cadeias alfa de Integrinas/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/metabolismo , Baço , Linfócitos T Reguladores/efeitos dos fármacos
18.
Biochim Biophys Acta Biomembr ; 1862(6): 183217, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061646

RESUMO

As the only exposed viral protein at the membrane surface of HIV, envelope glycoprotein gp120 is responsible for recognizing host cells and mediating virus-cell membrane fusion. Available structures of gp120 indicate that it exhibits two distinct conformational states, called closed and open states. Although experimental data demonstrates that CD4 binding stabilizes open state of gp120, detailed structural dynamics and kinetics of gp120 during this process remain elusive. Here, two open-state gp120 simulation systems, one without any ligands (ligand-free) and the other complexed with CD4 (CD4-bound), were subjected to microsecond-scale molecular dynamics simulations following the conformational transitions and allosteric pathways of gp120 evaluated by using the Markov state model and a network-based method, respectively. Our results provide an atomic-resolution description of gp120 conformational transitions, suggesting that gp120 is intrinsically dynamic from the open state to closed state, whereas CD4 binding blocks these transitions. Consistent with experimental structures, five metastable conformations with different orientations of the V1/V2 region and V3 loop have been extracted. The binding of CD4 significantly enhances allosteric communications from the CD4-binding site to V3 loop and ß20-21 hairpin, resulting in high-affinity interactions with coreceptors and activation of the conformational transitions switcher, respectively. This study will facilitate the structural understanding of the CD4-binding effects on conformational transitions and allosteric pathways of gp120.


Assuntos
Regulação Alostérica/fisiologia , Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Sítios de Ligação , Proteína gp120 do Envelope de HIV/química , Humanos , Cadeias de Markov , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica
19.
Cell ; 180(3): 471-489.e22, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004464

RESUMO

Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.


Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Antígenos CD4/metabolismo , Células CHO , Estudos de Coortes , Cricetulus , Epitopos/imunologia , Feminino , Células HEK293 , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mutação , Ligação Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética
20.
Clin Sci (Lond) ; 134(2): 123-138, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31922185

RESUMO

Type 2 inflammation and eosinophilic infiltration are prominent pathologic features of chronic rhinosinusitis with nasal polyps (CRSwNP). The purpose of the present study was to determine the roles of Tregs in controlling type 2 inflammation and inhibiting eosinophilic infiltration in CRSwNP. A total of 134 nasal polyps, 67 ostiomeatal complex from chronic rhinosinusitis (CRS) and 62 normal nasal tissues from controls were collected to study the enumeration and function of Tregs cells and the expressions of cytokine profiles via immunofluorescence staining, flow cytometry, qRT-PCR, ELISA, and/or H&E staining. The effects of Tregs on type2 and type3 inflammations were determined in an eosinophilic chronic sinusitis (ECRS) mice model. It was confirmed that the CRSwNP displayed the features of Th2 and Th17 cells-mediated inflammation, accompanying by an increased level of eosinophilic infiltration and the eosinophil cationic protein (ECP), with a decreased frequency of Treg cells. Furthermore, the percentages of CD4+CD25+CD127lowTreg and CD4+CD25+Foxp3+Treg were only decreased in the polyps of CRSwNP but not in the paired peripheral blood. The CRSwNP possessed the decreased Nrp1+Tregs, Helios+Treg, and low TGF-ß and interleukin (IL)-10 expressions in Tregs. The ECRS mice showed similar inflammatory characteristics to CRSwNP patients. The adoptive transfer of CD4+CD25+Foxp3+ Treg cells significantly decreased the inflammatory cytokines, eosinophilic chemotactic factors in the mucosa of the ECRS mice without alteration of the immune balance in the peripheral blood and spleen. In conclusion, CRSwNP showed high type 2 and type3 inflammation and defective Tregs. The induced regulatory T cell (iTreg) may correct the imbalance between immune tolerance and effect via limiting the eosinophil recruitment of mucosa in CRSwNP.


Assuntos
Eosinófilos/imunologia , Inflamação/imunologia , Mucosa Nasal/imunologia , Mucosa Nasal/patologia , Sinusite/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Animais , Grupo com Ancestrais do Continente Asiático , Antígenos CD4/metabolismo , Doença Crônica , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Interleucina-10/biossíntese , Masculino , Camundongos Endogâmicos BALB C , Pólipos Nasais/sangue , Pólipos Nasais/complicações , Pólipos Nasais/genética , Pólipos Nasais/imunologia , Rinite/sangue , Rinite/complicações , Rinite/genética , Rinite/imunologia , Sinusite/sangue , Sinusite/complicações , Sinusite/genética , Células Th17/imunologia , Células Th2/imunologia , Fator de Crescimento Transformador beta/biossíntese
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