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1.
Sci Transl Med ; 4(139): 139ra83, 2012 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-22723463

RESUMO

Dengue virus (DENV) is a mosquito-borne flavivirus that affects 2.5 billion people worldwide. There are four dengue serotypes (DENV1 to DENV4), and infection with one elicits lifelong immunity to that serotype but offers only transient protection against the other serotypes. Identification of the protective determinants of the human antibody response to DENV is a vital requirement for the design and evaluation of future preventative therapies and treatments. Here, we describe the isolation of a neutralizing antibody from a DENV1-infected patient. The human antibody 14c10 (HM14c10) binds specifically to DENV1. HM14c10 neutralizes the virus principally by blocking virus attachment; at higher concentrations, a post-attachment step can also be inhibited. In vivo studies show that the HM14c10 antibody has antiviral activity at picomolar concentrations. A 7 Å resolution cryoelectron microscopy map of Fab fragments of HM14c10 in a complex with DENV1 shows targeting of a discontinuous epitope that spans the adjacent surface of envelope protein dimers. As found previously, a human antibody specific for the related West Nile virus binds to a similar quaternary structure, suggesting that this could be an immunodominant epitope. These findings provide a structural and molecular context for durable, serotype-specific immunity to DENV infection.


Assuntos
Anticorpos Neutralizantes/uso terapêutico , Antivirais/uso terapêutico , Vírus da Dengue/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/farmacologia , Antivirais/farmacologia , Microscopia Crioeletrônica , Dengue/tratamento farmacológico , Vírus da Dengue/imunologia , Vírus da Dengue/ultraestrutura , Epitopos/imunologia , Humanos , Camundongos
2.
J Virol ; 86(5): 2817-25, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22205730

RESUMO

Foxp3(+) CD4(+) regulatory T cells (Tregs) represent a highly suppressive T cell subset with well-characterized immunosuppressive effects during immune homeostasis and chronic infections, although the role of these cells in acute viral infections is poorly understood. The present study sought to examine the induction of Foxp3(+) CD4(+) Tregs in a nonlethal murine model of pulmonary viral infection by the use of the prototypical respiratory virus influenza A. We establish that influenza A virus infection results in a robust Foxp3(+) CD4(+) T cell response and that regulatory T cell induction at the site of inflammation precedes the effector T cell response. Induced Foxp3(+) CD4(+) T cells are highly suppressive ex vivo, demonstrating that influenza virus-induced Foxp3(+) CD4(+) T cells are phenotypically regulatory. Influenza A virus-induced regulatory T cells proliferate vigorously in response to influenza virus antigen, are disseminated throughout the site of infection and primary and secondary lymphoid organs, and retain Foxp3 expression in vitro, suggesting that acute viral infection is capable of inducing a foreign-antigen-specific Treg response. The ability of influenza virus-induced regulatory T cells to suppress antigen-specific CD4(+) and CD8(+) T cell proliferation and cytokine production correlates closely to their ability to respond to influenza virus antigens, suggesting that virus-induced Tregs are capable of attenuating effector responses in an antigen-dependent manner. Collectively, these data demonstrate that primary acute viral infection is capable of inducing a robust, antigen-responsive, and suppressive regulatory T cell response.


Assuntos
Antígenos Virais/imunologia , Fatores de Transcrição Forkhead/imunologia , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/imunologia , Linfócitos T Reguladores/imunologia , Animais , Feminino , Fatores de Transcrição Forkhead/genética , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/genética , Influenza Humana/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
3.
J Cell Physiol ; 226(2): 397-406, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20665705

RESUMO

Airway epithelium functions not only as a physical barrier, but also a regulator of lung inflammation. IFN-γ plays a critical role in airway inflammation associated with respiratory viral infection. We investigated differential protein profiling in IFN-γ-stimulated normal human bronchial epithelial cells (HBEC) using a 2-dimensional gel electrophoresis followed by MALDI-TOF-MS/MS. IFN-γ markedly stimulated apolipoprotein L2 (ApoL2) protein expression in normal HBEC. ApoL2 mRNA expression was also elevated in normal human lung fibroblasts and smooth muscle cells stimulated with IFN-γ, in lung tissues from an IFN-γ-predominant influenza A virus-infected mouse lung injury model, and in cancer lung tissues from human patients. Normal HBEC showed strong resistance to IFN-γ-induced cytotoxicity. ApoL2 knockdown by siRNA promoted IFN-γ-induced cytotoxicity as revealed by a significant drop in cell viability using MTT and CyQUANT NF cell proliferation assays, and a marked increase in hypodiploid sub-G1 cell population in cell cycle analysis. Furthermore, depletion of ApoL2 facilitated IFN-γ-induced membrane damage and chromatin condensation as observed in Hoechst and propidium iodide-double staining and in transmission electron microscopy, and DNA fragmentation using a DNA laddering assay, in a caspase-dependent manner. Our results reveal a novel function for ApoL2 in conferring anti-apoptotic ability of human bronchial epithelium to the cytotoxic effects of IFN-γ, in maintaining airway epithelial layer integrity.


Assuntos
Apolipoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Brônquios/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Interferon gama/farmacologia , Lipoproteínas HDL/metabolismo , Mucosa Respiratória/citologia , Animais , Apolipoproteínas/genética , Apolipoproteínas L , Células Cultivadas , Fragmentação do DNA , Células Epiteliais/citologia , Humanos , Lipoproteínas HDL/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Espectrometria de Massas , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
4.
Nat Med ; 16(5): 586-91, 1p following 591, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20400961

RESUMO

Autoimmune diseases are common and debilitating, but their severe manifestations could be reduced if biomarkers were available to allow individual tailoring of potentially toxic immunosuppressive therapy. Gene expression-based biomarkers facilitating such tailoring of chemotherapy in cancer, but not autoimmunity, have been identified and translated into clinical practice. We show that transcriptional profiling of purified CD8(+) T cells, which avoids the confounding influences of unseparated cells, identifies two distinct subject subgroups predicting long-term prognosis in two autoimmune diseases, antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), a chronic, severe disease characterized by inflammation of medium-sized and small blood vessels, and systemic lupus erythematosus (SLE), characterized by autoantibodies, immune complex deposition and diverse clinical manifestations ranging from glomerulonephritis to neurological dysfunction. We show that the subset of genes defining the poor prognostic group is enriched for genes involved in the interleukin-7 receptor (IL-7R) pathway and T cell receptor (TCR) signaling and those expressed by memory T cells. Furthermore, the poor prognostic group is associated with an expanded CD8(+) T cell memory population. These subgroups, which are also found in the normal population and can be identified by measuring expression of only three genes, raise the prospect of individualized therapy and suggest new potential therapeutic targets in autoimmunity.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunossupressores/uso terapêutico , Linfócitos T/imunologia , Anticorpos Anticitoplasma de Neutrófilos/imunologia , Autoimunidade/imunologia , Estudos de Coortes , Relação Dose-Resposta a Droga , Expressão Gênica , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-7/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Prognóstico , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Vasculite/tratamento farmacológico , Vasculite/imunologia
5.
Mol Cell ; 34(5): 603-11, 2009 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-19524539

RESUMO

Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap(4)A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap(4)A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap(4)A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap(4)A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap(4)A production in immunologically stimulated mast cells, thus implying that LysRS is a key mediator in gene regulation.


Assuntos
Regulação da Expressão Gênica , Imunidade Celular/genética , Lisina-tRNA Ligase/fisiologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Fosfatos de Dinucleosídeos/biossíntese , Humanos , Lisina-tRNA Ligase/metabolismo , Sistema de Sinalização das MAP Quinases , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Fosforilação , Ratos , Serina/metabolismo
6.
Blood ; 114(2): 318-27, 2009 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-19420354

RESUMO

Receptors for the fragment crystallizable region of immunoglobulin-G (FcgammaRs) play an important role in linking the humoral and cellular arms of the immune response. In this study, we present a comprehensive functional comparison of 2 human Fc-receptors, FcgammaRI and FcgammaRIIa. Activation of FcgammaRI results in a novel signaling cascade that links phospholipase D1 to sphingosine kinase-1 in U937 cells and primary human monocytes. This induces the expression of proinflammatory mediators and is associated with trafficking of immune complexes into human leukocyte antigen-DM positive antigen-processing compartments coupled with improved MHC class II-mediated antigen presentation to T lymphocytes. In contrast, activation of FcgammaRIIa elicits signaling through phospholipase Cgamma1, resulting in increases in intracellular calcium, activation of nicotinamide adenine dinucleotide phosphate-oxidative burst, and differential membrane trafficking combined with impaired antigen presentation and proinflammatory cytokine expression. These data provide a mechanistic insight into the disparate activities associated with Fc receptors in immunity, namely, reinforcement of immune responses through stimulation of proinflammatory signaling and antigen presentation, versus the maintenance of immunologic homeostasis through the noninflammatory clearance of immune complexes.


Assuntos
Sinalização do Cálcio , Membrana Celular/imunologia , Membrana Celular/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Apresentação de Antígeno/imunologia , Células Cultivadas , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Isoenzimas/metabolismo , Estresse Oxidativo , Fosfolipase C gama/metabolismo , Ligação Proteica , Proteína Quinase C/metabolismo , Transporte Proteico , Receptores de IgG/genética
7.
Am J Respir Crit Care Med ; 179(8): 657-65, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19201922

RESUMO

RATIONALE: Persistent activation of nuclear factor (NF)-kappaB has been associated with the development of asthma. Andrographolide, the principal active component of the medicinal plant Andrographis paniculata, has been shown to inhibit NF-kappaB activity. OBJECTIVES: We hypothesized that andrographolide may attenuate allergic asthma via inhibition of the NF-kappaB signaling pathway. METHODS: BALB/c mice sensitized and challenged with ovalbumin (OVA) developed airway inflammation. Bronchoalveolar lavage fluid was assessed for total and differential cell counts, and cytokine and chemokine levels. Serum IgE levels were also determined. Lung tissues were examined for cell infiltration and mucus hypersecretion, and the expression of inflammatory biomarkers. Airway hyperresponsiveness was monitored by direct airway resistance analysis. MEASUREMENTS AND MAIN RESULTS: Andrographolide dose-dependently inhibited OVA-induced increases in total cell count, eosinophil count, and IL-4, IL-5, and IL-13 levels recovered in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. It attenuated OVA-induced lung tissue eosinophilia and airway mucus production, mRNA expression of E-selectin, chitinases, Muc5ac, and inducible nitric oxide synthase in lung tissues, and airway hyperresponsiveness to methacholine. In normal human bronchial epithelial cells, andrographolide blocked tumor necrosis factor-alpha-induced phosphorylation of inhibitory kappaB kinase-beta, and downstream inhibitory kappaB alpha degradation, p65 subunit of NF-kappaB phosphorylation, and p65 nuclear translocation and DNA-binding activity. Similarly, andrographolide blocked p65 nuclear translocation and DNA-binding activity in the nuclear extracts from lung tissues of OVA-challenged mice. CONCLUSIONS: Our findings implicate a potential therapeutic value of andrographolide in the treatment of asthma and it may act by inhibiting the NF-kappaB pathway at the level of inhibitory kappaB kinase-beta activation.


Assuntos
Asma/tratamento farmacológico , Diterpenos/administração & dosagem , NF-kappa B/antagonistas & inibidores , Animais , Antiasmáticos/administração & dosagem , Anti-Inflamatórios/administração & dosagem , Asma/imunologia , Asma/metabolismo , Brônquios/efeitos dos fármacos , Brônquios/patologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Feminino , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/antagonistas & inibidores , Ovalbumina/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos
8.
Pharmacol Ther ; 121(2): 123-31, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18940198

RESUMO

While it is well established that CD4(+) T lymphocytes play a crucial role in the initiation, progression and persistence of asthma, the role of CD8(+) T cells is less understood. CD8(+) T cells form functionally similar subsets which exhibit similar cytokine profiles as Th1 and Th2 cells, known as Tc1 and Tc2. Evidence from animal studies suggest that CD8(+) T cells are capable of regulating IgE production through the induction of IL-12 and IL-18 production in dendritic cells, and that CD8(+) T cells may act to moderate Th2 polarisation within the localised lymph nodes during allergic sensitisation. Such findings have led to the suggestion that Th1 polarising, CD8(+) T cell-inducing vaccines would inhibit the development of airway hyperresponsiveness (AHR) and Th2 cell infiltration. Despite these positive findings, the role of CD8(+) T cells within the lung remains poorly understood. While CD8(+) T cells, particularly those expressing the Tc1 phenotype, are capable of moderating inflammation and suppressing AHR, it has been postulated that Tc2 CD8(+) T cells predominate within established asthma and may act to amplify the inappropriate immune response which defines the condition. Within the clinic, the association between CD8(+) T cells and asthma is almost universally defined as injurious, further suggesting a prejudicial role for these cells within the established disease. CD8(+) T cells may be a valuable potential target for therapeutic intervention, either by potentiating their regulatory effects prior to the development of sensitisation, or through suppressing their pro-inflammatory properties within established atopy.


Assuntos
Antiasmáticos/farmacologia , Asma/imunologia , Linfócitos T CD8-Positivos/imunologia , Animais , Asma/tratamento farmacológico , Asma/fisiopatologia , Ensaios Clínicos como Assunto , Citocinas/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Pulmão/imunologia , Pulmão/fisiopatologia , Fenótipo
11.
J Am Soc Nephrol ; 15(7): 1877-82, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15213276

RESUMO

It was hypothesized that pentoxifylline might improve the response to recombinant human erythropoietin (rh-Epo) in anemic renal failure patients. Sixteen patients with ESRD and rh-Epo-resistant anemia, defined by a hemoglobin of <10.7 g/dl for 6 mo before treatment and a rh-Epo dose of > or =12,000 IU/wk, were recruited. They were treated with oral pentoxifylline 400 mg o.d. for 4 mo. Ex vivo T cell generation of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) from the patients was assessed before treatment and 6 to 8 wk after therapy. A total of 12 of 16 patients completed the study. Before therapy, the 12 patients' mean hemoglobin concentration was 9.5 +/- 0.9 g/dl. After 4 mo of pentoxifylline treatment, the mean hemoglobin concentration increased to 11.7 +/- 1.0 g/dl (P = 0.0001). Baseline ex vivo T cell expression of TNF-alpha decreased from 58% +/- 11% to 31% +/- 23% (P = 0.0007) after therapy. Likewise, IFN-gamma expression decreased from 31% +/- 10% to 13% +/- 10% (P = 0.0002). Pentoxifylline therapy may significantly improve the hemoglobin response in patients with previously rh-Epo-resistant anemia in renal failure. This may occur due to inhibition of proinflammatory cytokine production, which could interfere with the effectiveness of rh-Epo.


Assuntos
Anemia/complicações , Resistência a Medicamentos , Eritropoetina/farmacologia , Hemoglobinas/biossíntese , Pentoxifilina/farmacologia , Insuficiência Renal/complicações , Adulto , Idoso , Citocinas/biossíntese , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Sequestradores de Radicais Livres/farmacologia , Hemoglobinas/metabolismo , Humanos , Inflamação , Interferon gama/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/metabolismo , Linfócitos T/metabolismo , Fatores de Tempo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo
12.
J Am Soc Nephrol ; 14(7): 1776-84, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12819237

RESUMO

Resistance to recombinant human erythropoietin occurs in a small but important proportion of hemodialysis patients. This may be due to increased immune activation because pro-inflammatory cytokines inhibit erythropoiesis in vitro. Using FACScan flow cytometry, the proportion of PMA/ionomycin-stimulated T cells expressing cytokines ex vivo was compared in 18 poor responders to erythropoietin, 14 good responders to erythropoietin, and 14 normal controls. CD4(+) T cells from poor responders expressed more interferon-gamma (IFN-gamma; 19 +/- 6%) compared with good responders (11 +/- 6%, P < 0.001) and controls (12 +/- 6%, P < 0.01). Similarly, CD4+ T cells from poor responders expressed more tumor necrosis factor-alpha (TNF-alpha; poor responders: 51 +/- 19% versus good responders: 27 +/- 15% [P < 0.01] and controls: 30 +/- 19% [P < 0.01]). CD4+ expression of IL-10 was also enhanced (poor responders: 1.6 +/- 1.1% versus good responders: 0.7 +/- 0.6% [P < 0.05] and controls: 0.5 +/- 0.2% [P < 0.01]). Likewise, CD4+ expression of interleukin-13 (IL-13) was increased (poor responders: 4.4 +/- 4.2% versus good responders: 1.6 +/- 1.7% [P < 0.05] and controls: 1.6 +/- 1.5% [P < 0.05]). CD8+ T cells from poor responders also showed enhanced expression of cytokines. For IFN-gamma, poor responder expression was 48 +/- 20% compared with 31 +/- 17% (P < 0.05) for good responders and 23 +/- 15% (P < 0.01) for controls. TNF-alpha expression for poor responders was 41 +/- 21% versus 25 +/- 14% for good responders (P < 0.05) and 21 +/- 15% for controls (P < 0.01). IL-10 expression for poor responders was 2.0 +/- 1.2% (good responders: 0.7 +/- 0.6% [P < 0.01]; controls: 0.5 +/- 0.2% [P < 0.001]). These data indicate that T cells from poor responders are in an enhanced activation state possibly as a result of chronic inflammation. In the absence of any other cause (such as iron deficiency), the overproduction of cytokines may account for hyporesponsiveness to erythropoietic therapy in patients with renal failure.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Eritropoetina/uso terapêutico , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-13/biossíntese , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Adulto , Idoso , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Divisão Celular , Separação Celular , Citocinas/metabolismo , Eritropoese , Feminino , Citometria de Fluxo , Humanos , Inflamação , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Ionomicina/metabolismo , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Proteínas Recombinantes/uso terapêutico , Reprodutibilidade dos Testes , Fatores de Tempo , Resultado do Tratamento
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