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1.
Science ; 329(5999): 1667-71, 2010 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-20929851

RESUMO

Tissue maintenance and homeostasis can be achieved through the replacement of dying cells by differentiating precursors or self-renewal of terminally differentiated cells or relies heavily on cellular longevity in poorly regenerating tissues. Regulatory T cells (T(reg) cells) represent an actively dividing cell population with critical function in suppression of lethal immune-mediated inflammation. The plasticity of T(reg) cells has been actively debated because it could factor importantly in protective immunity or autoimmunity. By using inducible labeling and tracking of T(reg) cell fate in vivo, or transfers of highly purified T(reg) cells, we have demonstrated notable stability of this cell population under physiologic and inflammatory conditions. Our results suggest that self-renewal of mature T(reg) cells serves as a major mechanism of maintenance of the T(reg) cell lineage in adult mice.


Assuntos
Linhagem da Célula , Linfócitos T Reguladores/fisiologia , Animais , Autoimunidade/imunologia , Proliferação de Células , Citocinas/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Técnicas de Introdução de Genes , Homeostase , Inflamação/imunologia , Contagem de Leucócitos , Listeria monocytogenes , Listeriose/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/imunologia , Tamoxifeno/farmacologia
2.
Immunity ; 28(4): 546-58, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18387831

RESUMO

The regulatory T (Treg) cells restrain immune responses through suppressor-function elaboration that is dependent upon expression of the transcription factor Foxp3. Despite a critical role for Treg cells in maintaining lympho-myeloid homeostasis, it remains unclear whether a single mechanism or multiple mechanisms of Treg cell-mediated suppression are operating in vivo and how redundant such mechanisms might be. Here we addressed these questions by examining the role of the immunomodulatory cytokine IL-10 in Treg cell-mediated suppression. Analyses of mice in which the Treg cell-specific ablation of a conditional IL-10 allele was induced by Cre recombinase knocked into the Foxp3 gene locus showed that although IL-10 production by Treg cells was not required for the control of systemic autoimmunity, it was essential for keeping immune responses in check at environmental interfaces such as the colon and lungs. Our study suggests that Treg cells utilize multiple means to limit immune responses. Furthermore, these mechanisms are likely to be nonredundant, in that a distinct suppressor mechanism most likely plays a prominent and identifiable role at a particular tissue and inflammatory setting.


Assuntos
Mediadores da Inflamação/fisiologia , Interleucina-10/fisiologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/prevenção & controle , Colite/genética , Colite/imunologia , Colite/prevenção & controle , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/fisiologia , Mediadores da Inflamação/metabolismo , Integrases/genética , Interleucina-10/deficiência , Interleucina-10/genética , Proteínas Luminescentes/genética , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Pele/imunologia , Pele/metabolismo , Pele/patologia , Linfócitos T Reguladores/patologia
3.
Nature ; 453(7192): 236-40, 2008 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-18368049

RESUMO

T helper cells that produce IL-17 (T(H)17 cells) promote autoimmunity in mice and have been implicated in the pathogenesis of human inflammatory diseases. At mucosal surfaces, T(H)17 cells are thought to protect the host from infection, whereas regulatory T (T(reg)) cells control immune responses and inflammation triggered by the resident microflora. Differentiation of both cell types requires transforming growth factor-beta (TGF-beta), but depends on distinct transcription factors: RORgammat (encoded by Rorc(gammat)) for T(H)17 cells and Foxp3 for T(reg) cells. How TGF-beta regulates the differentiation of T cells with opposing activities has been perplexing. Here we demonstrate that, together with pro-inflammatory cytokines, TGF-beta orchestrates T(H)17 cell differentiation in a concentration-dependent manner. At low concentrations, TGF-beta synergizes with interleukin (IL)-6 and IL-21 (refs 9-11) to promote IL-23 receptor (Il23r) expression, favouring T(H)17 cell differentiation. High concentrations of TGF-beta repress IL23r expression and favour Foxp3+ T(reg) cells. RORgammat and Foxp3 are co-expressed in naive CD4+ T cells exposed to TGF-beta and in a subset of T cells in the small intestinal lamina propria of the mouse. In vitro, TGF-beta-induced Foxp3 inhibits RORgammat function, at least in part through their interaction. Accordingly, lamina propria T cells that co-express both transcription factors produce less IL-17 (also known as IL-17a) than those that express RORgammat alone. IL-6, IL-21 and IL-23 relieve Foxp3-mediated inhibition of RORgammat, thereby promoting T(H)17 cell differentiation. Therefore, the decision of antigen-stimulated cells to differentiate into either T(H)17 or T(reg) cells depends on the cytokine-regulated balance of RORgammat and Foxp3.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Interleucina-17/metabolismo , Receptores do Ácido Retinoico/antagonistas & inibidores , Receptores dos Hormônios Tireóideos/antagonistas & inibidores , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-17/biossíntese , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo
4.
Nat Rev Immunol ; 7(6): 443-53, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17525753

RESUMO

In the immune system, transforming growth factor-beta (TGFbeta) affects multiple cell lineages by either promoting or opposing their differentiation, survival and proliferation. Understanding the cellular mechanisms of TGFbeta-mediated regulation is complicated due to a broad distribution of TGFbeta receptors on the surface of different immune-cell types. Recent studies using in vivo genetic approaches revealed a critical role for TGFbeta signalling in T cells in restraining fatal autoimmune lesions. Here, we review recent advances in our understanding of a role for TGFbeta signalling in the regulation of T-cell differentiation in the thymus and in the periphery, with a particular emphasis on TGFbeta-mediated control of self-reactive T cells.


Assuntos
Autoimunidade , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/imunologia , Humanos , Proteínas Smad/imunologia , Proteínas Smad/metabolismo , Linfócitos T/citologia
5.
J Exp Med ; 204(4): 865-77, 2007 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-17403936

RESUMO

Arachidonic acid metabolites, the eicosanoids, are key mediators of allergen-induced airway inflammation and remodeling in asthma. The availability of free arachidonate in cells for subsequent eicosanoid biosynthesis is controlled by phospholipase A(2)s (PLA(2)s), most notably cytosolic PLA(2)-alpha. 10 secreted PLA(2)s (sPLA(2)s) have also been identified, but their function in eicosanoid generation is poorly understood. We investigated the role of group X sPLA(2) (sPLA(2)-X), the sPLA(2) with the highest in vitro cellular phospholipolysis activity, in acute and chronic mouse asthma models in vivo. The lungs of sPLA(2)-X(-/-) mice, compared with those of sPLA(2)-X(+/+) littermates, had significant reduction in ovalbumin-induced infiltration by CD4(+) and CD8(+) T cells and eosinophils, goblet cell metaplasia, smooth muscle cell layer thickening, subepithelial fibrosis, and levels of T helper type 2 cell cytokines and eicosanoids. These data direct attention to sPLA(2)-X as a novel therapeutic target for asthma.


Assuntos
Alérgenos/imunologia , Asma/enzimologia , Asma/imunologia , Modelos Animais de Doenças , Fosfolipases A/metabolismo , Animais , Asma/genética , Asma/patologia , Citocinas/metabolismo , Eicosanoides/metabolismo , Regulação Enzimológica da Expressão Gênica , Fosfolipases A2 do Grupo X , Inflamação/enzimologia , Inflamação/genética , Inflamação/imunologia , Metaplasia/enzimologia , Metaplasia/patologia , Camundongos , Camundongos Knockout , Fosfolipases A/deficiência , Fosfolipases A/genética , Fosfolipases A2 , Células Th2/enzimologia
6.
Mol Cell Biol ; 25(3): 1089-99, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15657435

RESUMO

Animal cells counteract oxidative stress and electrophilic attack through coordinated expression of a set of detoxifying and antioxidant enzyme genes mediated by transcription factor Nrf2. In unstressed cells, Nrf2 appears to be sequestered in the cytoplasm via association with an inhibitor protein, Keap1. Here, by using the yeast two-hybrid screen, human Keap1 has been identified as a partner of the nuclear protein prothymosin alpha. The in vivo and in vitro data indicated that the prothymosin alpha-Keap1 interaction is direct, highly specific, and functionally relevant. Furthermore, we showed that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal that is important for its inhibitory action. Prothymosin alpha was able to liberate Nrf2 from the Nrf2-Keap1 inhibitory complex in vitro through competition with Nrf2 for binding to the same domain of Keap1. In vivo, the level of Nrf2-dependent transcription was correlated with the intracellular level of prothymosin alpha by using prothymosin alpha overproduction and mRNA interference approaches. Our data attribute to prothymosin alpha the role of intranuclear dissociator of the Nrf2-Keap1 complex, thus revealing a novel function for prothymosin alpha and adding a new dimension to the molecular mechanisms underlying expression of oxidative stress-protecting genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Estresse Oxidativo/genética , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Timosina/análogos & derivados , Timosina/metabolismo , Transativadores/metabolismo , Ativação Transcricional/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo/fisiologia , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Ativação Transcricional/fisiologia , Células Tumorais Cultivadas , Técnicas do Sistema de Duplo-Híbrido
7.
Plant Cell ; 16(1): 157-71, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14660804

RESUMO

To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene-mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.


Assuntos
Endopeptidases/metabolismo , Tabaco/enzimologia , Agrobacterium tumefaciens/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Endopeptidases/genética , Ativação Enzimática , Proteínas de Fluorescência Verde , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/virologia , Tabaco/genética , Tabaco/virologia , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Vírus do Mosaico do Tabaco/crescimento & desenvolvimento
8.
Exp Cell Res ; 284(2): 211-23, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12651154

RESUMO

Human prothymosin alpha is a proliferation-related nuclear protein undergoing caspase-mediated fragmentation in apoptotic cells. We show here that caspase-3 is the principal executor of prothymosin alpha fragmentation in vivo. In apoptotic HeLa cells as well as in vitro, caspase-3 cleaves prothymosin alpha at one major carboxy terminal (DDVD(99)) and several suboptimal sites. Prothymosin alpha cleavage at two amino-terminal sites (AAVD(6) and NGRD(31)) contributes significantly to the final pattern of prothymosin alpha fragmentation in vitro and could be detected to occur in apoptotic cells. The major caspase cleavage at D(99) disrupts the nuclear localization signal of prothymosin alpha, which leads to a profound alteration in subcellular localization of the truncated protein. By using a set of anti-prothymosin alpha monoclonal antibodies, we were able to observe nuclear escape and cell surface exposure of endogenous prothymosin alpha in apoptotic, but not in normal, cells. We demonstrate also that ectopic production of human prothymosin alpha and its mutants with nuclear or nuclear-cytoplasmic localization confers increased resistance of HeLa cells toward the tumor necrosis factor-induced apoptosis.


Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Células Eucarióticas/metabolismo , Fragmentos de Peptídeos/metabolismo , Precursores de Proteínas/biossíntese , Transporte Proteico/fisiologia , Timosina/análogos & derivados , Timosina/biossíntese , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos/fisiologia , Anticorpos Monoclonais , Apoptose/efeitos dos fármacos , Caspase 3 , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Células HeLa , Humanos , Mutação/genética , Precursores de Proteínas/antagonistas & inibidores , Precursores de Proteínas/genética , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Timosina/antagonistas & inibidores , Timosina/genética
9.
J Immunol Methods ; 266(1-2): 185-96, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12133636

RESUMO

To overcome poor immunogenicity of prothymosin alpha, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin alpha antibodies in mice immunized with free human prothymosin alpha, with prothymosin alpha coupled to different carriers and with prothymosin alpha fused to green fluorescent protein was assessed. Fusing prothymosin alpha to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin alpha antibodies. From these studies, two highly specific anti-prothymosin alpha monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin alpha molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin alpha that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin alpha fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti-prothymosin alpha antibodies obtained were suitable for precipitation of human prothymosin alpha from HeLa cell lysates and for immunolocalization of the endogenous prothymosin alpha within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against 'problematic' proteins.


Assuntos
Anticorpos Monoclonais/imunologia , Precursores de Proteínas/imunologia , Timosina/análogos & derivados , Timosina/imunologia , Animais , Especificidade de Anticorpos , Mapeamento de Epitopos , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação Puntual , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade da Espécie , Timosina/química , Timosina/genética
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