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1.
EMBO J ; 36(16): 2353-2372, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28701484

RESUMO

Mature differentiated macrophages can self-maintain by local proliferation in tissues and can be extensively expanded in culture under specific conditions, but the mechanisms of this phenomenon remain only partially defined. Here, we show that SIRT1, an evolutionary conserved regulator of life span, positively affects macrophage self-renewal ability in vitro and in vivo Overexpression of SIRT1 during bone marrow-derived macrophage differentiation increased their proliferative capacity. Conversely, decrease of SIRT1 expression by shRNA inactivation, CRISPR/Cas9 mediated deletion and pharmacological inhibition restricted macrophage self-renewal in culture. Furthermore, pharmacological SIRT1 inhibition in vivo reduced steady state and cytokine-induced proliferation of alveolar and peritoneal macrophages. Mechanistically, SIRT1 inhibition negatively regulated G1/S transition, cell cycle progression and a network of self-renewal genes. This included inhibition of E2F1 and Myc and concomitant activation of FoxO1, SIRT1 targets mediating cell cycle progression and stress response, respectively. Our findings indicate that SIRT1 is a key regulator of macrophage self-renewal that integrates cell cycle and longevity pathways. This suggests that macrophage self-renewal might be a relevant parameter of ageing.


Assuntos
Proliferação de Células , Autorrenovação Celular , Macrófagos/fisiologia , Sirtuína 1/metabolismo , Animais , Ciclo Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Camundongos , Sirtuína 1/genética
2.
Porcine Health Manag ; 3: 10, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28496988

RESUMO

BACKGROUND: Acute outbreaks of Actinobacillus pleuropneumoniae (APP) require rapid, effective, parenteral antimicrobial treatment. The efficacy and safety of a single, short-acting, high dose of marbofloxacin (Forcyl® swine 160 mg/mL) compared with 1 or 2 doses of 7.5 mg/kg enrofloxacin in APP outbreaks in European farms was studied. METHODS: A controlled, randomised block, blinded, multicentre, field study was conducted on four farms with acute respiratory disease associated with APP. Animals with clinical signs of respiratory disease were allocated similarly to intramuscular treatments of either a single dose 8 mg/kg marbofloxacin on day 0 or, 7.5 mg/kg enrofloxacin (Baytril 1nject®) on day 0 and again on day 2, if clinical signs had not improved. RESULTS: The results were similar for intention to treat (242 pigs) and per protocol populations (239 pigs). On day 0, all pigs had pyrexia (means, 40.6 °C), moderate to severe clinical signs (depression, cough, dyspnoea). Following treatment, animals improved rapidly and on day 7, clinical signs were absent or mild in all pigs and mean temperatures for each treatment were <39.5 °C (P > 0.05). The primary efficacy criterion, animals cured, for marbofloxacin and enrofloxacin was 81.8 and 81.4% on day 7, and 84.2 and 82.2% on day 21, respectively. Results for cure, respiratory disease removals and mortalities, and relapses were compared using confidence intervals and confirmed that marbofloxacin was non-inferior to enrofloxacin (P > 0.05). There were no significant treatment differences in live weight gains, adverse events and injection site reactions (<2.5% animals) (P > 0.05). Significantly more animals developed concurrent disorders in the enrofloxacin (7.5%) than marbofloxacin (0.0%) group (P < 0.01). On day 0, the MIC90 values of APP for marbofloxacin and enrofloxacin were 0.06 µg/mL for APP, less than the clinical breakpoints. CONCLUSIONS: Marbofloxacin (single dose of 8 mg/kg) and enrofloxacin (1 or 2 doses of 7.5 mg/kg) were clinically safe and effective in the treatment of clinical respiratory disease associated predominantly with APP in four European commercial, fattening pig herds.

3.
Science ; 353(6301): aad8670, 2016 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-27338705

RESUMO

Microglia, the resident myeloid cells of the central nervous system, play important roles in life-long brain maintenance and in pathology. Despite their importance, their regulatory dynamics during brain development have not been fully elucidated. Using genome-wide chromatin and expression profiling coupled with single-cell transcriptomic analysis throughout development, we found that microglia undergo three temporal stages of development in synchrony with the brain--early, pre-, and adult microglia--which are under distinct regulatory circuits. Knockout of the gene encoding the adult microglia transcription factor MAFB and environmental perturbations, such as those affecting the microbiome or prenatal immune activation, led to disruption of developmental genes and immune response pathways. Together, our work identifies a stepwise microglia developmental program integrating immune response pathways that may be associated with several neurodevelopmental disorders.


Assuntos
Encéfalo/embriologia , Homeostase/fisiologia , Microglia/citologia , Neurogênese/imunologia , Animais , Barreira Hematoencefálica/embriologia , Barreira Hematoencefálica/imunologia , Encéfalo/imunologia , Cromatina/metabolismo , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Código das Histonas , Homeostase/genética , Imunidade/genética , Fator de Transcrição MafB/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/imunologia , Células Mieloides/citologia , Neurogênese/genética , Análise de Célula Única
4.
Science ; 351(6274): aad5510, 2016 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-26797145

RESUMO

Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell-specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.


Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Células-Tronco Embrionárias/citologia , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica , Macrófagos/citologia , Animais , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Redes Reguladoras de Genes , Fator de Transcrição MafB/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-maf/metabolismo , Análise de Célula Única , Ativação Transcricional
5.
Immunity ; 31(2): 197-208, 2009 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-19682930

RESUMO

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Transtornos Linfoproliferativos/imunologia , Proteínas de Membrana/imunologia , Fosfoproteínas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Transferência Adotiva , Animais , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Transtornos Linfoproliferativos/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mutação , Fosfoproteínas/genética , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia
6.
Arterioscler Thromb Vasc Biol ; 29(5): 746-53, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19229070

RESUMO

OBJECTIVES: During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration. Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown. METHODS AND RESULTS: TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes. Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. CONCLUSIONS: Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation.


Assuntos
Quimiotaxia/imunologia , Inflamação/imunologia , Monócitos/imunologia , Antígeno CD146/imunologia , Linhagem Celular Tumoral , Células Endoteliais/fisiologia , Humanos , Fator de Necrose Tumoral alfa/imunologia , Veias Umbilicais/citologia
7.
J Immunol ; 180(3): 1565-75, 2008 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-18209052

RESUMO

Mutant mice where tyrosine 136 of linker for activation of T cells (LAT) was replaced with a phenylalanine (Lat(Y136F) mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that produce massive amounts of Th2 cytokines and trigger severe inflammation and autoantibodies. We analyzed whether the Lat(Y136F) pathology constitutes a bona fide autoimmune disorder dependent on TCR specificity. Using adoptive transfer experiments, we demonstrated that the expansion and uncontrolled Th2-effector function of Lat(Y136F) CD4 cells are not triggered by an MHC class II-driven, autoreactive process. Using Foxp3EGFP reporter mice, we further showed that nonfunctional Foxp3(+) regulatory T cells are present in Lat(Y136F) mice and that pathogenic Lat(Y136F) CD4 T cells were capable of escaping the control of infused wild-type Foxp3(+) regulatory T cells. These results argue against a scenario where the Lat(Y136F) pathology is primarily due to a lack of functional Foxp3(+) regulatory T cells and suggest that a defect intrinsic to Lat(Y136F) CD4 T cells leads to a state of TCR-independent hyperactivity. This abnormal status confers Lat(Y136F) CD4 T cells with the ability to trigger the production of Abs and of autoantibodies in a TCR-independent, quasi-mitogenic fashion. Therefore, despite the presence of autoantibodies causative of severe systemic disease, the pathological conditions observed in Lat(Y136F) mice unfold in an Ag-independent manner and thus do not qualify as a genuine autoimmune disorder.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doenças Autoimunes/imunologia , Transtornos Linfoproliferativos/imunologia , Proteínas de Membrana/genética , Fosfoproteínas/genética , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Animais , Autoanticorpos/sangue , Doenças Autoimunes/genética , Antígenos CD4/análise , Proliferação de Células , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Interleucina-7/metabolismo , Transtornos Linfoproliferativos/genética , Camundongos , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T/imunologia
8.
Immunity ; 22(5): 643-54, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15894281

RESUMO

Langerhans cells (LCs) are prominent dendritic cells (DCs) in epithelia, but their role in immunity is poorly defined. To track and discriminate LCs from dermal DCs in vivo, we developed knockin mice expressing enhanced green fluorescent protein (EGFP) under the control of the langerin (CD207) gene. By using vital imaging, we showed that most EGFP(+) LCs were sessile under steady-state conditions, whereas skin inflammation induced LC motility and emigration to lymph nodes (LNs). After skin immunization, dermal DCs arrived in LNs first and colonized areas distinct from slower migrating LCs. LCs reaching LNs under steady-state or inflammatory conditions expressed similar levels of costimulatory molecules. Langerin and EGFP were also expressed on thymic DCs and on blood-derived, CD8alpha(+) DCs from all secondary lymphoid organs. By using a similar knockin strategy involving a diphtheria toxin receptor (DTR) fused to EGFP, we demonstrated that LCs were dispensable for triggering hapten-specific T cell effectors through skin immunization.


Assuntos
Células de Langerhans/citologia , Células de Langerhans/imunologia , Linfonodos/citologia , Pele/citologia , Animais , Antígenos de Superfície/genética , Movimento Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Dermatite de Contato/imunologia , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Proteínas de Fluorescência Verde/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Células de Langerhans/metabolismo , Lectinas Tipo C/genética , Linfonodos/imunologia , Lectinas de Ligação a Manose/genética , Camundongos , Camundongos Transgênicos , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Pele/imunologia , Pele/lesões , Baço/citologia , Baço/imunologia , Timo/citologia , Timo/imunologia
9.
J Virol ; 78(14): 7410-7, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15220414

RESUMO

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i). an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii). a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii). an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.


Assuntos
Antivirais/farmacologia , Técnicas de Química Combinatória , Desenho de Fármacos , Biblioteca de Peptídeos , Vírus da Raiva/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Espectrometria de Massas/métodos , Chaperonas Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Fosfoproteínas/efeitos dos fármacos , Fosfoproteínas/metabolismo , Vírus da Raiva/genética , Vírus da Raiva/fisiologia , Transcrição Genética , Proteínas Estruturais Virais/efeitos dos fármacos , Proteínas Estruturais Virais/metabolismo
10.
Biologicals ; 31(1): 9-16, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12623055

RESUMO

Quality control of human rabies vaccines performed by National Control Laboratories (NCLs) prior to marketing vaccines batches requires in vivo and in vitro potency assays as requested by the relevant European Pharmacopoeia monographs, OMCLs guidelines and WHO technical recommendations. The aim of the present study was to check the suitability of an enzyme-linked immunosorbent assay (ELISA) using a virus neutralizing monoclonal antibody, directed to the rabies virus glycoprotein, to monitor the consistency of the lot to lot rabies vaccines production. Furthermore, this work was implemented to establish in house specifications for the glycoprotein content.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Vacinas Antirrábicas/normas , Vacinas de Produtos Inativados/normas , Relação Dose-Resposta Imunológica , Humanos , Controle de Qualidade , Vacinas Antirrábicas/administração & dosagem , Vacinas de Produtos Inativados/administração & dosagem
11.
Virus Res ; 91(2): 181-7, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12573496

RESUMO

The oligomeric structure and the fusion activity of lyssavirus glycoprotein (G) was studied by comparing G from Mokola virus (GMok) and rabies virus (PV strain) (GPV), which are highly divergent lyssaviruses. G expressed at the surface of BSR cells upon either plasmid transfection or virus infection are shown to be mainly trimeric after cross-linking experiments. However, solubilization by a detergent (CHAPS) and analysis in sucrose sedimentation gradient evidenced that GMok trimer is less stable than GPV trimer. A chimeric glycoprotein (G Mok-PV) associating the N-terminal half of GMok to the C-terminal half part of GPV formed trimers with an intermediate stability, indicating that the G C-terminal domain is essential in trimer stability. A cell to cell fusion assay revealed that GMok (and not G Mok-PV) was able to induce fusion at a higher pH (0.5 pH unit) than GPV. Such differences in the oligomeric structure stability and in the fusion activity of lyssavirus glycoproteins may partly account for the previously reported differences of their immunogenic and pathogenic properties.


Assuntos
Antígenos Virais , Glicoproteínas/química , Lyssavirus/patogenicidade , Fusão de Membrana , Vírus da Raiva/patogenicidade , Proteínas do Envelope Viral/química , Animais , Fusão Celular , Linhagem Celular , Cricetinae , Dimerização , Glicoproteínas/metabolismo , Concentração de Íons de Hidrogênio , Lyssavirus/metabolismo , Vírus da Raiva/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Proteínas do Envelope Viral/metabolismo
12.
Geneva; World Health Organization; 1996-11. (World health, 49, 6).
em Inglês | WHO IRIS | ID: who-330546
13.
In. Kita, Etsuko. Final report of the research project on a study on the health and prospective medical assistance for affected persons. Tokyo, Japan. Ministory of Health and Welfare;Japan. Government of Japan, Mar. 1996. p.79-101, ilus, tab.
Monografia em Inglês | Desastres | ID: des-10933
16.
Genéve; Comité International de la Croix-Rouge; s.f. 261 p. ilus.
Monografia em Inglês | Desastres | ID: des-3008
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