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1.
Diagn Cytopathol ; 48(11): 1107-1110, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32472960

RESUMO

Adamantinoma-like Ewing Sarcoma (ALES) is a rare subtype of Ewing sarcoma family of tumors (EFTs) which are defined by their EWSR1 gene rearrangements. We present a case of a 15-year old female with a swelling in her anterior neck of 4 months duration which had recently begun to rapidly grow in size. Fine needle aspiration showed a small blue round cell tumor with immunoreactivity for cytokeratin, CD99 and FLI1. Material for molecular testing was available on the resection specimen. Demonstration of t(11;22) (EWS-FLI1) was helpful in establishing the diagnosis.


Assuntos
Adamantinoma/diagnóstico , Neoplasias de Cabeça e Pescoço/diagnóstico , Sarcoma de Ewing/diagnóstico , Glândula Tireoide/patologia , Antígeno 12E7/imunologia , Adamantinoma/patologia , Adolescente , Biomarcadores Tumorais/imunologia , Biópsia por Agulha Fina/métodos , Diagnóstico Diferencial , Feminino , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Hibridização in Situ Fluorescente , Queratinas/imunologia , Proteínas de Fusão Oncogênica/análise , Proteína Proto-Oncogênica c-fli-1/análise , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteína EWS de Ligação a RNA/análise , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/patologia , Sarcoma de Ewing/cirurgia , Glândula Tireoide/cirurgia , Tireoidectomia
2.
Cells ; 9(3)2020 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-32183259

RESUMO

The transcription factor Friend leukemia integration 1 (Fli-1) regulates the expression of numerous cytokines and chemokines and alters the progression of lupus nephritis in humans and in the MRL/MpJ-Faslpr (MRL/lpr) mouse model. Th17-mediated immune responses are notably important as they promote ongoing inflammation. The purpose of this study is to determine the impact of Fli-1 on expression of interleukin-17A (IL-17A) and the infiltration of immune cells into the kidney. IL-17A concentrations were measured by ELISA in sera collected from MRL/lpr Fli-1-heterozygotes (Fli-1+/-) and MRL/lpr Fli-1+/+ control littermates. Expression of IL-17A and related proinflammatory mediators was measured by real-time polymerase chain reaction (RT-PCR). Immunofluorescence staining was performed on renal tissue from MRL/lpr Fli-1+/- and control littermates using anti-CD3, anti-CD4, and anti-IL-17A antibodies to detect Th17 cells and anti-CCL20 and anti-CD11b antibodies to identify CCL20+ monocytes. The expression of IL-17A in renal tissue was significantly reduced; this was accompanied by decreases in expression of IL-6, signal transducer and activator of transcription 3 (STAT3), and IL-1ß. Likewise, we detected fewer CD3+IL-17+ and CD4+IL-17+ cells in renal tissue of MLR/lpr Fli-1+/- mice and significantly fewer CCL20+CD11b+ monocytes. In conclusion, partial deletion of Fli-1 has a profound impact on IL-17A expression and on renal histopathology in the MRL/lpr mouse.


Assuntos
Interleucina-17/metabolismo , Leucócitos/metabolismo , Nefrite Lúpica/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos MRL lpr
3.
Mol Immunol ; 107: 123-131, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30738249

RESUMO

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Megacariócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Subunidade p45 do Fator de Transcrição NF-E2/genética , Subunidade p45 do Fator de Transcrição NF-E2/imunologia , Selectina-P/genética , Selectina-P/imunologia , Ploidias , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/imunologia , Acetato de Tetradecanoilforbol/farmacologia
4.
Mol Immunol ; 108: 1-7, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30739075

RESUMO

Our previous data demonstrated that Friend leukemia virus integration 1 (Fli-1), an ETS transcription factor, governs pericyte loss and vascular dysfunction in cecal ligation and puncture-induced murine sepsis by regulating essential pyroptosis markers including caspase-1. However, whether Fli-1 regulates caspase-1 expression levels in vitro and how Fli-1 regulates caspase-1 remain unknown. Our present work further demonstrated that overexpressed Fli-1 significantly increased caspase-1 and IL-18 expression levels in cultured mouse lung pericytes. Bacterial outer membrane vesicles (OMVs) have been found to induce cell pyroptosis through transferring LPS intracellularly. Using OMVs to induce an in vitro model of pyroptosis, we observed that OMVs significantly increased protein levels of Fli-1 in mouse lung pericytes. Furthermore, knockdown of Fli-1 by siRNA blocked OMVs-induced caspase-1, caspase-11 and IL-18 expression levels. As caspase-1 was predicted as a potential target of Fli-1, we cloned murine caspase-1 promoter into a luciferase construct. Our data demonstrate for the first time that Fli-1 regulates caspase-1 expression by directly binding to its promoter regions measured by chromatin immunoprecipitation (ChIP) assay and luciferase reporter system. In summary, our findings demonstrated a novel role and mechanism of Fli-1 in regulating caspase-1 expression in lung pericytes.


Assuntos
Caspase 1/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/farmacologia , Caspase 1/genética , Escherichia coli K12/química , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Interleucina-18/genética , Interleucina-18/imunologia , Pulmão , Camundongos , Pericitos , Proteína Proto-Oncogênica c-fli-1/genética
5.
Infect Genet Evol ; 49: 212-220, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28119029

RESUMO

FLI1 (Friend leukemia virus integration 1) and IL6 (interleukin 6; IL-6) are associated with Leishmania braziliensis susceptibility. Cutaneous lesions show exaggerated matrix metalloproteinase 1 (MMP1). In other skin diseases, FLI1 promoter methylation reduces FLI1 expression, and low FLI1 down-regulates MMP1. IL-6 increases FLI1 expression. We hypothesized that epigenetic regulation of FLI1 in cutaneous leishmaniasis, together with IL-6, might determine MMP1 expression. While generally low (<10%), percent FLI1 promoter methylation was lower (P=0.001) in lesion biopsies than normal skin. Contrary to expectation, a strong positive correlation occurred between FLI1 methylation and gene expression in lesions (r=0.98, P=0.0005) and in IL-6-treated L. braziliensis-infected macrophages (r=0.99, P=0.0004). In silico analysis of the FLI1 promoter revealed co-occurring active H3K27ac and repressive DNA methylation marks to enhance gene expression. FLI1 expression was enhanced between 3 and 24hour post infection in untreated (P=0.0002) and IL-6-treated (P=0.028) macrophages. MMP1 was enhanced in lesion biopsies (P=0.0002), induced (P=0.007) in infected macrophages, but strongly inhibited by IL-6. No correlations occurred between FLI1 and MMP1 expression in lesions or infected macrophages (with/without IL-6). We conclude that MMP1 is regulated by factors other than FLI1, and that the influence of IL-6 on MMP1 was independent of its effect on FLI1.


Assuntos
Epigênese Genética , Interações Hospedeiro-Patógeno , Interleucina-6/genética , Leishmaniose Cutânea/genética , Metaloproteinase 1 da Matriz/genética , Proteína Proto-Oncogênica c-fli-1/genética , Adolescente , Adulto , Criança , Metilação de DNA , Feminino , Regulação da Expressão Gênica , Histonas/genética , Histonas/imunologia , Humanos , Interleucina-6/imunologia , Leishmania braziliensis/patogenicidade , Leishmania braziliensis/fisiologia , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Metaloproteinase 1 da Matriz/imunologia , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/imunologia , Pele/imunologia , Pele/patologia
6.
Mol Immunol ; 81: 59-66, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27889620

RESUMO

Mammalian cells produce inflammatory cytokines and chemokines in response to innate immune signals and their expression is tightly regulated. Chemokine (C-X-C motif) ligand 2 (CXCL2), also known as macrophage inflammatory protein 2-alpha (MIP2-alpha), is an inflammatory chemokine belonging to the CXC chemokine family. CXCL2 is chemotactic for neutrophils and elevated expression of CXCL2 is associated with many inflammatory and autoimmune diseases. The Fli-1 gene belongs to the large Ets transcription factor family, whose members regulate a wide variety of cellular functions including the immune response. In this study, we demonstrate that endothelial cells transfected with Fli-1 specific siRNA produce significantly less CXCL2 compared to cells transfected with control siRNA after stimulation by the Toll-like receptor (TLR) 4 ligands, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-α). The production of CXCL2 in endothelial cells stimulated with LPS stimulation is dose-dependent. We found that Fli-1 binds to the CXCL2 promoter as established by Chromatin immunoprecipitation (ChIP) assay. Transient transfection assays show that Fli-1 drives transcription from the CXCL2 promoter in a dose-dependent manner and Fli-1 regulates the expression of CXCL2 largely by directly binding to the promoter. Targeted knockdown and transient transfection experiments suggest that both Fli-1 and the p65 subunit of NF-κB affect the activation of CXCL2 in an additive manner. These results indicate that Fli-1 is a novel, critical transcription factor that regulates the expression of the inflammatory chemokine CXCL2.


Assuntos
Quimiocina CXCL2/biossíntese , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Animais , Imunoprecipitação da Cromatina , Células Endoteliais/imunologia , Ensaio de Imunoadsorção Enzimática , Técnicas de Silenciamento de Genes , Humanos , Immunoblotting , Camundongos , Proteína Proto-Oncogênica c-fli-1/imunologia , Reação em Cadeia da Polimerase em Tempo Real
7.
Mol Immunol ; 71: 184-191, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26900987

RESUMO

IL-27 is an important regulator of TLR4-activated innate immune. The mechanism by which IL-27 production is regulated in TLR4-activated innate immune remains largely unclear. Here we show that expression of transcription factor Fli-1 at protein level is increased in macrophages following LPS stimulation. Fli-1 overexpression increases LPS-activated IL-27 production in macrophages. Consistently, Fli-1 knockdown inhibits LPS-induced IL-27 production in macrophages. Chromatin immunoprecipitation (ChIP) assay reveals that Fli-1 binds the promoter of IL-27 p28 subunit. Further experiments manifest that Fli-1 binds the region between -250 and -150 bp upstream of the transcriptional start site of p28 gene and increases p28 gene promoter-controlled transcription. These results demonstrate that Fli-1 positively regulates IL-27 production in TLR4-activated immune response by promoting transcription of IL-27 p28 gene.


Assuntos
Regulação da Expressão Gênica/imunologia , Interleucina-27/biossíntese , Macrófagos/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Animais , Imunoprecipitação da Cromatina , Células HEK293 , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Immunoblotting , Interleucina-27/genética , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/genética , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Transfecção
8.
Int J Biochem Cell Biol ; 67: 92-100, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26043891

RESUMO

Systemic sclerosis (SSc) is a heterogeneous and life-threatening autoimmune disease characterized by damage to small blood vessels, interruption of immune homeostasis and ultimately, fibrosis. Currently, the mechanisms involved in SSc pathogenesis remain unknown. An increasing amount of data shows that, via certain signaling pathways, epigenetic mechanisms, including DNA methylation, histone modification, and miRNAs, are closely related to the three primary processes that characterize SSc: vascular abnormalities, activation of immune system, and excessive extracellular matrix deposition. In the clinical setting, identification of molecules and biomarkers for determining disease severity, predicting disease progression and assessing response to treatment remains challenging. In this review, we aim to summarize the key epigenetic mechanisms involved in the pathogenesis of SSc. Certain cytokines or molecules, such as CD40, CD70, and Fli-1, are expressed at varying rates in SSc due to epigenetic modification and play important roles in SSc. It is therefore likely that these molecules may be biomarkers for SSc. In addition, epigenetic changes of certain genes, including Fli-1, BMPRII, CD11a, Foxp3, and eNOS, influence the expression of these genes to ultimately result in an anti-fibrotic effect. The influence that epigenetics has on SSc pathogenesis suggests that epigenetics-targeting drugs may have potential therapeutic effects against SSc. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/análogos & derivados , Epigênese Genética , Inibidores de Histona Desacetilases/uso terapêutico , Processamento de Proteína Pós-Traducional , Escleroderma Sistêmico/genética , Azacitidina/uso terapêutico , Biomarcadores/metabolismo , Ligante CD27/genética , Ligante CD27/imunologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Metilação de DNA , Decitabina , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Fibrose , Histonas/genética , Histonas/imunologia , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , MicroRNAs/imunologia , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/imunologia , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Transdução de Sinais
9.
Int J Biochem Cell Biol ; 67: 86-91, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26055516

RESUMO

Systemic sclerosis (SSc) is a multisystem connective tissue disease featured by immune abnormalities, vasculopathy and tissue fibrosis with unknown etiology. A series of studies on disease-susceptibility genes and twins have demonstrated the association of genetic factors with autoimmunity and disease severity and the contribution of environmental factors to the induction of clinical features in this disease. Friend leukemia virus integration 1 (Fli1), a member of Ets transcription factor family, is epigenetically suppressed in the lesional skin of SSc patients, suggesting that Fli1 is a potential predisposing factor of SSc reflecting the influence of environmental factors. Consistent with this idea, Fli1 deficiency induces SSc-like phenotypes in dermal fibroblasts and dermal microvascular endothelial cells in vivo and in vitro at molecular levels. Furthermore, Fli1 haploinsufficiency recapitulates tissue fibrosis, vascular activation and inflammation characteristic of SSc to a greater extent in bleomycin-treated mice. Importantly, bosentan, a dual endothelin receptor antagonist with a potential disease-modifying effect on SSc vasculopathy, reverses the expression of Fli1 protein by increasing its protein stability. Therefore, Fli1 may serve as a predisposing factor of SSc and can be a promising therapeutic target of this incurable and devastating disease. This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.


Assuntos
Epigênese Genética , Predisposição Genética para Doença , Proteína Proto-Oncogênica c-fli-1/genética , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/genética , Sulfonamidas/farmacologia , Animais , Bleomicina/antagonistas & inibidores , Bleomicina/farmacologia , Bosentana , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/patologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica c-fli-1/agonistas , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/imunologia , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia , Células Th2/patologia
10.
Exp Hematol ; 43(2): 125-36, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25448492

RESUMO

Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12(+) thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12(+) thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.


Assuntos
Anticorpos Monoclonais/farmacologia , Plaquetas/citologia , Fígado/citologia , Baço/citologia , Difosfato de Adenosina/farmacologia , Animais , Anticorpos Monoclonais/biossíntese , Biomarcadores/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Cloreto de Cálcio/farmacologia , Células Cultivadas , Colágeno/farmacologia , Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/imunologia , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Agregação Plaquetária , Glicoproteína IIb da Membrana de Plaquetas/genética , Glicoproteína IIb da Membrana de Plaquetas/imunologia , Ploidias , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/imunologia , Baço/efeitos dos fármacos , Baço/imunologia , Trombina/farmacologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/imunologia , Trombocitopenia/patologia , Trombopoese , Xenopus laevis/genética , Xenopus laevis/imunologia
11.
Nat Commun ; 5: 5797, 2014 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-25504335

RESUMO

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukaemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to the Fli1 silencing-dependent collagen induction, the simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B-cell activation and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.


Assuntos
Autoanticorpos/biossíntese , Linfócitos B/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Proteína Proto-Oncogênica c-fli-1/genética , Escleroderma Sistêmico/genética , Pele/metabolismo , Animais , Autoimunidade , Linfócitos B/imunologia , Linfócitos B/patologia , Vasos Sanguíneos/imunologia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Estudos de Casos e Controles , Colágeno/agonistas , Colágeno/genética , Colágeno/imunologia , Fator de Crescimento do Tecido Conjuntivo/agonistas , Fator de Crescimento do Tecido Conjuntivo/genética , Fator de Crescimento do Tecido Conjuntivo/imunologia , Modelos Animais de Doenças , Epigênese Genética , Fibroblastos/imunologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/imunologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Proteína Proto-Oncogênica c-fli-1/antagonistas & inibidores , Proteína Proto-Oncogênica c-fli-1/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Transdução de Sinais , Pele/imunologia , Pele/patologia
12.
J Immunol ; 193(6): 2661-8, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25098295

RESUMO

The friend leukemia insertion site 1 (Fli-1) transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival, and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus-prone NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1-specific small interfering RNA compared with controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant-negative transcription factor for Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225-bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation.


Assuntos
Quimiocina CCL5/genética , DNA/química , Células Endoteliais/imunologia , Proteína Proto-Oncogênica c-fli-1/genética , Ativação Transcricional/genética , Células 3T3 , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular , Quimiocina CCL5/biossíntese , DNA/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células Endoteliais/citologia , Inflamação/genética , Inflamação/imunologia , Rim/citologia , Rim/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Transgênicos , Nefrite/genética , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/imunologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Transfecção
13.
Arthritis Rheumatol ; 66(12): 3436-44, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25155007

RESUMO

OBJECTIVE: The Fli-1 transcription factor is implicated in the pathogenesis of systemic lupus erythematosus (SLE), both in humans and in animal models. Dysregulation of interleukin-6 (IL-6) is also associated with SLE. The purpose of this study was to investigate whether Fli-1 directly regulates the expression of IL-6. METHODS: Sera were collected from wild-type and Fli-1-heterozygous (Fli-1(+/-) ) MRL/lpr mice, and the concentration of IL-6 was measured by enzyme-linked immunosorbent assay (ELISA). Expression of IL-6 in the kidney was measured by real-time polymerase chain reaction analysis. T cells were isolated from wild-type and Fli-1(+/-) MRL/lpr mice and stimulated with CD3/CD28 beads, and the concentration of IL-6 in the supernatants was measured by ELISA. MS1 endothelial cells were transfected with Fli-1 and control small interfering RNA, and the production of IL-6 was compared after lipopolysaccharide stimulation. A chromatin immunoprecipitation (ChIP) assay was performed to determine whether Fli-1 binds to the IL-6 promoter region. Transient transfections with the NIH3T3 cell line were performed to examine whether Fli-1 regulates the expression of IL-6. RESULTS: Fli-1(+/-) MRL/lpr mice had significantly decreased IL-6 levels in sera and reduced expression of IL-6 in kidneys as compared to their wild-type littermates. T cells isolated from Fli-1(+/-) MRL/lpr mice produced less IL-6 than did those from wild-type mice. Inhibiting the expression of Fli-1 in endothelial cells resulted in reduced production of IL-6. The ChIP assay revealed direct binding of Fli-1 to 3 regions within the IL-6 promoter. Fli-1 activated transcription from the IL-6 promoter in a dose-dependent manner. CONCLUSION: The direct regulation of IL-6 expression by Fli-1 represents one possible mechanism for the protective effect of decreased Fli-1 expression in lupus.


Assuntos
Regulação da Expressão Gênica , Interleucina-6/genética , Lúpus Eritematoso Sistêmico/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Mensageiro/análise , Linfócitos T/metabolismo , Animais , Autoanticorpos , Imunoprecipitação da Cromatina , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Heterozigoto , Interleucina-6/imunologia , Interleucina-6/metabolismo , Rim/metabolismo , Lipopolissacarídeos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Células NIH 3T3 , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteína Proto-Oncogênica c-fli-1/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
14.
Eur J Immunol ; 44(9): 2617-24, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24935715

RESUMO

Friend leukemia integration 1 (Fli-1) is a member of the Ets transcription factor family and is expressed during T-cell development; however, the role Fli-1 plays in early T-cell differentiation has not been elucidated. In this report, we demonstrate that in mouse, Fli-1 overexpression retards the CD4(-) CD8(-) double-negative (DN) to CD4(+) CD8(+) double-positive (DP) transition by deregulating normal DN thymocyte development. Specifically, Fli-1 expression moderates the DN2 and DN3 developmental transitions. We further show that Fli-1 overexpression partially mimics strong TCR signals in developing DN thymocytes and thereby enhances γδ T-cell development. Conversely, Fli-1 knockdown by small hairpin RNA reverses the lineage bias from γδ T cells and directs DN cells to the αß lineage by attenuating TCR signaling. Therefore, Fli-1 plays a critical role in both the DN2 to DN3 transition and αß/γδ lineage commitment.


Assuntos
Proteína Proto-Oncogênica c-fli-1/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Timócitos/imunologia , Animais , Células Cultivadas , Camundongos , Proteína Proto-Oncogênica c-fli-1/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais/genética , Linfócitos T/citologia , Timócitos/citologia
15.
Int Immunopharmacol ; 21(2): 336-41, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24861249

RESUMO

An increasing number of T-cell epitopes derived from various tumor-associated antigens have been reported, and they proved to play significant roles for tumor rejection both in vivo and in vitro. Over 85% of Ewing's sarcoma family of tumors (ESFTs) express tumor-specific chimeric protein EWS/FLI-1, making it an attractive target for therapeutic cytotoxic T-lymphocyte responses. Here, we identified a novel peptide epitope derived from the EWS/FLI-1 protein and demonstrated that effectors induced by the peptide could specifically secrete IFN-γ and lyse the tumor cell line of EWS/FLI-1-positive and HLA-matched cells. In addition, mice treated with dendritic cells pulsed with the EWS/FLI-1 epitope were able to reject a lethal tumor inoculation of the Ewing's sarcoma A673 cells. Therefore, these data provide evidence for the use of the EWS/FLI-l peptide epitope in T cell-based immunotherapeutic concepts against Ewing's sarcoma cell in vitro and in vivo.


Assuntos
Antineoplásicos/imunologia , Células Dendríticas/imunologia , Proteínas de Fusão Oncogênica/imunologia , Peptídeos/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteína EWS de Ligação a RNA/imunologia , Sarcoma de Ewing/imunologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Epitopos de Linfócito T/imunologia , Humanos , Interferon gama/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Linfócitos T Citotóxicos/imunologia
16.
Mod Pathol ; 27(4): 496-501, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24072183

RESUMO

Epithelioid sarcoma is a rare, aggressive keratin-positive sarcoma that co-expresses CD34 in 50% of cases and may mimic an angiosarcoma. Recently, we have observed one case of epithelioid sarcoma that labeled for ERG, an ETS family regulatory transcription factor, which is considered to be a reliable marker for vascular differentiation. We investigated the prevalence of nuclear expression of ERG and FLI1, a homologous transcription factor, in these tumors. A formalin-fixed paraffin-embedded tissue microarray of 37 epithelioid sarcomas was examined. Immunohistochemistry was performed using anti-ERG monoclonal antibody to the N-terminus, anti-ERG monoclonal antibody to the C-terminus and anti-FLI1 monoclonal antibody. Comparison was made with CD34, CD31, and D2-40 labeling. The extent of immunoreactivity was graded according to the percentage of positive tumor cell nuclei (0: no staining; 1+: <5%; 2+: 5-25%; 3+: 26-50%; 4+: 51-75%; and 5+: 76-100%), and the intensity of staining was graded as weak, moderate, or strong. Nuclear staining for the N-terminus of ERG was seen in 19 out of 28 cases: 10 with diffuse(4 to 5+) strong/moderate labeling; 1 with 2+ moderate labeling and 8 with weak labeling (1 to 4+, 2 each). Focal staining for the C-terminus of ERG was seen in only 1 out of 29 cases (2+ moderate). FLI1 labeling was seen in nearly all (28 out of 30) cases: 16 with diffuse (5+) predominantly moderate labeling, and 8 cases with diffuse(5+) weak labeling. The remainder had variable moderate (1 to 3+) or weak (1 to 4+) FLI1 staining. CD34 was positive in 22 out of 30 cases and D2-40 was found to be positive in 22 out of 31 cases. All cases were negative for CD31 (0 out of 30). Epithelioid sarcoma can label with antibodies to the N-terminus of ERG, FLI1, and D2-40, which may cause diagnostic confusion for a vascular tumor. A panel of other antibodies including SMARCB1 and CD31 should be used in evaluating these tumors. ERG antibody selection is also critical, as those directed against the C-terminus are less likely to label epithelioid sarcoma.


Assuntos
Biomarcadores Tumorais/análise , Proteína Proto-Oncogênica c-fli-1/análise , Sarcoma/química , Transativadores/análise , Anticorpos Monoclonais , Anticorpos Monoclonais Murinos , Especificidade de Anticorpos , Antígenos CD34/análise , Núcleo Celular/química , Núcleo Celular/patologia , Reações Cruzadas , Humanos , Imuno-Histoquímica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Valor Preditivo dos Testes , Proteína Proto-Oncogênica c-fli-1/imunologia , Reprodutibilidade dos Testes , Sarcoma/patologia , Análise Serial de Tecidos , Transativadores/imunologia , Regulador Transcricional ERG
17.
Am J Clin Pathol ; 139(6): 771-9, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23690120

RESUMO

Ewing family tumors (EFTs) and prostate carcinomas are characterized by rearrangement of ETS genes, most commonly FLI1 (EFTs) and ERG (prostate carcinomas). Previously, we characterized an antibody against ERG (EPR3864) for detecting ERG-rearranged prostate carcinoma. Because EPR3864 also cross-reacts with FLI1, we evaluated the usefulness of EPR3864 for discriminating EFTs from other small round blue cell tumors (SRBCTs) with immunohistochemistry. Of 57 evaluable EFTs, 47 (82%) demonstrated at least moderate, diffuse, nuclear ERG/FLI1 staining (including 89% and 100% of cases with confirmed EWSR1:FLI1 and EWSR1:ERG fusions, respectively), of which 1, 3, and 43 showed negative, cytoplasmic, or membranous CD99 staining, respectively. Among other SRBCTs (61 cases, 7 types), at least moderate, diffuse, nuclear EPR3864 staining was seen in all precursor B-lymphoblastic lymphomas/leukemias and subsets of Burkitt lymphomas (10%) and synovial sarcomas (45%). In summary, EPR3864 may be useful in detecting EWSR1:FLI1 and EWSR1:ERG rearranged EFTs in addition to prostate carcinomas.


Assuntos
Anticorpos Monoclonais , Proteínas de Ligação a Calmodulina/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteínas de Ligação a RNA/imunologia , Transativadores/imunologia , Antígeno 12E7 , Adolescente , Adulto , Antígenos CD/análise , Moléculas de Adesão Celular/análise , Feminino , Humanos , Imuno-Histoquímica , Masculino , Proteínas de Neoplasias , Neoplasias da Próstata/diagnóstico , Proteína EWS de Ligação a RNA , Sarcoma de Ewing/diagnóstico , Análise Serial de Tecidos , Regulador Transcricional ERG
18.
Med Oncol ; 29(5): 3421-30, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22562156

RESUMO

To predict B cell epitope of Ewing's sarcoma EWS/FLI-l fusion protein and to analyze its antigenicity and immunogenicity. Comprehensive algorithms were applied to predict the possible B cell epitopes of EWS/FLI-l fusion protein. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis were performed to identify the synthesized epitope peptides, ELISA assays and Western blot to detect the antigenicity, and the immunogenicity of epitope peptides. Three B cell epitopes were screened out, and HPLC and MS analysis confirmed all three synthesized epitope peptides were demandable. ELISA assays verified all three epitope peptides could prime intense antigen-antibody reaction and induce ideal antibody titers after immunization to the New Zealand white rabbit. However, Western blot confirmed that antiserum of one of these epitope peptides could not recognize EWS/FLI-1 protein. Two B cell epitopes, PQDGNKPTETSQPQ and DPDEVARRWGQRKS, derived from EWS/FLI-l protein, are identified to have potential antigenicity and immunogenicity.


Assuntos
Algoritmos , Antígenos de Neoplasias/química , Epitopos de Linfócito B/imunologia , Proteínas de Fusão Oncogênica/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteína EWS de Ligação a RNA/imunologia , Sarcoma de Ewing/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/imunologia , Western Blotting , Vacinas Anticâncer/imunologia , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B/química , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/química , Estrutura Secundária de Proteína , Proteína Proto-Oncogênica c-fli-1/química , Proteína EWS de Ligação a RNA/química , Coelhos
19.
Anticancer Res ; 29(11): 4489-96, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20032396

RESUMO

BACKGROUND: Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still unsatisfying. New treatment strategies have to be developed. MATERIALS AND METHODS: A hypoxanthine/aminopterin/thymidine (HAT)-sensitive EFT cell line was developed by repetitive treatment of the EFT cell line SK-N-MC with 8'-azaguanine (8AG). By using DNA microarrays, the gene expression profile of this cell line was characterized. Immunostimulatory activity was assessed by mixed lymphocyte/tumour cell culture (MLTC). Artificial fusion of tumour cells and dendritic cells was visualized by flow cytometry. RESULTS: After selection of 8AG-resistant cells, a cell line with high sensitivity for treatment with HAT was obtained. Expression of the X chromosome inactivation specific transcript XIST was higher in HAT-sensitive cells. Nevertheless, HAT-sensitive cells retained the EFT-associated gene expression profile. Moreover, in the presence of HAT, it was possible to use these cells without irradiation as stimulatory cells in MLTC or as fusion partner for dendritic cells. CONCLUSION: HAT-sensitive EFT cells might be an interesting tool for the development of new immunotherapeutic approaches for the treatment of EFT.


Assuntos
Aminopterina/farmacologia , Hipoxantina/farmacologia , Imunoterapia Ativa/métodos , Sarcoma de Ewing/imunologia , Timidina/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Azaguanina/farmacologia , Proteínas de Ligação a Calmodulina/biossíntese , Proteínas de Ligação a Calmodulina/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/imunologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/imunologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia
20.
Cancer Res ; 66(20): 9862-9, 2006 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17047047

RESUMO

The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.


Assuntos
Fragmentos de Imunoglobulinas/imunologia , Proteínas de Fusão Oncogênica/imunologia , Proteína Proto-Oncogênica c-fli-1/imunologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Epitopos/imunologia , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Neoplasias Hepáticas , Dados de Sequência Molecular , Neuroblastoma , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Dobramento de Proteína , Proteína Proto-Oncogênica c-fli-1/química , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA , Ativação Transcricional , Transfecção
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