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1.
Immunity ; 52(1): 151-166.e6, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31924474

RESUMO

In addition to helper and regulatory potential, CD4+ T cells also acquire cytotoxic activity marked by granzyme B (GzmB) expression and the ability to promote rejection of established tumors. Here, we examined the molecular and cellular mechanisms underpinning the differentiation of cytotoxic CD4+ T cells following immunotherapy. CD4+ transfer into lymphodepleted animals or regulatory T (Treg) cell depletion promoted GzmB expression by tumor-infiltrating CD4+, and this was prevented by interleukin-2 (IL-2) neutralization. Transcriptional analysis revealed a polyfunctional helper and cytotoxic phenotype characterized by the expression of the transcription factors T-bet and Blimp-1. While T-bet ablation restricted interferon-γ (IFN-γ) production, loss of Blimp-1 prevented GzmB expression in response to IL-2, suggesting two independent programs required for polyfunctionality of tumor-reactive CD4+ T cells. Our findings underscore the role of Treg cells, IL-2, and Blimp-1 in controlling the differentiation of cytotoxic CD4+ T cells and offer a pathway to enhancement of anti-tumor activity through their manipulation.


Assuntos
Granzimas/imunologia , Neoplasias/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Humanos , Interferon gama/imunologia , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/citologia , Microambiente Tumoral/imunologia
2.
Insect Sci ; 27(1): 14-21, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31246335

RESUMO

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. Here, we describe a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, we could manipulate omb expression levels at a moderate level. It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas do Tecido Nervoso/genética , Proteínas com Domínio T/genética , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais , Proteínas com Domínio T/metabolismo
3.
Immunity ; 51(6): 1088-1101.e5, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31732168

RESUMO

The B cell response to Ehrlichia muris is dominated by plasmablasts (PBs), with few-if any-germinal centers (GCs), yet it generates protective immunoglobulin M (IgM) memory B cells (MBCs) that express the transcription factor T-bet and harbor V-region mutations. Because Ehrlichia prominently infects the liver, we investigated the nature of liver B cell response and that of the spleen. B cells within infected livers proliferated and underwent somatic hypermutation (SHM). Vh-region sequencing revealed trafficking of clones between the spleen and liver and often subsequent local clonal expansion and intraparenchymal localization of T-bet+ MBCs. T-bet+ MBCs expressed MBC subset markers CD80 and PD-L2. Many T-bet+ MBCs lacked CD11b or CD11c expression but had marginal zone (MZ) B cell phenotypes and colonized the splenic MZ, revealing T-bet+ MBC plasticity. Hence, liver and spleen are generative sites of B cell responses, and they include V-region mutation and result in liver MBC localization.


Assuntos
Linfócitos B/imunologia , Ehrlichia/imunologia , Ehrlichiose/imunologia , Imunoglobulina M/imunologia , Fígado/imunologia , Baço/imunologia , Animais , Antígeno B7-1/biossíntese , Região Variável de Imunoglobulina/genética , Memória Imunológica/imunologia , Fígado/citologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 2 Ligante de Morte Celular Programada 1/biossíntese , Hipermutação Somática de Imunoglobulina/genética , Baço/citologia , Proteínas com Domínio T/metabolismo
4.
DNA Cell Biol ; 38(12): 1470-1479, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31633376

RESUMO

Vascular smooth muscle cells (VSMCs) of ascending aorta and TBX18+ sinus node both originated from the second heart field. The study explored whether ascending aortic smooth muscle cells in vitro could be reprogrammed into pacemaker-like cells with human TBX18. In the study, VSMCs were infected with TBX18, and then cocultured with neonatal rat ventricular cardiomyocytes (NRVMs) in vitro. By overexpressing TBX18, the transfected VSMCs expressed high levels of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), insulin gene enhancer binding protein 1, and human dwarf homeobox gene SHOX2, cardiac troponin I, and low level of connexin 43. In addition, funny current (If) was recorded by patch clamp appeared the time and voltage dependence in TBX18 group, which the amplitude of If density was from -5.164 ± 0.662 pA/pF to -0.765 ± 0.358 pA/pF (n = 14). Furthermore, TBX18-transfected VSMCs coupled with NRVMs showed typical action potential of pacemaker-like cells and the beating rate was faster (178.00 ± 7.55 bpm, p < 0.05) compared with other groups. In conclusion, our study indicated that transcription factor TBX18 could reprogram VSMCs into pacemaker-like cells in vitro.


Assuntos
Aorta/citologia , Reprogramação Celular , Músculo Liso Vascular/citologia , Miócitos Cardíacos/citologia , Marca-Passo Artificial , Proteínas com Domínio T/metabolismo , Animais , Animais Recém-Nascidos , Aorta/metabolismo , Diferenciação Celular , Células Cultivadas , Masculino , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-Dawley
5.
Nat Commun ; 10(1): 3946, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477701

RESUMO

Cerebral cortex expansion is a hallmark of mammalian brain evolution; yet, how increased neurogenesis is coordinated with structural and functional development remains largely unclear. The T-box protein TBR2/EOMES is preferentially enriched in intermediate progenitors and supports cortical neurogenesis expansion. Here we show that TBR2 regulates fine-scale spatial and circuit organization of excitatory neurons in addition to enhancing neurogenesis in the mouse cortex. TBR2 removal leads to a significant reduction in neuronal, but not glial, output of individual radial glial progenitors as revealed by mosaic analysis with double markers. Moreover, in the absence of TBR2, clonally related excitatory neurons become more laterally dispersed and their preferential synapse development is impaired. Interestingly, TBR2 directly regulates the expression of Protocadherin 19 (PCDH19), and simultaneous PCDH19 expression rescues neurogenesis and neuronal organization defects caused by TBR2 removal. Together, these results suggest that TBR2 coordinates neurogenesis expansion and precise microcircuit assembly via PCDH19 in the mammalian cortex.


Assuntos
Caderinas/genética , Córtex Cerebral/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Proteínas com Domínio T/genética , Animais , Caderinas/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos Knockout , Camundongos Transgênicos , Interferência de RNA , Sinapses/metabolismo , Proteínas com Domínio T/metabolismo
6.
PLoS Genet ; 15(8): e1008301, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31412026

RESUMO

We investigated whether Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.


Assuntos
Síndrome de DiGeorge/patologia , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/metabolismo , Animais , Região Branquial/embriologia , Síndrome de DiGeorge/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Endoderma/embriologia , Feminino , Fatores de Transcrição Forkhead/genética , Coração/embriologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Miocárdio/patologia , Proteínas com Domínio T/genética
7.
Nat Commun ; 10(1): 3859, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455769

RESUMO

Induction of long-lived antibody responses during infection or vaccination is often essential for subsequent protection, but the relative contributions of T follicular helper (Tfh) cells and T helper 1 (Th1) cells for induction of antigen specific antibody responses to viruses are unclear. Here, we establish an acute Zika virus (ZIKV) infection model in immunocompetent mice, and show that ZIKV infection elicits robust Th1-like Tfh cell and protective antibody responses. While these Th1-like Tfh cells share phenotypic and transcriptomic profiles with both Tfh and Th1 cells, they also have unique surface markers and gene expression characteristics, and are dependent on T-bet for their development. Th1-like Tfh cells, but not Th1 cells, are essential for class switching of ZIKV-specific IgG2c antibodies and maintenance of long-term neutralizing antibody responses. Our study suggests that specific modulation of the Th1-like Tfh cell response during infection or vaccination may augment the induction of antiviral antibody response to ZIKV and other viruses.


Assuntos
Switching de Imunoglobulina/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Infecção por Zika virus/imunologia , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Interações entre Hospedeiro e Microrganismos/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos , Camundongos Knockout , Transdução de Sinais/imunologia , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Vero , Infecção por Zika virus/virologia
8.
Nat Commun ; 10(1): 3807, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444346

RESUMO

Pioneer transcription factors are characterized by having the unique property of enabling the opening of closed chromatin sites, for implementation of cell fates. We previously found that the pioneer Pax7 specifies melanotrope cells through deployment of an enhancer repertoire, which allows binding of Tpit, a nonpioneer factor that determines the related lineages of melanotropes and corticotropes. Here, we investigate the relation between these two factors in the pioneer mechanism. Cell-specific gene expression and chromatin landscapes are defined by scRNAseq and chromatin accessibility profiling. We find that in vivo deployment of the melanotrope enhancer repertoire and chromatin opening requires both Pax7 and Tpit. In cells, binding of heterochromatin targets by Pax7 is independent of Tpit but Pax7-dependent chromatin opening requires Tpit. The present work shows that pioneer core properties are limited to the ability to recognize heterochromatin targets and facilitate nonpioneer binding. Chromatin opening per se may be provided through cooperation with nonpioneer factors.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição PAX7/metabolismo , Proteínas com Domínio T/metabolismo , Animais , Linhagem Celular Tumoral , Corticotrofos/fisiologia , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Masculino , Melanotrofos/fisiologia , Camundongos Knockout , Fator de Transcrição PAX7/genética , Ligação Proteica/genética , Análise de Sequência de RNA , Análise de Célula Única , Proteínas com Domínio T/genética
9.
Immunogenetics ; 71(7): 489-499, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31297569

RESUMO

Epigenetic modifications have been shown to be important for immune cell differentiation by regulating gene transcription. However, the role and mechanism of histone methylation in the development and differentiation of iNKT cells in rheumatoid arthritis (RA) mice have yet to be deciphered. The DBA/1 mouse RA model was established by using a modified GPI mixed peptide. We demonstrated that total peripheral blood, thymus, and spleen iNKT cells in RA mice decreased significantly, while iNKT1 in the thymus and spleen was increased significantly. PLZF protein and PLZF mRNA levels were significantly decreased in thymus DP T cells, while T-bet protein and mRNA were significantly increased in thymus iNKT cells. We found a marked accumulation in H3K27me3 around the promoter regions of the signature gene Zbtb16 in RA mice thymus DP T cells, and an accumulation of H3K4me3 around the promoters of the Tbx21 gene in iNKT cells. The expression levels of UTX in the thymus of RA mice were significantly reduced. The changes in the above indicators were particularly significant in the progressive phase of inflammation (11 days after modeling) and the peak phase of inflammation (14 days after modeling) in RA mice. Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels (H3K27me3 and H3K4me3) in the promoters of key genes Zbtb16 (encoding PLZF) and Tbx21 (encoding T-bet). Decreased UTX of thymus histone demethylase levels resulted in the accumulation of H3K27me3 modification.


Assuntos
Artrite Reumatoide/patologia , Lisina/metabolismo , Células T Matadoras Naturais/patologia , Regiões Promotoras Genéticas , Timo/fisiologia , Animais , Artrite Experimental/patologia , Diferenciação Celular , Epigênese Genética , Regulação da Expressão Gênica , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Metilação , Camundongos Endogâmicos DBA , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Baço/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
10.
Artif Cells Nanomed Biotechnol ; 47(1): 3021-3028, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31334674

RESUMO

Identification of specific cell markers is crucial for recognizing functionally healthy nucleus pulposus (NP) cells. The objective of this study was to investigate the role of CD24 expression in adult human NP cells. Cells were retrieved from NP tissues of 20 patients (aged 17-44) operated on for lumbar disc herniation. Based on CD24 expression, NP cells were separated by sorting and then used to examine phenotypic behavior, the effects of culture conditions and cellular senescence pathway related proteins. CD24 expression was positive in 35.5 ± 3.7% (range 9.1-65.2%) of NP cells. Consistently, normoxic expansion and serial passages in monolayers decreased percentage positivity for CD24 in NP cells. CD24- NP cells showed a markedly decreased GSK-3ß activity and increased mitogen-activated protein kinase phosphorylation accompanying by an increased ß-catenin expression. Higher levels of matrix metalloproteinases, as well as lower levels of ACAN and COL2 in CD24- cells, indicated the breakdown and reduced the formation of key extracellular matrix components. CD24+ NP cells presented a more favorable phenotype while CD24- cells showed a more prominent cellular senescence fate. CD24 in NP cells may be a surrogate marker of healthy cells, in the cell-based therapeutic treatment of degenerative disc disorders.


Assuntos
Antígeno CD24/genética , Antígeno CD24/metabolismo , Senescência Celular , Regulação da Expressão Gênica , Núcleo Pulposo/citologia , Fenótipo , Adolescente , Adulto , Feminino , Proteínas Fetais/metabolismo , Humanos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Virais/metabolismo , Proteínas com Domínio T/metabolismo , Adulto Jovem
11.
Int J Mol Sci ; 20(14)2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31311130

RESUMO

Gender affects cancer susceptibility. Currently, there are only a few studies on Y chromosome-linked long noncoding RNAs (lncRNAs), and the potential association between lncRNAs and cancers in males has not been fully elucidated. Here, we examined the expression of testis-specific transcript Y-linked 15 (TTTY15) in 37 males with non-small cell lung cancer (NSCLC), and performed circular chromosome conformation capture with next-generation sequencing to determine the genomic interaction regions of the TTTY15 gene. Our results showed that the expression levels of TTTY15 were lower in NSCLC tissues. Lower TTTY15 expression levels were associated with Tumor-Node-Metastasis (TNM) stage. A TTTY15 knockdown promoted malignant transformation of NSCLC cells. Based on the bioinformatics analysis of circular chromosome conformation capture data, we found that T-box transcription factor 4 (TBX4) may be a potential target gene of TTTY15. The RNA immunoprecipitation and chromatin immunoprecipitation results showed that TTTY15 may interact with DNA (cytosine-5)-methyltransferase 3A (DNMT3A), and the TTTY15 knockdown increased the binding of DNMT3A to the TBX4 promoter. We concluded that low TTTY15 expression correlates with worse prognosis among patients with NSCLC. TTTY15 promotes TBX4 expression via DNMT3A-mediated regulation. The identification of lncRNAs encoded by male-specific genes may help to identify potential targets for NSCLC therapy.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células , RNA Longo não Codificante/genética , Proteínas de Plasma Seminal/metabolismo , Proteínas com Domínio T/genética , Idoso , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , RNA Longo não Codificante/metabolismo , Proteínas de Plasma Seminal/genética , Proteínas com Domínio T/metabolismo
12.
Zhonghua Gan Zang Bing Za Zhi ; 27(7): 541-546, 2019 Jul 20.
Artigo em Chinês | MEDLINE | ID: mdl-31357781

RESUMO

Objective: To study the correlation between the level of T-bet expression and liver damage in peripheral plasma cells of patients with autoimmune hepatitis (AIH) in order to provide reference for the study of pathogenesis and development of diseases. Methods: The peripheral venous blood and clinical examination data of 29 cases with AIH and 6 healthy volunteers were collected. The percentage of subpopulations of peripheral blood B cells and the proportion of T-bet(+) cells in each subgroup were detected by flow cytometry. Plasma cells (CD19(+)CD10(-)CD27(hi)CD38(hi)), primary B cells (CD19(+)CD10(-)CD27(-)IgD(+)), transitional B cells (CD19(+)CD10(+)), and memory B cells (CD19(+)CD10(-)CD27(+)IgD(-)) were the included subsets of B cells. Serum immunoglobulin G (IgG) and alanine aminotransferase (ALT) levels, the proportion of B cells in peripheral blood subsets and IgG level, the proportion of T-bet(+) cells in each subset and the proportion of T-bet(+) plasma cells in each subset in B cells, the proportion of T-bet(+) plasma cells and the level of serum ALT were analyzed for correlation analysis. Statistical analysis was performed using two independent sample t-tests and linear regression. Results: The serum IgG level of AIH patients with abnormal ALT (19.47 ± 1.039)g/L was significantly higher than that of normal ALT patients (15.5 ± 1.069)g/L, and the difference was statistically significant (t = 2.65, P < 0.05). The percentage of peripheral plasma cells in B cells of AIH patients (2.80 ± 0.14) % was higher than that of healthy volunteers (0.73 ± 0.09) %, and the difference was statistically significant (P < 0.01). The percentage of T-bet(+) cells in peripheral plasma cells of AIH patients (23.54 ± 1.61) % was higher than that of healthy volunteers (6.59±0.59) % , and the difference was statistically significant (P < 0.01). The correlation analysis showed that the proportion of T-bet(+) cells in peripheral plasma cells of AIH patients was positively correlated with the proportion of plasma cells to B cells (r = 0.224 7, P < 0.01), and the percentage of peripheral plasma cells to B cells was positively correlated with the level of serum IgG (r = 0.299 1, P < 0.01). Serum IgG level was correlated with the level of ALT, reflecting an indicator of liver damage (t = 2.65, P < 0.05). Conclusion: The increase of T-bet expression in the peripheral plasma cells of AIH patients is associated with liver damage, which is a new mechanism of AIH pathogenesis and disease progression.


Assuntos
Hepatite Autoimune/patologia , Plasmócitos/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos B/metabolismo , Citometria de Fluxo , Humanos
13.
J Neurooncol ; 144(1): 65-77, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31240525

RESUMO

BACKGROUND: Chordoma is a rare refractory neoplasm that arises from the embryological remnants of the notochord, which is incurable using any multimodality therapy. Vascular endothelial growth factor (VEGF) is a potent activator of angiogenesis that is strongly associated with the tumor-immune microenvironment. These factors have not been elucidated for chordomas. METHODS: To evaluate the characteristics of vascular and tumor cells in chordoma, we first analyzed the expression of VEGF receptor (VEGFR) 1, VEGFR2, CD34, and Brachyury in a cell line and 54 tumor tissues. Patients with primary skull base chordomas were divided into the following two groups as per the tumor growth rate: patients with slow progression (SP: < 3 mm/year) and those with rapid progression (RP: ≥ 3 mm/year). Thus, the expressions of VEGF-A, VEGFR 1, and VEGFR2 on tumor cells; tumor infiltrative immune cells, including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs); and immune-checkpoint molecules (PD-1/PD-L1) were analyzed with the clinical courses, especially in a comparison between the two groups. RESULTS: In chordomas, both VEGFR1 and VEGFR2 were strongly expressed not only on vascular endothelial cells, but also on tumor cells. The recurrent cases showed significantly higher VEGFR1 expressions on tumor cells than the primary cases. The expression of VEGF-A was significantly higher in RP than that in SP group. The numbers of CD163+ TAMs and Foxp3+ Tregs were higher in RP than that in SP group. CONCLUSIONS: Expression of VEGFR1 and VEGFR2 on tumor cells and immunosuppressive tumor-microenvironment were related to tumor growth in patients with chordomas.


Assuntos
Biomarcadores Tumorais/metabolismo , Cordoma/patologia , Recidiva Local de Neoplasia/patologia , Neoplasias da Base do Crânio/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adolescente , Adulto , Idoso , Antígenos CD34/metabolismo , Cordoma/metabolismo , Cordoma/cirurgia , Feminino , Proteínas Fetais/metabolismo , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/cirurgia , Prognóstico , Neoplasias da Base do Crânio/metabolismo , Neoplasias da Base do Crânio/cirurgia , Proteínas com Domínio T/metabolismo , Adulto Jovem
14.
Nat Immunol ; 20(8): 980-991, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31209406

RESUMO

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.


Assuntos
Imunidade Inata/imunologia , Interferon gama/metabolismo , Interleucinas/metabolismo , Mucosa Intestinal/imunologia , Linfócitos/imunologia , Tonsila Palatina/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Criança , Pré-Escolar , Humanos , Fator de Transcrição Ikaros/metabolismo , Mucosa Intestinal/citologia , Linfócitos/classificação , Linfócitos/citologia , Camundongos , Proteínas com Domínio T/metabolismo
15.
Nat Med ; 25(5): 784-791, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31061540

RESUMO

Owing to recent medical and technological advances in neonatal care, infants born extremely premature have increased survival rates1,2. After birth, these infants are at high risk of hypoxic episodes because of lung immaturity, hypotension and lack of cerebral-flow regulation, and can develop a severe condition called encephalopathy of prematurity3. Over 80% of infants born before post-conception week 25 have moderate-to-severe long-term neurodevelopmental impairments4. The susceptible cell types in the cerebral cortex and the molecular mechanisms underlying associated gray-matter defects in premature infants remain unknown. Here we used human three-dimensional brain-region-specific organoids to study the effect of oxygen deprivation on corticogenesis. We identified specific defects in intermediate progenitors, a cortical cell type associated with the expansion of the human cerebral cortex, and showed that these are related to the unfolded protein response and changes. Moreover, we verified these findings in human primary cortical tissue and demonstrated that a small-molecule modulator of the unfolded protein response pathway can prevent the reduction in intermediate progenitors following hypoxia. We anticipate that this human cellular platform will be valuable for studying the environmental and genetic factors underlying injury in the developing human brain.


Assuntos
Lesões Encefálicas/etiologia , Hipóxia Encefálica/etiologia , Modelos Neurológicos , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Hipóxia Encefálica/metabolismo , Hipóxia Encefálica/patologia , Lactente Extremamente Prematuro , Recém-Nascido , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Neurogênese/genética , Neurogênese/fisiologia , Organoides/metabolismo , Organoides/patologia , Proteínas com Domínio T/metabolismo , Resposta a Proteínas não Dobradas
16.
Biol Res ; 52(1): 27, 2019 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-31054579

RESUMO

BACKGROUND: To assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatio-temporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR. RESULTS: In the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.


Assuntos
Malformações Anorretais/genética , Etilenotioureia/farmacologia , Regulação da Expressão Gênica/genética , Proteínas com Domínio T/genética , Animais , Malformações Anorretais/induzido quimicamente , Western Blotting , Feminino , Imuno-Histoquímica , Gravidez , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Proteínas com Domínio T/metabolismo
17.
Iran J Allergy Asthma Immunol ; 18(2): 190-199, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31066255

RESUMO

Contrasting studies are reported on the induction of IL-10 and IFN-γ via chitin microparticles (CMPs) during immune stimulation. Our previous studies have shown marked protection among CMP treated Leishmania-infected mice via regulated IL-10/IFN-γ response, at the present study, once more, examined the inconsistent responses regarding the immunologic response of CMPS. To verify whether CMPs could indeed up-regulate IL-10/IFN-γ axis, isolated spleen cells from the myelin oligodendrocyte glycoprotein (MOG) induced experimental autoimmune encephalomyelitis (EAE) mice were cultured in the presence of MOG peptide and/or CMPs. The effects of CMPs on IL-10, IFN-γ and IL-17 production were evaluated by Enzyme-linked Immunosorbent Assay (ELISA). Moreover, GATA binding protein 3 (Gata3), T-box transcription factor TBX21 (Tbx21), and RAR-related orphan receptor gamma (RORγT) expressions (real-time PCR) were investigated. MOG alone stimulated the production of IFN-γ (p≤0.004) but not, IL-10 (p≤0.140). MOG/chitin stimulation resulted in a significant increase in IFN-γ and IL-10 levels, respectively; (p≤0.004 and p≤0.003) rather than MOG. Additionally, the expression of Tbx21 (p≤0.001), but not Gata3 (p≤0.08), was increased in the MOG/chitin-treated spleen cells. All in all, CMP supports Gata3 independent IL-10 production and promotes Tbx21 dependent IFN-γ induction. These results, alongside our previous data, indicate that CMPs has particular adjuvant effects.


Assuntos
Micropartículas Derivadas de Células/metabolismo , Quitina/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Baço/patologia , Adjuvantes Imunológicos , Animais , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/patologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Imunomodulação , Interferon gama/metabolismo , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
18.
Breast Cancer Res Treat ; 176(3): 569-577, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31069590

RESUMO

PURPOSE: T-box transcription factor 21 (T-bet), which is the master regulator of effector T-cell activation, is derived by stimulation of T-cell receptors. In this study, we focused on T-bet and examined the function of activated T cells. METHODS: This study included 242 patients with primary triple-negative breast cancer (TNBC) who underwent resection without neoadjuvant chemotherapy between January 2004 and December 2014. The immunohistochemistry scoring for CD8 and T-bet expression on tumor-infiltrating lymphocytes (TILs) was defined as ≥ 30 per 6.25 × 10-3 mm2. RESULTS: Of the 242 TNBC cases, CD8 was positively expressed in 127 (52.5%) tumors, and T-bet was positively expressed in 67 (27.7%) tumors. T-bet expression was significantly correlated with CD8 expression (p < 0.0001). Patients with T-bet+ tumors had longer overall survival (OS) compared with patients with T-bet- tumors (p = 0.047). The combination of CD8+ and T-bet+ was associated with a better recurrence-free survival (RFS) and OS compared to CD8+/T-bet- tumors (p = 0.037 and p = 0.024, respectively). Adjuvant chemotherapy provided significantly greater benefit to patients with T-bet+ tumors (p = 0.031 for RFS, p = 0.0003 for OS). Multivariate analysis revealed that T-bet expression on TILs was an independent and positive prognostic indicator (HR = 0.36, 95% confidence interval (CI) 0.12-0.94, p = 0.037 for RFS, HR = 0.30, 95% CI 0.07-0.95, p = 0.039 for OS). CONCLUSIONS: OS was significantly improved for patients with high T-bet-expressing TILs in TNBC. Thus, T-bet may be a predictive indicator for survival and various immunotherapy strategies in TNBC.


Assuntos
Linfócitos do Interstício Tumoral/metabolismo , Proteínas com Domínio T/metabolismo , Subpopulações de Linfócitos T/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/terapia , Carga Tumoral
19.
PLoS One ; 14(5): e0214873, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31086358

RESUMO

BACKGROUNDS: Reducing toxicants transplacental rates could contribute to the prevention of congenital heart defects (CHDs). Placental P-glycoprotein (P-gp) plays a vital role in fetal toxicants exposure and subsequently affects the risk of toxicants-induced birth defects. However, data on the role of placental P-gp in decreasing toxicants-induced cardiac anomalies is extremely limited. This study aimed to explore the protective role of placental P-gp in reducing the risk of Di-(2-ethylhexyl)-phthalate (DEHP) induced cardiac anomalies in mice. METHODS: The C57BL mice were randomly divided into four groups: the vehicle group (corn oil, n = 10), 500mg/Kg DEHP group (n = 15), 3mg/Kg verapamil group (n = 10) and 500mg/Kg DEHP & 3mg/Kg verapamil group (n = 20). Pregnant dams in different group received respective intervention by gavage once daily from E6.5-14.5. Maternal weights were monitored every day and samples were collected at E15.5. HE staining was used to examine fetal cardiac malformations. Real-time quantitative PCR (RT-qPCR) and Western-Blot were applied to detect Nkx2.5/Gata4/Tbx5/Mef2c/Chf1 mRNA and protein expression, respectively. The mRNA expression of peroxisome proliferator-activated receptor γ (PPARγ) was also determined using RT-qPCR. RESULTS: Co-administration of verapamil and DEHP significantly elevated fetal cardiac malformation rates, in comparison with the DEHP group, the verapamil group and the vehicle group. Different phenotypes of cardiac anomalies, including septal defects and ventricular myocardium noncompaction, were noted both in the DEHP group and the DEHP & verapamil group. The ventricular myocardium noncompaction appeared to be more severe in the DEHP & verapamil group. Fetal cardiac PPARγ mRNA expression was notably increased and Gata4/Mef2c/Chf1 expression was markedly decreased in the DEHP & verapamil group. CONCLUSION: Placental P-gp inhibition enhances susceptibility to DEHP induced cardiac malformations in mice.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Dietilexilftalato/toxicidade , Coração Fetal/efeitos dos fármacos , Cardiopatias Congênitas/patologia , Placenta/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Animais , Feminino , Coração Fetal/metabolismo , Coração Fetal/patologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Cardiopatias Congênitas/induzido quimicamente , Cardiopatias Congênitas/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Exposição Materna , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/genética , PPAR gama/metabolismo , Gravidez , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Verapamil/farmacologia
20.
Dev Cell ; 49(4): 643-650.e3, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31112700

RESUMO

It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.


Assuntos
Imunidade Inata/imunologia , Morfolinos/imunologia , Morfolinos/metabolismo , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Imunidade Inata/fisiologia , Oligonucleotídeos Antissenso/genética , Processamento de RNA , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcriptoma/genética , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética
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