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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.
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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 , Interleucina 22RESUMO
Skeletal muscle is a dynamic tissue the size of which can be remodeled through the concerted actions of various cues. Here, we investigated the skeletal muscle transcriptional program and identified key tissue-specific regulatory genetic elements. Our results show that Myod1 is bound to numerous skeletal muscle enhancers in collaboration with the glucocorticoid receptor (GR) to control gene expression. Remarkably, transcriptional activation controlled by these factors occurs through direct contacts with the promoter region of target genes, via the CpG-bound transcription factor Nrf1, and the formation of Ctcf-anchored chromatin loops, in a myofiber-specific manner. Moreover, we demonstrate that GR negatively controls muscle mass and strength in mice by down-regulating anabolic pathways. Taken together, our data establish Myod1, GR and Nrf1 as key players of muscle-specific enhancer-promoter communication that orchestrate myofiber size regulation.
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Cromatina/metabolismo , Elementos Facilitadores Genéticos , Músculo Esquelético/metabolismo , Proteína MyoD/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Linhagem Celular , Cromatina/genética , Sequenciamento de Cromatina por Imunoprecipitação , Regulação da Expressão Gênica/genética , Histonas/genética , Histonas/metabolismo , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Força Muscular/genética , Músculo Esquelético/fisiologia , Proteína MyoD/genética , Mioblastos/metabolismo , Fator 1 Nuclear Respiratório/genética , Receptores de Glucocorticoides/genética , Proteínas RecombinantesRESUMO
OBJECTIVE: Neutrophils are typically the most abundant leucocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. METHODS: We performed RNA sequencing of neutrophils from healthy human blood, arthritic blood and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. RESULTS: Blood neutrophils from healthy donors and patients with active arthritis showed largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN-γ), as well as to tumour necrosis factor, interleukin-6 and hypoxia, in both humans and mice. Mass cytometry confirmed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN-γ and prolonged culture. CONCLUSIONS: Circulating neutrophils from patients with arthritis resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN-γ response and ageing as complementary drivers of the synovial fluid neutrophil phenotype.
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Artrite , Neutrófilos , Envelhecimento , Animais , Artrite/metabolismo , Humanos , Interferon gama/metabolismo , Camundongos , Neutrófilos/metabolismo , Fenótipo , Líquido Sinovial/metabolismoRESUMO
OBJECTIVES: Advances in immunotherapy by blocking TNF have remarkably improved treatment outcomes for Rheumatoid arthritis (RA) patients. Although treatment specifically targets TNF, the downstream mechanisms of immune suppression are not completely understood. The aim of this study was to detect biomarkers and expression signatures of treatment response to TNF inhibition. METHODS: Peripheral blood mononuclear cells (PBMCs) from 39 female patients were collected before anti-TNF treatment initiation (day 0) and after 3 months. The study cohort included patients previously treated with MTX who failed to respond adequately. Response to treatment was defined based on the EULAR criteria and classified 23 patients as responders and 16 as non-responders. We investigated differences in gene expression in PBMCs, the proportion of cell types and cell phenotypes in peripheral blood using flow cytometry and the level of proteins in plasma. Finally, we used machine learning models to predict non-response to anti-TNF treatment. RESULTS: The gene expression analysis in baseline samples revealed notably higher expression of the gene EPPK1 in future responders. We detected the suppression of genes and proteins following treatment, including suppressed expression of the T cell inhibitor gene CHI3L1 and its protein YKL-40. The gene expression results were replicated in an independent cohort. Finally, machine learning models mainly based on transcriptomic data showed high predictive utility in classifying non-response to anti-TNF treatment in RA. CONCLUSIONS: Our integrative multi-omics analyses identified new biomarkers for the prediction of response, found pathways influenced by treatment and suggested new predictive models of anti-TNF treatment in RA patients.
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Antirreumáticos , Artrite Reumatoide , Antirreumáticos/metabolismo , Antirreumáticos/uso terapêutico , Artrite Reumatoide/diagnóstico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Biomarcadores , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Aprendizado de Máquina , Metotrexato/metabolismo , Metotrexato/uso terapêutico , Resultado do Tratamento , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Defining clinical phenotypes provides opportunities for new diagnostics and may provide insights into early intervention and disease prevention. There is increasing evidence that patient-derived health data may contain information that complements traditional methods of clinical phenotyping. The utility of these data for defining meaningful phenotypic groups is of great interest because social media and online resources make it possible to query large cohorts of patients with health conditions. METHODS: We evaluated the degree to which patient-reported categorical data is useful for discovering subclinical phenotypes and evaluated its utility for discovering new measures of disease severity, treatment response and genetic architecture. Specifically, we examined the responses of 1961 patients with inflammatory bowel disease to questionnaires in search of sub-phenotypes. We applied machine learning methods to identify novel subtypes of Crohn's disease and studied their associations with drug responses. RESULTS: Using the patients' self-reported information, we identified two subpopulations of Crohn's disease; these subpopulations differ in disease severity, associations with smoking, and genetic transmission patterns. We also identified distinct features of drug response for the two Crohn's disease subtypes. These subtypes show a trend towards differential genotype signatures. CONCLUSION: Our findings suggest that patient-defined data can have unplanned utility for defining disease subtypes and may be useful for guiding treatment approaches.
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Doença de Crohn , Doenças Inflamatórias Intestinais , Doença de Crohn/diagnóstico , Doença de Crohn/tratamento farmacológico , Doença de Crohn/genética , Genótipo , Humanos , Fenótipo , Inquéritos e QuestionáriosRESUMO
Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in innate immune signaling by Toll-like receptors (TLRs), and loss of IRAK4 activity in mice and humans increases susceptibility to bacterial infections and causes defects in TLR and IL1 ligand sensing. However, the mechanism by which IRAK4 activity regulates the production of downstream inflammatory cytokines is unclear. Using transcriptomic and biochemical analyses of human monocytes treated with a highly potent and selective inhibitor of IRAK4, we show that IRAK4 kinase activity controls the activation of interferon regulatory factor 5 (IRF5), a transcription factor implicated in the pathogenesis of multiple autoimmune diseases. Following TLR7/8 stimulation by its agonist R848, chemical inhibition of IRAK4 abolished IRF5 translocation to the nucleus and thus prevented IRF5 binding to and activation of the promoters of inflammatory cytokines in human monocytes. We also found that IKKß, an upstream IRF5 activator, is phosphorylated in response to the agonist-induced TLR signaling. Of note, IRAK4 inhibition blocked IKKß phosphorylation but did not block the nuclear translocation of NFκB, which was surprising, given the canonical role of IKKß in phosphorylating IκB to allow NFκB activation. Moreover, pharmacological inhibition of either IKKß or the serine/threonine protein kinase TAK1 in monocytes blocked TLR-induced cytokine production and IRF5 translocation to the nucleus, but not nuclear translocation of NFκB. Taken together, our data suggest a mechanism by which IRAK4 activity regulates TAK1 and IKKß activation, leading to the nuclear translocation of IRF5 and induction of inflammatory cytokines in human monocytes.
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Quinase I-kappa B/metabolismo , Fatores Reguladores de Interferon/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Modelos Imunológicos , Monócitos/metabolismo , Receptor 7 Toll-Like/agonistas , Receptor 8 Toll-Like/agonistas , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Células Cultivadas , Biologia Computacional , Citocinas/agonistas , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/química , Fatores Reguladores de Interferon/agonistas , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/química , MAP Quinase Quinase Quinases/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Análise de Célula Única , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismoRESUMO
BACKGROUND: Genome-wide association studies have identified numerous genetic regions that influence cross-sectional lung function. Longitudinal decline in lung function also includes a heritable component but the genetic determinants have yet to be defined. OBJECTIVES: We aimed to determine whether regions associated with cross-sectional lung function were also associated with longitudinal decline and to seek novel variants which influence decline. METHODS: We analysed genome-wide data from 4167 individuals from the Busselton Health Study cohort, who had undergone spirometry (12â 695 observations across eight time points). A mixed model was fitted and weighted risk scores were calculated for the joint effect of 26 known regions on baseline and longitudinal changes in FEV1 and FEV1/FVC. Potential additional regions of interest were identified and followed up in two independent cohorts. RESULTS: The 26 regions previously associated with cross-sectional lung function jointly showed a strong effect on baseline lung function (p=4.44×10-16 for FEV1/FVC) but no effect on longitudinal decline (p=0.160 for FEV1/FVC). This was replicated in an independent cohort. 39 additional regions of interest (48 variants) were identified; these associations were not replicated in two further cohorts. CONCLUSIONS: Previously identified genetic variants jointly have a strong effect on cross-sectional lung function in adults but little or no effect on the rate of decline of lung function. It is possible that they influence COPD risk through lung development. Although no genetic variants have yet been associated with lung function decline at stringent genome-wide significance, longitudinal change in lung function is heritable suggesting that there is scope for future discoveries.
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Variação Genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Respiração/genética , Adulto , Estudos Transversais , Progressão da Doença , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Testes de Função Respiratória , Medição de Risco , Fatores de Risco , Espirometria , Fatores de Tempo , Austrália OcidentalRESUMO
Chronic obstructive pulmonary disease (COPD) is a leading cause of global morbidity and mortality and, whilst smoking remains the single most important risk factor, COPD risk is heritable. Of 26 independent genomic regions showing association with lung function in genome-wide association studies, eleven have been reported to show association with airflow obstruction. Although the main risk factor for COPD is smoking, some individuals are observed to have a high forced expired volume in 1 second (FEV1) despite many years of heavy smoking. We hypothesised that these "resistant smokers" may harbour variants which protect against lung function decline caused by smoking and provide insight into the genetic determinants of lung health. We undertook whole exome re-sequencing of 100 heavy smokers who had healthy lung function given their age, sex, height and smoking history and applied three complementary approaches to explore the genetic architecture of smoking resistance. Firstly, we identified novel functional variants in the "resistant smokers" and looked for enrichment of these novel variants within biological pathways. Secondly, we undertook association testing of all exonic variants individually with two independent control sets. Thirdly, we undertook gene-based association testing of all exonic variants. Our strongest signal of association with smoking resistance for a non-synonymous SNP was for rs10859974 (P = 2.34 × 10(-4)) in CCDC38, a gene which has previously been reported to show association with FEV1/FVC, and we demonstrate moderate expression of CCDC38 in bronchial epithelial cells. We identified an enrichment of novel putatively functional variants in genes related to cilia structure and function in resistant smokers. Ciliary function abnormalities are known to be associated with both smoking and reduced mucociliary clearance in patients with COPD. We suggest that genetic influences on the development or function of cilia in the bronchial epithelium may affect growth of cilia or the extent of damage caused by tobacco smoke.
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Cílios/fisiologia , Exoma , Proteínas/fisiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fumar/fisiopatologia , Adolescente , Adulto , Idoso , Estudos de Coortes , Humanos , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Doença Pulmonar Obstrutiva Crônica/genética , Locos de Características Quantitativas , Adulto JovemRESUMO
Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic inflammation and is mediated by multiple immune cell types. In this work, we aimed to determine the relevance of changes in cell proportions in peripheral blood mononuclear cells (PBMCs) during the development of disease and following treatment. Samples from healthy blood donors, newly diagnosed RA patients, and established RA patients that had an inadequate response to MTX and were about to start tumor necrosis factor inhibitors (TNFi) treatment were collected before and after 3 months of treatment. We used in parallel a computational deconvolution approach based on RNA expression and flow cytometry to determine the relative cell-type frequencies. Cell-type frequencies from deconvolution of gene expression indicate that monocytes (both classical and non-classical) and CD4+ cells (Th1 and Th2) were increased in RA patients compared to controls, while NK cells and B cells (naïve and mature) were significantly decreased in RA patients. Treatment with MTX caused a decrease in B cells (memory and plasma cell), and a decrease in CD4 Th cells (Th1 and Th17), while treatment with TNFi resulted in a significant increase in the population of B cells. Characterization of the RNA expression patterns found that most of the differentially expressed genes in RA subjects after treatment can be explained by changes in cell frequencies (98% and 74% respectively for MTX and TNFi).
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Antirreumáticos , Artrite Reumatoide , Humanos , Antirreumáticos/uso terapêutico , Leucócitos Mononucleares/metabolismo , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/diagnóstico , Linfócitos T CD4-Positivos/metabolismo , RNARESUMO
Background: Methotrexate (MTX) is the first line treatment for rheumatoid arthritis (RA), but failure of satisfying treatment response occurs in a significant proportion of patients. Here we present a longitudinal multi-omics study aimed at detecting molecular and cellular processes in peripheral blood associated with a successful methotrexate treatment of rheumatoid arthritis. Methods: Eighty newly diagnosed patients with RA underwent clinical assessment and donated blood before initiation of MTX, and 3 months into treatment. Flow cytometry was used to describe cell types and presence of activation markers in peripheral blood, the expression of 51 proteins was measured in serum or plasma, and RNA sequencing was performed in peripheral blood mononuclear cells (PBMC). Response to treatment after 3 months was determined using the EULAR response criteria. We assessed the changes in biological phenotypes during treatment, and whether these changes differed between responders and non-responders with regression analysis. By using measurements from baseline, we also tried to find biomarkers of future MTX response or, alternatively, to predict MTX response. Results: Among the MTX responders, (Good or Moderate according to EULAR treatment response classification, n = 60, 75%), we observed changes in 29 partly overlapping cell types proportions, levels of 13 proteins and expression of 38 genes during treatment. These changes were in most cases suppressions that were stronger among responders compared to non-responders. Within responders to treatment, we observed a suppression of FOXP3 gene expression, reduction of immunoglobulin gene expression and suppression of genes involved in cell proliferation. The proportion of many HLA-DR expressing T-cell populations were suppressed in all patients irrespective of clinical response, and the proportion of many IL21R+ T-cells were reduced exclusively in non-responders. Using only the baseline measurements we could not detect any biomarkers or prediction models that could predict response to MTX. Conclusion: We conclude that a deep molecular and cellular phenotyping of peripheral blood cells in RA patients treated with methotrexate can reveal previously not recognized differences between responders and non-responders during 3 months of treatment with MTX. This may contribute to the understanding of MTX mode of action and explain non-responsiveness to MTX therapy.
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BACKGROUND: Patient-derived organoid (PDO) models offer potential to transform drug discovery for inflammatory bowel disease (IBD) but are limited by inconsistencies with differentiation and functional characterization. We profiled molecular and cellular features across a range of intestinal organoid models and examined differentiation and establishment of a functional epithelial barrier. METHODS: Patient-derived organoids or monolayers were generated from control or IBD patient-derived colon or ileum and were molecularly or functionally profiled. Biological or technical replicates were examined for transcriptional responses under conditions of expansion or differentiation. Cell-type composition was determined by deconvolution of cell-associated gene signatures and histological features. Differentiated control or IBD-derived monolayers were examined for establishment of transepithelial electrical resistance (TEER), loss of barrier integrity in response to a cocktail of interferon (IFN)-γ and tumor necrosis factor (TNF)-α, and prevention of cytokine-induced barrier disruption by the JAK inhibitor, tofacitinib. RESULTS: In response to differentiation media, intestinal organoids and monolayers displayed gene expression patterns consistent with maturation of epithelial cell types found in the human gut. Upon differentiation, both colon- and ileum-derived monolayers formed functional barriers, with sustained TEER. Barrier integrity was compromised by inflammatory cytokines IFN-γ and TNF-α, and damage was inhibited in a dose-dependent manner by tofacitinib. CONCLUSIONS: We describe the generation and characterization of human colonic or ileal organoid models capable of functional differentiation to mature epithelial cell types. In monolayer culture, these cells formed a robust epithelial barrier with sustained TEER and responses to pharmacological modulation. Our findings demonstrate that control and IBD patient-derived organoids possess consistent transcriptional and functional profiles that can enable development of epithelial-targeted therapies.
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Doenças Inflamatórias Intestinais , Intestinos , Organoides , Humanos , Citocinas/metabolismo , Células Epiteliais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Organoides/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Intestinos/fisiologiaRESUMO
Skeletal muscle atrophy can be a consequence of many diseases, environmental insults, inactivity, age, and injury. Atrophy is characterized by active degradation, removal of contractile proteins, and a reduction in muscle fiber size. Animal models have been extensively used to identify pathways that lead to atrophic conditions. We used genome-wide expression profiling analyses and quantitative PCR to identify the molecular changes that occur in two clinically relevant mouse models of muscle atrophy: hindlimb casting and Achilles tendon laceration (tenotomy). Gastrocnemius muscle samples were collected 2, 7, and 14 days after casting or injury. The total amount of muscle loss, as measured by wet weight and muscle fiber size, was equivalent between models on day 14, although tenotomy resulted in a more rapid induction of muscle atrophy. Furthermore, tenotomy resulted in the regulation of significantly more mRNA transcripts then did casting. Analysis of the regulated genes and pathways suggest that the mechanisms of atrophy are distinct between these models. The degradation following casting was ubiquitin-proteasome mediated, while degradation following tenotomy was lysosomal and matrix-metalloproteinase mediated, suggesting a possible role for autophagy. These data suggest that there are multiple mechanisms leading to muscle atrophy and that specific therapeutic agents may be necessary to combat atrophy resulting from different conditions.
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Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Tendão do Calcâneo/lesões , Tendão do Calcâneo/metabolismo , Animais , Perfilação da Expressão Gênica , Membro Posterior/lesões , Membro Posterior/metabolismo , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TenotomiaRESUMO
Ectopic expression of recombinant human bone morphogenetic protein 2 (rhBMP2) induces osteogenesis, while ectopic expression of rhBMP12 and rhBMP13 induces the formation of tendon-like tissue. Despite their different in vivo activities, all three ligands bound to the type I bone morphogenic protein receptors (BMPRs), activin receptor-like kinase (ALK)-3 and ALK6, and to the type II BMPRs, activin receptor type-2A, activin receptor type-2B, and BMPR2, with similar affinities. Treatment of C3H10T1/2 cells with rhBMP2 activated SMAD signaling and induced expression of osteoblast markers including osteocalcin mRNA (Ocn). In contrast, treatment with rhBMP12 or rhBMP13 resulted in a dose-dependent induction of a tendon-specific gene (Thbs4) expression with no detectable activation of SMAD 1, 5, and 8. Differential regulation of Thbs4 and Ocn has potential utility as an in vitro biomarker for induction of tenogenic signaling. Such an assay also permits the ability to distinguish between the activities of different BMPs and may prove useful in studies on the molecular mechanisms of BMP tenogenic activity.
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Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Fator 6 de Diferenciação de Crescimento/metabolismo , Fatores de Diferenciação de Crescimento/metabolismo , Receptores de Ativinas/metabolismo , Animais , Proteína Morfogenética Óssea 2/farmacologia , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Fator 6 de Diferenciação de Crescimento/biossíntese , Fator 6 de Diferenciação de Crescimento/farmacologia , Fatores de Diferenciação de Crescimento/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Osteocalcina/biossíntese , Osteocalcina/genética , Osteogênese/efeitos dos fármacos , Reação em Cadeia da Polimerase , Ligação Proteica , RNA Mensageiro/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Tendões/metabolismo , Trombospondinas/biossínteseRESUMO
Leydig cells are the testosterone-producing cells in the adult male. Adult Leydig cells (ALCs) develop from stem Leydig cells (SLCs) through at least two intermediate cells, progenitor Leydig cells (PLCs) and immature Leydig cells (ILCs). Microarray gene expression was used to identify the transcriptional changes that occur with the differentiation of SLCs to PLCs and, thus, with the entry of SLCs into the Leydig cell lineage; to comprehensively examine differentiation through the development of ALCs; and to relate the pattern of gene expression in SLCs to that in a well-established stem cell, bone marrow stem cells (BSCs). We show that the pattern of gene expression by SLCs was more similar to the expression by BSCs, an established stem cell outside the male reproductive tract, than to any of the cells in the Leydig cell developmental lineage. These results indicated that the SLCs have many of the molecular characteristics of other stem cells. Pathway analysis indicated that development of Leydig cells from SLCs to PLCs was associated with decreased expression of genes related to adhesion and increased expression of genes related to steroidogenesis. Gene expression changes between PLCs and ILCs and between ILCs and ALCs were relatively minimal, suggesting that these cells are highly similar. In contrast, gene expression changes between SLCs and ALCs were quite distinct.
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Células-Tronco Adultas/metabolismo , Diferenciação Celular , Células Intersticiais do Testículo/metabolismo , Animais , Células da Medula Óssea/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Células Intersticiais do Testículo/citologia , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Chronic tendon injuries, also known as tendinopathies, are common among professional and recreational athletes. These injuries result in a significant amount of morbidity and health care expenditure, yet little is known about the molecular mechanisms leading to tendinopathy. METHODS: We have used histological evaluation and molecular profiling to determine gene expression changes in 23 human patients undergoing surgical procedures for the treatment of chronic tendinopathy. RESULTS: Diseased tendons exhibit altered extracellular matrix, fiber disorientation, increased cellular content and vasculature, and the absence of inflammatory cells. Global gene expression profiling identified 983 transcripts with significantly different expression patterns in the diseased tendons. Global pathway analysis further suggested altered expression of extracellular matrix proteins and the lack of an appreciable inflammatory response. CONCLUSIONS: Identification of the pathways and genes that are differentially regulated in tendinopathy samples will contribute to our understanding of the disease and the development of novel therapeutics.
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Traumatismos em Atletas/genética , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/fisiologia , Predisposição Genética para Doença/genética , Traumatismos dos Tendões/genética , Tendões/metabolismo , Adulto , Idoso , Traumatismos em Atletas/fisiopatologia , Traumatismos em Atletas/cirurgia , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Traumatismos dos Tendões/diagnóstico , Traumatismos dos Tendões/fisiopatologia , Tendões/fisiopatologiaRESUMO
Mammalian spermatogenesis is a complex biological process that occurs within a highly organized tissue, the seminiferous epithelium. The coordinated maturation of spermatogonia, spermatocytes, and spermatids suggests the existence of precise programs of gene expression in these cells and in their neighboring somatic Sertoli cells. The objective of this study was to identify the genes that execute these programs. Rat seminiferous tubules at stages I, II-III, IV-V, VI, VIIa,b, VIIc,d, VIII, IX-XI, XII, and XIII-XIV of the cycle were isolated by microdissection, whereas Sertoli cells, spermatogonia plus early spermatocytes, pachytene spermatocytes, and round spermatids were purified from enzymatically dispersed testes. Microarray analysis by using Rat Genome 230 2.0 arrays identified 16,971 probe sets that recognized testicular transcripts, and 398 of these were identified as testis-specific. Expression of 1,286 probe sets were found to differ at least 4-fold between two cell types and also across the stages of the cycle. Pathway and annotated cluster analyses of those probe sets predicted that entire biological pathways and processes are regulated cyclically in specific cells. Important among these are the cell cycle, DNA repair, and embryonic neuron development. Taken together, these data indicate that stage-regulated gene expression is a widespread and fundamental characteristic of spermatogenic cells and Sertoli cells.
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Regulação da Expressão Gênica no Desenvolvimento , Células de Sertoli/metabolismo , Espermátides/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Animais , Ciclo Celular/genética , Células Cultivadas , Reparo do DNA/genética , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Masculino , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Epitélio Seminífero/metabolismoRESUMO
OBJECTIVE: The objectives of this study were to assess the 1-year persistence to methotrexate (MTX) initiated as the first ever conventional synthetic disease-modifying antirheumatic drug in new-onset rheumatoid arthritis (RA) and to investigate the marginal gains and robustness of the results by increasing the number and nature of covariates and by using data-driven, instead of hypothesis-based, methods to predict this persistence. METHODS: Through the Swedish Rheumatology Quality Register, linked to other data sources, we identified a cohort of 5475 patients with new-onset RA in 2006-2016 who were starting MTX monotherapy as their first disease-modifying antirheumatic drug. Data on phenotype at diagnosis and demographics were combined with increasingly detailed data on medical disease history and medication use in four increasingly complex data sets (48-4162 covariates). We performed manual model building using logistic regression. We also performed five different machine learning (ML) methods and combined the ML results into an ensemble model. We calculated the area under the receiver operating characteristic curve (AUROC) and made calibration plots. We trained on 90% of the data, and tested the models on a holdout data set. RESULTS: Of the 5475 patients, 3834 (70%) remained on MTX monotherapy 1 year after treatment start. Clinical RA disease activity and baseline characteristics were most strongly associated with the outcome. The best manual model had an AUROC of 0.66 (95% confidence interval [CI] 0.60-0.71). For the ML methods, Lasso regression performed best (AUROC = 0.67; 95% CI 0.62-0.71). CONCLUSION: Approximately two thirds of patients with early RA who start MTX remain on this therapy 1 year later. Predicting this persistence remains a challenge, whether using hypothesis-based or ML models, and may yet require additional types of data.
RESUMO
Crohn's disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30+γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated TH17 but decreased CD8+T, γδT, TFH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8+, as well as reduced CD4+T cells with an elevated TH17 over Treg/TFH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets.
Assuntos
Doença de Crohn/imunologia , Linfócitos Intraepiteliais/imunologia , Análise de Célula Única/métodos , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Doença de Crohn/patologia , Perfilação da Expressão Gênica/métodos , Humanos , Linfócitos Intraepiteliais/metabolismo , Contagem de Linfócitos , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismoRESUMO
Liver X receptors (LXRalpha and -beta) are liposensors that exert their metabolic effects by orchestrating the expression of macrophage genes involved in lipid metabolism and inflammation. LXRs are also expressed in other tissues, including skin, where their natural oxysterol ligands induce keratinocyte differentiation and improve epidermal barrier function. To extend the potential use of LXR ligands to dermatological indications, we explored the possibility of using LXR as a target for skin aging. We demonstrate that LXR signaling is down-regulated in cell-based models of photoaging, i.e. UV-activated keratinocytes and TNFalpha-activated dermal fibroblasts. We show that a synthetic LXR ligand inhibits the expression of cytokines and metalloproteinases in these in vitro models, thus indicating its potential in decreasing cutaneous inflammation associated with the etiology of photoaging. Furthermore, a synthetic LXR ligand induces the expression of differentiation markers, ceramide biosynthesis enzymes, and lipid synthesis and transport genes in keratinocytes. Remarkably, LXRbeta-null mouse skin showed some of the molecular defects that are observed in chronologically aged human skin. Finally, we demonstrate that a synthetic LXR agonist inhibits UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the ability of an LXR ligand to modulate multiple pathways underlying the etiology of skin aging suggests that LXR is a novel target for developing potential therapeutics for photoaging and chronological skin aging indications.