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1.
Chemosphere ; 251: 126642, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32345545

RESUMO

The ubiquitous presence of aluminum in the environment leads to a high likelihood of human exposure. Neurotoxicity of the trivalent cationic form of this metal (Al3+) occurs in the central nervous system via accumulation of Al in cells of neural origin, including neural progenitor cells (NPCs). NPCs play a key role in the development and regeneration of the brain throughout life; therefore, this metal may contribute to neuropathological conditions. Here, we evaluated the effects of different Al3+ concentrations (0-50 µM) on the purinergic system of NPCs isolated from embryonic telencephalons, cultured as neurospheres. Al3+ adhered to the cell surface of neurospheres reducing extracellular ATP release, as well as ATP, ADP, and AMP hydrolysis by NTPDase and 5'-nucleotidase, respectively. In addition, impaired nucleotide release by Al3+ reduced P2Y1 and adenosine A2A receptors expression in differentiated neurospheres. These receptors are crucial for NPC proliferation during brain development and self-repair against external stimuli, such as metal exposure. Thus, Al3+ represents an environmental agent linked to neurodegeneration through alterations in the ATP-signalling pathway, proving to be a potential mechanism associated with NPC proliferation and brain degeneration.


Assuntos
Alumínio/toxicidade , 5'-Nucleotidase , Trifosfato de Adenosina/metabolismo , Alumínio/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Sistema Nervoso Central/metabolismo , Proteínas Ligadas por GPI , Humanos , Transdução de Sinais/efeitos dos fármacos , Células-Tronco , Testes de Toxicidade
2.
Muscle Nerve ; 61(5): 570-574, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32035011

RESUMO

Herein we report a case of sporadic inclusion-body myositis (sIBM) occurring at an unusually young age in a patient with primary Sjögren syndrome, and use the case to explore possible shared mechanisms for disease susceptibility. Possible factors may include the association of both conditions with the 8.1 ancestral haplotype; the presence of anti-cN1A antibodies, which, although considered specific for sIBM, are also seen in pSS; and the shared association with T-cell large granular lymphocyte leukemia (T-LGLL). Further evaluation of this patient did in fact reveal underlying T-LGLL and mechanisms by which T cells in sIBM may escape immune regulation and contribute to disease phenotype are explored. Despite myofiber infiltration with CD8-positive T cells in sIBM, and, although sIBM is traditionally considered treatment-refractory, we report a significant response to the anti-CD20 monoclonal antibody, rituximab, and discuss possible mechanisms by which this response may be mediated.


Assuntos
5'-Nucleotidase/imunologia , Autoanticorpos/imunologia , Leucemia Linfocítica Granular Grande/imunologia , Miosite de Corpos de Inclusão/imunologia , Síndrome de Sjogren/imunologia , Adulto , Azatioprina/uso terapêutico , Linfócitos T CD8-Positivos/patologia , Feminino , Antígenos HLA/genética , Haplótipos , Humanos , Hidroxicloroquina/uso terapêutico , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Leucemia Linfocítica Granular Grande/complicações , Imagem por Ressonância Magnética , Metotrexato/uso terapêutico , Músculo Esquelético/diagnóstico por imagem , Músculo Esquelético/patologia , Miosite de Corpos de Inclusão/complicações , Miosite de Corpos de Inclusão/patologia , Miosite de Corpos de Inclusão/terapia , Prednisolona/uso terapêutico , Rituximab/uso terapêutico , Síndrome de Sjogren/complicações , Síndrome de Sjogren/terapia
3.
Nat Commun ; 11(1): 515, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980601

RESUMO

CD73, an ecto-5'-nucleotidase (NT5E), serves as an immune checkpoint by generating adenosine (ADO), which suppresses immune activation through the A2A receptor. Elevated CD73 levels in tumor tissues correlate with poor clinical outcomes. However, the crucial source of CD73 activity within the tumor microenvironment remains unspecified. Here, we demonstrate that cancer-associated fibroblasts (CAFs) constitute the prominent CD73hi population in human colorectal cancers (CRCs) and two CD73- murine tumor models, including a modified CRC. Clinically, high CAF abundancy in CRC tissues correlates strongly with elevated CD73 activity and poor prognosis. Mechanistically, CAF-CD73 expression is enhanced via an ADO-A2B receptor-mediated feedforward circuit triggered by tumor cell death, which enforces the CD73-checkpoint. Simultaneous inhibition of A2A and A2B pathways with CD73-neutralization synergistically enhances antitumor immunity in CAF-rich tumors. Therefore, the strategic and effective targeting of both the A2B-mediated ADO-CAF-CD73 feedforward circuit and A2A-mediated immune suppression is crucial for improving therapeutic outcomes.


Assuntos
5'-Nucleotidase/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Receptor A2B de Adenosina/metabolismo , Adenosina/metabolismo , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Progressão da Doença , Sinergismo Farmacológico , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Testes de Neutralização , Transcriptoma/genética , Resultado do Tratamento , Microambiente Tumoral , Regulação para Cima
4.
Mol Cell Biochem ; 463(1-2): 1-11, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31531757

RESUMO

We reported previously that the rat submandibular gland is able to release nanovesicles capable to hydrolyse millimolar concentrations of ATP, ADP and AMP in vitro. Here, we show that rat saliva also contains nanovesicles with the ability to hydrolyse ATP. Our aim was to identify and characterize vesicular nucleotidases by using kinetic, immunological and in silico approaches. Nucleotidase activity in the absence or presence of specific inhibitors allowed us to assume the participation of NTPDase1, -2 and -3, together with ecto-5'-nucleotidase, confirmed using specific antibodies. At neutral pH, initial ATPase activity would be mostly due to NTPDase2, which was thereafter inactivated, leaving NTPDase1 and NTPDase3 to hydrolyse ATP and ADP with an efficacy ATPase/ADPase around 2. Ecto-5'nucleotidase would be mainly responsible for AMP hydrolysis and adenosine accumulation. We proposed a kinetic model for NTPDase2 as a tool to isolate and analyse the turnover of this enzyme in the presence of different ATP concentrations, including those expected in extracellular media. Our study characterizes the ectonucleotidases carried by extracellular vesicles which contribute to modulate ATP and adenosine concentrations in the oral cavity, essential players in purinergic signalling.


Assuntos
5'-Nucleotidase/metabolismo , Vesículas Extracelulares/metabolismo , Boca/metabolismo , Pirofosfatases/metabolismo , Saliva/enzimologia , Proteínas e Peptídeos Salivares/metabolismo , Transdução de Sinais , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ratos , Ratos Wistar
5.
Blood ; 135(1): 41-55, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31697823

RESUMO

To study the mechanisms of relapse in acute lymphoblastic leukemia (ALL), we performed whole-genome sequencing of 103 diagnosis-relapse-germline trios and ultra-deep sequencing of 208 serial samples in 16 patients. Relapse-specific somatic alterations were enriched in 12 genes (NR3C1, NR3C2, TP53, NT5C2, FPGS, CREBBP, MSH2, MSH6, PMS2, WHSC1, PRPS1, and PRPS2) involved in drug response. Their prevalence was 17% in very early relapse (<9 months from diagnosis), 65% in early relapse (9-36 months), and 32% in late relapse (>36 months) groups. Convergent evolution, in which multiple subclones harbor mutations in the same drug resistance gene, was observed in 6 relapses and confirmed by single-cell sequencing in 1 case. Mathematical modeling and mutational signature analysis indicated that early relapse resistance acquisition was frequently a 2-step process in which a persistent clone survived initial therapy and later acquired bona fide resistance mutations during therapy. In contrast, very early relapses arose from preexisting resistant clone(s). Two novel relapse-specific mutational signatures, one of which was caused by thiopurine treatment based on in vitro drug exposure experiments, were identified in early and late relapses but were absent from 2540 pan-cancer diagnosis samples and 129 non-ALL relapses. The novel signatures were detected in 27% of relapsed ALLs and were responsible for 46% of acquired resistance mutations in NT5C2, PRPS1, NR3C1, and TP53. These results suggest that chemotherapy-induced drug resistance mutations facilitate a subset of pediatric ALL relapses.


Assuntos
Biomarcadores Tumorais/genética , Metotrexato/uso terapêutico , Mutagênese/efeitos dos fármacos , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , 5'-Nucleotidase/genética , Antimetabólitos Antineoplásicos/uso terapêutico , Criança , Análise Mutacional de DNA , Feminino , Seguimentos , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , Receptores de Glucocorticoides/genética , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genética
6.
Arterioscler Thromb Vasc Biol ; 40(1): 61-71, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31619062

RESUMO

OBJECTIVE: CD73 is an ectonucleotidase which catalyzes the conversion of AMP (adenosine monophosphate) to adenosine. Adenosine has been shown to be anti-inflammatory and vasorelaxant. The impact of ectonucleotidases on age-dependent atherosclerosis remains unclear. Our aim was to investigate the role of CD73 in age-dependent accumulation of atherosclerosis. Approach and results: Mice doubly deficient in CD73 and ApoE (apolipoprotein E; (cd73-/-/apoE-/-) were generated, and the extent of aortic atherosclerotic plaque was compared with apoE-/- controls at 12, 20, 32, and 52 weeks. By 12 weeks of age, cd73-/-/apoE-/- mice exhibited a significant increase in plaque (1.4±0.5% of the total vessel surface versus 0.4±0.1% in apoE-/- controls, P<0.005). By 20 weeks of age, this difference disappeared (2.9±0.4% versus 3.3±0.7%). A significant reversal in phenotype emerged at 32 weeks (9.8±1.2% versus 18.3±1.4%; P<0.0001) and persisted at the 52 week timepoint (22.4±2.1% versus 37.0±2.1%; P<0.0001). The inflammatory response to aging was found to be comparable between cd73-/-/apoE-/- mice and apoE-/- controls. A reduction in lipolysis in CD73 competent mice was observed, even with similar plasma lipid levels (cd73-/-/apoE-/- versus apoE-/- at 12 weeks [16.2±0.7 versus 9.5±1.4 nmol glycerol/well], 32 weeks [24.1±1.5 versus 7.4±0.4 nmol/well], and 52 weeks [13.8±0.62 versus 12.7±2.0 nmol/well], P<0.001). CONCLUSIONS: At early time points, CD73 exerts a subtle antiatherosclerotic influence, but with age, the pattern reverses, and the presence of CD73 promoted suppression of lipid catabolism.


Assuntos
5'-Nucleotidase/genética , Aterosclerose/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA/genética , 5'-Nucleotidase/biossíntese , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo
7.
Am J Physiol Heart Circ Physiol ; 318(1): H189-H202, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31834840

RESUMO

We hypothesized that ranolazine-induced adenosine release is responsible for its beneficial effects in ischemic heart disease. Sixteen open-chest anesthetized dogs with noncritical coronary stenosis were studied at rest, during dobutamine stress, and during dobutamine stress with ranolazine. Six additional dogs without stenosis were studied only at rest. Regional myocardial function and perfusion were assessed. Coronary venous blood was drawn. Murine endothelial cells and cardiomyocytes were incubated with ranolazine and adenosine metabolic enzyme inhibitors, and adenosine levels were measured. Cardiomyocytes were also exposed to dobutamine and dobutamine with ranolazine. Modeling was employed to determine whether ranolazine can bind to an enzyme that alters adenosine stores. Ranolazine was associated with increased adenosine levels in the absence (21.7 ± 3.0 vs. 9.4 ± 2.1 ng/mL, P < 0.05) and presence of ischemia (43.1 ± 13.2 vs. 23.4 ± 5.3 ng/mL, P < 0.05). Left ventricular end-systolic wall stress decreased (49.85 ± 4.68 vs. 57.42 ± 3.73 dyn/cm2, P < 0.05) and endocardial-to-epicardial myocardial blood flow ratio tended to normalize (0.89 ± 0.08 vs. 0.76 ± 0.10, P = nonsignificant). Adenosine levels increased in cardiac endothelial cells and cardiomyocytes when incubated with ranolazine that was reversed when cytosolic-5'-nucleotidase (cN-II) was inhibited. Point mutation of cN-II aborted an increase in its specific activity by ranolazine. Similarly, adenosine levels did not increase when cardiomyocytes were incubated with dobutamine. Modeling demonstrated plausible binding of ranolazine to cN-II with a docking energy of -11.7 kcal/mol. We conclude that the anti-adrenergic and cardioprotective effects of ranolazine-induced increase in tissue adenosine levels, likely mediated by increasing cN-II activity, may contribute to its beneficial effects in ischemic heart disease.NEW & NOTEWORTHY Ranolazine is a drug used for treatment of angina pectoris in patients with ischemic heart disease. We discovered a novel mechanism by which this drug may exhibit its beneficial effects. It increases coronary venous levels of adenosine both at rest and during dobutamine-induced myocardial ischemia. Ranolazine also increases adenosine levels in endothelial cells and cardiomyocytes in vitro, by principally increasing activity of the enzyme cytosolic-5'-nucleotidase. Adenosine has well-known myocardial protective and anti-adrenergic properties that may explain, in part, ranolazine's beneficial effect in ischemic heart disease.


Assuntos
Adenosina/metabolismo , Fármacos Cardiovasculares/farmacologia , Estenose Coronária/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Ranolazina/farmacologia , 5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Animais , Sítios de Ligação , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/metabolismo , Células Cultivadas , Estenose Coronária/metabolismo , Estenose Coronária/fisiopatologia , Modelos Animais de Doenças , Cães , Hemodinâmica/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Miócitos Cardíacos/metabolismo , Ligação Proteica , Conformação Proteica , Ranolazina/química , Ranolazina/metabolismo , Relação Estrutura-Atividade , Regulação para Cima , Função Ventricular Esquerda/efeitos dos fármacos
8.
Virchows Arch ; 476(4): 569-576, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31853625

RESUMO

The immune system plays a key role in tumor surveillance and escape. Recently, CD73 has been proposed as a prognostic biomarker associated with disease-free survival and overall survival in triple negative breast cancer (TNBC). In this study, we investigated the role of both CD73 expression and stromal tumor-infiltrating lymphocytes (TILs) in predicting the pathologic response of TNBC to neoadjuvant chemotherapy (NACT). We retrospectively analyzed CD73 immunohistochemical expression and stromal TILs on 61 consecutive biopsies from patients who received standard NACT. Twenty-three patients (38%) achieved pathologic complete response (pCR). TILs were present in the majority of biopsies (93%) with percentages ranging from 2 to 80%. High TILs (≥ 50%) were found in 30% of cases, and in this group, pCR was achieved in 76.5% of cases. Levels of TILs were associated with a better pathologic response only at univariate analysis (p = 0.037). The median value of CD73 expression on tumor cells was 40%. In 32 (52.5%) basal biopsies, CD73 expression was below or equal to median value ("low CD73"). A pCR was obtained in 53% of cases with "low CD73" and in 21% with high CD73, and this was statistically different both at univariate (p = 0.011) and multivariate (p = 0.014) analysis.Our results suggest that CD73 expression better predicts the response to NACT than TILs in TNBC. Characterization of both TILs and microenvironment could be a promising approach to personalize treatment.


Assuntos
5'-Nucleotidase/metabolismo , Linfócitos do Interstício Tumoral/patologia , Terapia Neoadjuvante , Neoplasias de Mama Triplo Negativas/patologia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Feminino , Humanos , Linfócitos do Interstício Tumoral/metabolismo , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/terapia , Microambiente Tumoral/imunologia
9.
Methods Mol Biol ; 2041: 87-106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646482

RESUMO

Xenopus embryos are one of the most used animal models in developmental biology and are well suited for apprehending functions of signaling pathways during embryogenesis. To do so, it is necessary to be able to detect expression pattern of the key genes of these signaling pathways. Here we describe the whole-mount in situ hybridization technique to investigate the expression pattern of ectonucleotidases and purinergic receptors during embryonic development.


Assuntos
5'-Nucleotidase/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hibridização In Situ/métodos , Receptores Purinérgicos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , 5'-Nucleotidase/genética , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Feminino , Sondas RNA , Receptores Purinérgicos/genética , Transdução de Sinais , Proteínas de Xenopus/genética , Xenopus laevis/embriologia
10.
Methods Mol Biol ; 2041: 107-116, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646483

RESUMO

Studies on pathophysiology and the therapeutic potential of extracellular ATP and other purines represent an important and rapidly evolving field. The integral response of the cell is determined by multiple factors, including the release of endogenous ATP, co-expression of different types of nucleotide- and adenosine-selective receptors, as well as the specific makeup of ectoenzymes governing the duration and magnitude of purinergic signaling. Current findings support the presence of an extensive network of purine-converting ectoenzymes that are co-expressed to a variable extent among the mammalian tissues and share similarities in substrate specificity. Here, we describe a histochemical approach for simultaneous detection of ecto-nucleotidase and tissue-nonspecific alkaline phosphatase (TNAP) activities in the same tissue slice. Further employment of this technique for staining human palatine tonsil cryosections revealed selective distribution of the key ectoenzymes within certain tonsillar structures, including germinal centers and connective tissues (ecto-5'-nucleotidase/CD73), as well as interfollicular area (TNAP and NTPDase1/CD39).


Assuntos
5'-Nucleotidase/metabolismo , Trifosfato de Adenosina/metabolismo , Fosfatase Alcalina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Imuno-Histoquímica/métodos , Tonsila Palatina/enzimologia , Humanos
11.
Nat Med ; 26(1): 39-46, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31873309

RESUMO

Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a subset of patients with specific tumor types1,2. Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73hi macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73-/- mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies.


Assuntos
5'-Nucleotidase/metabolismo , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Glioblastoma/imunologia , Glioblastoma/terapia , Terapia de Alvo Molecular , Algoritmos , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Proteínas Ligadas por GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/diagnóstico por imagem , Glioblastoma/genética , Humanos , Imunoterapia , Linfócitos do Interstício Tumoral/imunologia , Macrófagos/metabolismo , Imagem por Ressonância Magnética , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo
12.
Int J Mol Sci ; 20(24)2019 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-31861118

RESUMO

Pseudoxanthoma elasticum is a rare disease mainly due to ABCC6 gene mutations and characterized by ectopic biomineralization and fragmentation of elastic fibers resulting in skin, cardiovascular and retinal calcifications. It has been recently described that pyrophosphate (a calcification inhibitor) deficiency could be the main cause of ectopic calcifications in this disease and in other genetic disorders associated to mutations of ENPP1 or CD73. Patients affected by Pseudoxanthoma Elasticum seem also prone to develop kidney stones originating from papillary calcifications named Randall's plaque, and to a lesser extent may be affected by nephrocalcinosis. In this narrative review, we summarize some recent discoveries relative to the pathophysiology of this mendelian disease responsible for both cardiovascular and renal papillary calcifications, and we discuss the potential implications of pyrophosphate deficiency as a promoter of vascular calcifications in kidney stone formers and in patients affected by chronic kidney disease.


Assuntos
Difosfatos/metabolismo , Cálculos Renais/metabolismo , Pseudoxantoma Elástico/metabolismo , Doenças Raras/metabolismo , Urolitíase/metabolismo , Calcificação Vascular/metabolismo , 5'-Nucleotidase/genética , Proteínas Ligadas por GPI/genética , Humanos , Cálculos Renais/complicações , Cálculos Renais/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Pseudoxantoma Elástico/complicações , Pseudoxantoma Elástico/genética , Doenças Raras/complicações , Doenças Raras/genética , Urolitíase/complicações , Urolitíase/genética , Calcificação Vascular/complicações , Calcificação Vascular/genética
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(11): 961-966, 2019 Nov.
Artigo em Chinês | MEDLINE | ID: mdl-31878990

RESUMO

Objective To investigate the levels and function status of CD4+CD25-FOXP3+ T cells (CD25- Tregs) in the peripheral blood and synovial fluid from rheumatoid arthritis (RA) patients, and their relationships with disease activity. Methods The study enrolled 60 RA patients and 69 healthy controls (HCs). Flow cytometry was used to analyze the percentage and phenotype of CD25- Tregs, and the results were analyzed by Mann-Whitey U test and Spearman correlation. Results The percentage of circulating CD25- Tregs in CD4+ cells was compared between RA patients and HCs. However, the percentage of CD25- Tregs in RA synovial fluid was significantly higher than that in the peripheral blood of RA patients and HCs. When RA patients were grouped according to their disease activity or clinical indicators, such as rheumatoid factor (RF), anti-cyclic citrullinated peptides (CCP) antibody, anti-mutated citrullinated vimentin (MCV) antibody and anti-keratin antibody (AKA), circulating CD25- Tregs percentage was not significantly different among the groups, and had no correlation with the levels of erythrocyte sedimentation rate (ESR) and C reactive protein (CRP). The expression of CD39 in CD25- Tregs in RA synovial fluid was significantly lower than that in the peripheral blood of HCs. And CD73 and TGF-ß1 expression in CD25- Tregs in RA synovial fluid were significantly lower than those in the peripheral blood of both RA patients and HCs. However, there was no significant difference in the expression of CTLA4 and IL-10 in CD25- Tregs among the groups. Conclusion The percentage of CD25- Tregs increases in RA synovial fluid. And the expression of CD39, CD73 and TGF-ß1 decrease in CD25- Tregs, suggesting that its inhibitory function may be defective, resulting in local inflammation not being effectively controlled.


Assuntos
Artrite Reumatoide/imunologia , Líquido Sinovial/citologia , Linfócitos T Reguladores/imunologia , 5'-Nucleotidase/metabolismo , Apirase/metabolismo , Estudos de Casos e Controles , Fatores de Transcrição Forkhead/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Fator de Crescimento Transformador beta1/metabolismo
14.
PLoS One ; 14(10): e0220094, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31652269

RESUMO

The success of immunotherapy treatment in oncology ushered a new modality for treating a wide variety of cancers. However, lack of effect in some patients made it imperative to identify other pathways that are exploited by cancer cells to circumvent immune surveillance, and possibly synergize immune checkpoint treatment in those cases. It has been recently recognized that adenosine levels increase significantly in the tumor microenvironment and that adenosine/adenosine receptors play a powerful role as immunosuppressive and attenuating several effector T cell functions. The two main enzymes responsible for generating adenosine in the microenvironment are the ectonucleotidases CD39 and CD73, the former utilizes both ATP and ADP and produces AMP while the latter utilizes AMP and generates adenosine. Thus, these two enzymes combined are the major source for the bulk of adenosine produced in the microenvironment. They were shown to be validated targets in oncology leading to several clinical trials that include small molecules as well as antibodies, showing positive and encouraging results in the preclinical arena. Towards the development of novel drugs to target these enzymes, we have developed a platform that can be utilized to monitor the activities of both enzymes in vitro (biochemical) as well as in cells (cell based) assays. We have developed very sensitive and homogenous assays that enabled us to monitor the activity of both enzymes and demonstrate selectivity of known inhibitors as well as monoclonal antibodies. This should speed up screening for novel inhibitors that might lead to more effective cancer therapy.


Assuntos
5'-Nucleotidase/metabolismo , Apirase/metabolismo , Membrana Celular/enzimologia , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Ligadas por GPI/metabolismo , Humanos , Células Jurkat , Solubilidade
15.
Semin Immunol ; 42: 101304, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31604539

RESUMO

Suppression of anti-tumor immunity is recognized as a critical step in the development of many types of cancers. Over the past decade, a multitude of immunosuppressive pathways occurring in the tumor microenvironment (TME) have been identified. Amongst them, the hydrolysis of extracellular ATP into adenosine by ecto-nucleotidases has been increasingly documented as new immune checkpoint pathway that can significantly impair anti-tumor immunity of multiple types of cancer. In this review, we summarize past and recent research on the ecto-nucleotidases CD39 and CD73, conducted by our group and others, that recently lead to the development and clinical testing of adenosine targeting agents for cancer immunotherapy.


Assuntos
5'-Nucleotidase/imunologia , Adenosina/imunologia , Antígenos CD/imunologia , Apirase/imunologia , Imunoterapia , Neoplasias/terapia , Animais , Humanos , Neoplasias/imunologia , Transdução de Sinais
16.
PLoS One ; 14(10): e0223892, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31634358

RESUMO

2'-Deoxyadenosine 5'-monophosphate (dAMP), a deoxyribonucleotide found in DNA, affects intestinal cell growth. The molecular mechanisms underlying gastrointestinal absorption of foreign DNA ingested along with food has hardly been investigated. The aim of this study was to investigate the mechanism underlying intestinal absorption of dAMP. The uptake of [3H]dAMP by Caco-2 cells was Na+- and pH-dependent and was inhibited by various nucleosides. In contrast, nitrobenzylthioinosine (NMBPR), an equilibrative nucleoside transporter inhibitor, showed little inhibitory effects on [3H]dAMP uptake. Additionally, human concentrative nucleoside transporter (CNT) 3, transiently expressed in COS-7 cells, mediated the uptake of [3H]dAMP. A kinetic study revealed that the Km value of CNT3-mediated uptake of dAMP (59.6 µM) was close to that of 2'-deoxyadenosine (dAdo) (56.3 µM), whereas the dAMP Vmax (15.6 pmol·mg protein-1min-1) was 500-fold lesser than the dAdo Vmax (7782 pmol·mg protein-1min-1). Further, [3H]dAMP uptake was greater in COS-7 cells expressing ecto-5'-nucleotidase/CD73 with CNT3 than in those expressing CNT3 alone. These data suggest that, although dAMP is a substrate of CNT3, it is dephosphorylated to dAdo by CD73 and is efficiently absorbed as dAdo from the intestinal lumen.


Assuntos
5'-Nucleotidase/metabolismo , Nucleotídeos de Desoxiadenina/metabolismo , Desoxiadenosinas/metabolismo , Intestinos/fisiologia , Proteínas de Membrana Transportadoras/metabolismo , Transporte Biológico , Células CACO-2 , Proteínas Ligadas por GPI/metabolismo , Humanos
17.
Int J Mol Sci ; 20(19)2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557947

RESUMO

The effector proteins secreted by a pathogen not only promote virulence and infection of the pathogen, but also trigger plant defense response. Therefore, these proteins could be used as important genetic resources for transgenic improvement of plant disease resistance. Magnaporthe oryzae systemic defense trigger 1 (MoSDT1) is an effector protein. In this study, we compared the agronomic traits and blast disease resistance between wild type (WT) and MoSDT1 overexpressing lines in rice. Under control conditions, MoSDT1 transgenic lines increased the number of tillers without affecting kernel morphology. In addition, MoSDT1 transgenic lines conferred improved blast resistance, with significant effects on the activation of callose deposition, reactive oxygen species (ROS) accumulation and cell death. On the one hand, overexpression of MoSDT1 could delay biotrophy-necrotrophy switch through regulating the expression of biotrophy-associated secreted protein 4 (BAS4) and Magnaporthe oryzaecell death inducing protein 1 (MoCDIP1), and activate plant defense response by regulating the expression of Bsr-d1, MYBS1, WRKY45, peroxidase (POD), heat shock protein 90 (HSP90), allenoxide synthase 2 (AOS2), phenylalanine ammonia lyase (PAL), pathogenesis-related protein 1a (PR1a) in rice. On the other hand, overexpression of MoSDT1 could increase the accumulation of some defense-related primary metabolites such as two aromatic amino acids (L-tyrosine and L-tryptohan), 1-aminocyclopropane carboxylic acid, which could be converted to ethylene, vanillic acid and L-saccharopine. Taken together, overexpression of MoSDT1 confers improved rice blast resistance in rice, through modulation of callose deposition, ROS accumulation, the expression of defense-related genes, and the accumulation of some primary metabolites.


Assuntos
5'-Nucleotidase/genética , Resistência à Doença/genética , Expressão Gênica , Magnaporthe/genética , Oryza/genética , Oryza/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , 5'-Nucleotidase/química , 5'-Nucleotidase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Magnaporthe/enzimologia , Oryza/enzimologia , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espécies Reativas de Oxigênio/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
BMC Cancer ; 19(1): 912, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31510956

RESUMO

BACKGROUND: Interaction between cancer cells and fibroblasts mediated by extracellular matrix metalloproteinase inducer (emmprin, CD147) is important in the invasion and proliferation of cancer cells. However, the exact mechanism of emmprin mediated stimulation of matrix metalloprotease-2 (MMP-2) production from fibroblasts has not been elucidated. Our previous studies using an inhibitory peptide against emmprin suggested the presence of a molecule on the cell membrane which forms a complex with emmprin. Here we show that CD73 expressed on fibroblasts interacts with emmprin and is a required factor for MMP-2 production in co-cultures of sarcoma cells with fibroblasts. METHODS: CD73 along with CD99 was identified by mass spectrometry analysis as an emmprin interacting molecule from a co-culture of cancer cells (epithelioid sarcoma cell line FU-EPS-1) and fibroblasts (immortalized fibroblasts cell line ST353i). MMP-2 production was measured by immunoblot and ELISA. The formation of complexes of CD73 with emmprin was confirmed by immunoprecipitation, and their co-localization in tumor cells and fibroblasts was shown by fluorescent immunostaining and proximity ligation assays. RESULTS: Stimulated MMP-2 production in co-culture of cancer cells and fibroblasts was completely suppressed by siRNA knockdown of CD73, but not by CD99 knockdown. MMP-2 production was not suppressed by CD73-specific enzyme inhibitor (APCP). However, MMP-2 production was decreased by CD73 neutralizing antibodies, suggesting that CD73-mediated suppression of MMP-2 production is non-enzymatic. In human epithelioid sarcoma tissues, emmprin was immunohistochemically detected to be mainly expressed in tumor cells, and CD73 was expressed in fibroblasts and tumor cells: emmprin and CD73 were co-localized predominantly on tumor cells. CONCLUSION: This study provides a novel insight into the role of CD73 in emmprin-mediated regulation of MMP-2 production.


Assuntos
5'-Nucleotidase/metabolismo , Basigina/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Técnicas de Cocultura , Fibroblastos , Proteínas Ligadas por GPI/metabolismo , Humanos , Imuno-Histoquímica , Espectrometria de Massas , Modelos Biológicos , Proteômica/métodos
19.
Immunol Lett ; 214: 55-64, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31479688

RESUMO

Rheumatoid arthritis (RA) is a classic inflammatory autoimmune disease. Local joint destruction and extra-articular manifestations of RA deeply compromise the life quality of the affected patients. RA immunopathogenesis depends on continuous immunogenic activation in which the purinergic system participates. The purinergic system comprises the signaling and metabolism of purines such as adenosine triphosphate (ATP) and adenosine. ATP signaling is involved in the activation and maintenance of the inflammatory state of RA through the activation of P2X7 and the production of cytokines, which orchestrate the pathogenesis of RA. The breakdown of ATP through the CD39/CD73 axis produces adenosine, which mostly inhibits the inflammatory process through activation of specific P1 receptors. Adenosine is hydrolyzed by adenosine deaminase (ADA) that interacts with other molecules playing additional roles in this disease. This review explores the release, metabolism, and the effects of binding of ATP and adenosine to their respective receptors in the context of RA, as well as their potential use as biomarkers and therapeutic targets.


Assuntos
Trifosfato de Adenosina/imunologia , Adenosina/imunologia , Artrite Reumatoide/imunologia , Transdução de Sinais/imunologia , 5'-Nucleotidase/imunologia , Animais , Apirase/imunologia , Artrite Reumatoide/patologia , Biomarcadores , Proteínas Ligadas por GPI/imunologia , Humanos , Receptores Purinérgicos P2X7/imunologia
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