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1.
Cells ; 10(9)2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34571872

RESUMO

There is increasing evidence for a link between inflammation and thrombosis. Following tissue injury, vascular endothelium becomes activated, losing its antithrombotic properties whereas inflammatory mediators build up a prothrombotic environment. Platelets are the first elements to be activated following endothelial damage; they participate in physiological haemostasis, but also in inflammatory and thrombotic events occurring in an injured tissue. While physiological haemostasis develops rapidly to prevent excessive blood loss in the endothelium activated by inflammation, hypoxia or by altered blood flow, thrombosis develops slowly. Activated platelets release the content of their granules, including ATP and ADP released from their dense granules. Ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1)/CD39 dephosphorylates ATP to ADP and to AMP, which in turn, is hydrolysed to adenosine by ecto-5'-nucleotidase (CD73). NTPDase1/CD39 has emerged has an important molecule in the vasculature and on platelet surfaces; it limits thrombotic events and contributes to maintain the antithrombotic properties of endothelium. The aim of the present review is to provide an overview of platelets as cellular elements interfacing haemostasis and inflammation, with a particular focus on the emerging role of NTPDase1/CD39 in controlling both processes.


Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Inflamação/complicações , Trombose/complicações , Animais , Humanos , Inflamação/sangue , Nucleotídeos/metabolismo , Ativação Plaquetária , Transdução de Sinais , Trombose/sangue
2.
Int J Mol Sci ; 22(18)2021 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-34575993

RESUMO

In previous studies using isolated, paced guinea pig left atria, we observed that FSCPX, known as a selective A1 adenosine receptor antagonist, paradoxically increased the direct negative inotropic response to A1 adenosine receptor agonists (determined using concentration/effect (E/c) curves) if NBTI, a nucleoside transport inhibitor, was present. Based on mathematical modeling, we hypothesized that FSCPX blunted the cardiac interstitial adenosine accumulation in response to nucleoside transport blockade, probably by inhibiting CD39 and/or CD73, which are the two main enzymes of the interstitial adenosine production in the heart. The goal of the present study was to test this hypothesis. In vitro CD39 and CD73 inhibitor assays were carried out; furthermore, E/c curves were constructed in isolated, paced rat and guinea pig left atria using adenosine, CHA and CPA (two A1 adenosine receptor agonists), FSCPX, NBTI and NBMPR (two nucleoside transport inhibitors), and PSB-12379 (a CD73 inhibitor), measuring the contractile force. We found that FSCPX did not show any inhibitory effect during the in vitro enzyme assays. However, we successfully reproduced the paradox effect of FSCPX in the rat model, mimicked the "paradox" effect of FSCPX with PSB-12379, and demonstrated the lipophilia of FSCPX, which could explain the negative outcome of inhibitor assays with CD39 and CD73 dissolved in a water-based solution. Taken together, these three pieces of indirect evidence are strong enough to indicate that FSCPX possesses an additional action besides the A1 adenosine receptor antagonism, which action may be the inhibition of an ectonucleotidase. Incidentally, we found that POM-1 inhibited CD73, in addition to CD39.


Assuntos
5'-Nucleotidase/antagonistas & inibidores , Antagonistas do Receptor A1 de Adenosina/farmacologia , Apirase/antagonistas & inibidores , Receptor A1 de Adenosina/metabolismo , Xantinas/farmacologia , 5'-Nucleotidase/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Cobaias , Masculino , Ratos , Ratos Wistar
3.
Chem Commun (Camb) ; 57(74): 9398-9401, 2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34528964

RESUMO

A tumor cell membrane-camouflaged therapeutic system was fabricated to eliminate tumors by embedding apyrase and glucose oxidase (GOx) into zeolitic imidazolate framework-8 (ZIF-8) nanoparticles for tumor-targeted metabolic therapy. Experimental results demonstrated that these functional nanoparticles could disturb the energy supply of tumor cells by depleting ATP and glucose and efficiently induce tumor cell death.


Assuntos
Apirase/metabolismo , Materiais Biomiméticos/metabolismo , Glucose Oxidase/metabolismo , Estruturas Metalorgânicas/metabolismo , Nanopartículas/metabolismo , Neoplasias/metabolismo , Trifosfato de Adenosina/metabolismo , Materiais Biomiméticos/química , Morte Celular/efeitos dos fármacos , Glucose/metabolismo , Humanos , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Nanopartículas/química , Neoplasias/tratamento farmacológico , Neoplasias/patologia
4.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576057

RESUMO

Stomatal regulation is crucial to reduce water consumption under drought conditions. Extracellular ATP (eATP) serves as a signaling agent in stomatal regulation; however, it is less known whether the eATP mediation of stomatal aperture is linked to apyrases (APYs), the principal enzymes that control the concentration of eATP. To clarify the role of APYs in stomatal control, PeAPY1 and PeAPY2 were isolated from Populus euphratica and transferred into Arabidopsis. Compared with the wild-type Arabidopsis and loss-of-function mutants (Atapy1 and Atapy2), PeAPY1- and PeAPY2-transgenic plants decreased stomatal aperture under mannitol treatment (200 mM, 2 h) and reduced water loss during air exposure (90 min). The role of apyrase in stomatal regulation resulted from its control in eATP-regulated stomatal movements and increased stomatal sensitivity to ABA. The bi-phasic dose-responses to applied nucleotides, i.e., the low ATP (0.3-1.0 mM)-promoted opening and high ATP (>2.0 mM)-promoted closure, were both restricted by P. euphratica apyrases. It is noteworthy that eATP at a low concentration (0.3 mM) counteracted ABA action in the regulation of stomatal aperture, while overexpression of PeAPY1 or PeAPY2 effectively diminished eATP promotion in opening, and consequently enhanced ABA action in closure. We postulate a speculative model of apyrase signaling in eATP- and ABA-regulated stomatal movements under drought.


Assuntos
Apirase/genética , Arabidopsis/genética , Plantas Geneticamente Modificadas/genética , Populus/enzimologia , Arabidopsis/crescimento & desenvolvimento , Secas , Regulação da Expressão Gênica de Plantas/genética , Estômatos de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Populus/genética , Estresse Fisiológico/genética
5.
Front Immunol ; 12: 668884, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34504486

RESUMO

Although the human immune response to cancer is naturally potent, it can be severely disrupted as a result of an immunosuppressive tumor microenvironment. Infiltrating regulatory T lymphocytes contribute to this immunosuppression by inhibiting proliferation of cytotoxic CD8+ T lymphocytes, which are key to an effective anti-cancer immune response. Other important contributory factors are thought to include metabolic stress caused by the local nutrient deprivation common to many solid tumors. Interleukin-33 (IL-33), an alarmin released in reaction to cell damage, and sphingosine-1-phosphate (S1P) are known to control cell positioning and differentiation of T lymphocytes. In an in vitro model of nutrient deprivation, we investigated the influence of IL-33 and S1P receptor 4 (S1P4) on the differentiation and migration of human CD8+ T lymphocytes. Serum starvation of CD8+ T lymphocytes induced a subset of CD8Low and IL-33 receptor-positive (ST2L+) cells characterized by enhanced expression of the regulatory T cell markers CD38 and CD39. Both S1P1 and S1P4 were transcriptionally regulated after stimulation with IL-33. Moreover, expression of the chemokine receptor CXCR4 was increased in CD8+ T lymphocytes treated with the selective S1P4 receptor agonist CYM50308. We conclude that nutrient deprivation promotes CD8Low T lymphocytes, contributing to an immunosuppressive microenvironment and a poor anti-cancer immune response by limiting cytotoxic effector functions. Our results suggest that S1P4 signaling modulation may be a promising target for anti-CXCR4 cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Receptores CXCR4/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Microambiente Tumoral , ADP-Ribosil Ciclase 1/metabolismo , Apirase/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Movimento Celular , Células Cultivadas , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1/metabolismo , Interleucina-33/farmacologia , Linfócitos do Interstício Tumoral/imunologia , Glicoproteínas de Membrana/metabolismo , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR4/genética , Transdução de Sinais , Receptores de Esfingosina-1-Fosfato/agonistas , Receptores de Esfingosina-1-Fosfato/genética , Microambiente Tumoral/imunologia , Regulação para Cima
6.
Immunity ; 54(9): 2024-2041.e8, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34473957

RESUMO

Sepsis results in elevated adenosine in circulation. Extracellular adenosine triggers immunosuppressive signaling via the A2a receptor (A2aR). Sepsis survivors develop persistent immunosuppression with increased risk of recurrent infections. We utilized the cecal ligation and puncture (CLP) model of sepsis and subsequent infection to assess the role of adenosine in post-sepsis immune suppression. A2aR-deficient mice showed improved resistance to post-sepsis infections. Sepsis expanded a subset of CD39hi B cells and elevated extracellular adenosine, which was absent in mice lacking CD39-expressing B cells. Sepsis-surviving B cell-deficient mice were more resistant to secondary infections. Mechanistically, metabolic reprogramming of septic B cells increased production of ATP, which was converted into adenosine by CD39 on plasmablasts. Adenosine signaling via A2aR impaired macrophage bactericidal activity and enhanced interleukin-10 production. Septic individuals exhibited expanded CD39hi plasmablasts and adenosine accumulation. Our study reveals CD39hi plasmablasts and adenosine as important drivers of sepsis-induced immunosuppression with relevance in human disease.


Assuntos
Adenosina/imunologia , Antígenos CD/imunologia , Apirase/imunologia , Tolerância Imunológica/imunologia , Macrófagos/imunologia , Plasmócitos/imunologia , Sepse/imunologia , Adenosina/metabolismo , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Reprogramação Celular/imunologia , Macrófagos/metabolismo , Camundongos , Plasmócitos/metabolismo , Receptor A2A de Adenosina/imunologia , Receptor A2A de Adenosina/metabolismo , Sepse/metabolismo
7.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360833

RESUMO

CD39 is an enzyme which is responsible, together with CD73, for a cascade converting adenosine triphosphate into adenosine diphosphate and cyclic adenosine monophosphate, ultimately leading to the release of an immunosuppressive form of adenosine in the tumor microenvironment. Here, we first review the environmental and genetic factors shaping CD39 expression. Second, we report CD39 functions in the T cell compartment, highlighting its role in regulatory T cells, conventional CD4+ T cells and CD8+ T cells. Finally, we compile a list of studies, from preclinical models to clinical trials, which have made essential contributions to the discovery of novel combinatorial approaches in the treatment of cancer.


Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/fisiologia , Apirase/genética , Apirase/imunologia , Apirase/fisiologia , Regulação da Expressão Gênica , Humanos , Linfócitos T/imunologia
8.
Cells ; 10(7)2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34359872

RESUMO

Ovarian cancer is the most lethal gynecological malignancy, with serous histotype as the most prevalent epithelial ovarian cancer (EOC). Peritoneal ascites is a frequent comorbidity in advanced EOC. EOC-associated ascites provide a reliable sampling source for studying lymphocytes directly from tumor environment. Herein, we carried out flow cytometry-based analysis to readdress issues on NK and T lymphocyte subsets in women with advanced EOC, additionally evaluating phenotypic modulation of their intracellular pathways involved in interleukin (IL)-2 and IL-15 signaling. Results depicted ascites as an inflammatory and immunosuppressive environment, presenting significantly (p < 0.0001) higher amounts of IL-6 and IL-10 than in the patients' blood, as well as significantly (p < 0.05) increased expression of checkpoint inhibitory receptors (programmed death protein-1, PD-1) and ectonucleotidase (CD39) on T lymphocytes. However, NK lymphocytes from EOC-associated ascites showed higher (p < 0.05) pS6 phosphorylation compared with NK from blood. Additionally, in vitro treatment of lymphocytes with IL-2 or IL-15 elicited significantly (p < 0.001) phosphorylation of the STAT5 protein in NK, CD3 and CD8 lymphocytes, both from blood and ascites. EOC-associated ascites had a significantly (p < 0.0001) higher proportion of NK CD56bright lymphocytes than blood, which, in addition, were more responsive (p < 0.05) to stimulation by IL-2 than CD56dim NK. EOC-associated ascites allow studies on lymphocyte phenotype modulation in the tumor environment, where inflammatory profile contrasts with the presence of immunosuppressive elements and development of cellular self-regulating mechanisms.


Assuntos
Ascite/imunologia , Antígeno CD56/imunologia , Cistadenocarcinoma Seroso/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Ovarianas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apirase/genética , Apirase/imunologia , Ascite/genética , Ascite/patologia , Antígeno CD56/genética , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunofenotipagem , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-15/genética , Interleucina-15/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células K562 , Células Matadoras Naturais/patologia , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia
9.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360901

RESUMO

The oxidative properties of nanomaterials arouse legitimate concerns about oxidative damage in biological systems. On the other hand, the undisputable benefits of nanomaterials promote them for biomedical applications; thus, the strategies to reduce oxidative potential are urgently needed. We aimed at analysis of nitrogen-containing carbon quantum dots (N-CQDs) in terms of their biocompatibility and internalization by different cells. Surprisingly, N-CQD uptake does not contribute to the increased oxidative stress inside cells and lacks cytotoxic influence even at high concentrations, primarily through protein corona formation. We proved experimentally that the protein coating effectively limits the oxidative capacity of N-CQDs. Thus, N-CQDs served as an immobilization support for three different enzymes with the potential to be used as therapeutics. Various kinetic parameters of immobilized enzymes were analyzed. Regardless of the enzyme structure and type of reaction catalyzed, adsorption on the nanocarrier resulted in increased catalytic efficiency. The enzymatic-protein-to-nanomaterial ratio is the pivotal factor determining the course of kinetic parameter changes that can be tailored for enzyme application. We conclude that the above properties of N-CQDs make them an ideal support for enzymatic drugs required for multiple biomedical applications, including personalized medical therapies.


Assuntos
Biocatálise , Carbono/química , Carbono/farmacologia , Nitrogênio/química , Nitrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Coroa de Proteína/metabolismo , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Células A549 , Animais , Apirase/química , Apirase/farmacologia , Catalase/química , Catalase/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Microambiente Celular/efeitos dos fármacos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HeLa , Humanos , Ratos , Espécies Reativas de Oxigênio/metabolismo , beta-Galactosidase/química , beta-Galactosidase/farmacologia
10.
Life Sci ; 282: 119826, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34265363

RESUMO

The immunosuppressive tumor microenvironment has been implicated in attenuating anti-tumoral immune responses and tumor growth in various cancers. Inhibitory immune checkpoints have been introduced as the primary culprits for developing the immunosuppressive tumor microenvironment. Therefore, a better understanding of the cross-talk between inhibitory immune checkpoints in the tumor microenvironment can pave the way for introducing novel approaches for treating affected patients. Growing evidence indicates that CD39 and CD73, as novel checkpoints, can transform adenosine triphosphate (ATP)-mediated pro-inflammatory tumor microenvironment into an adenosine-mediated immunosuppressive one via the purinergic signaling pathway. Indeed, enzymatic processes of CD39 and CD73 have crucial roles in adjusting the extent, intensity, and chemical properties of purinergic signals. This study aims to review the biological function of CD39 and CD73 and shed light on their significance in regulating anti-tumoral immune responses in various cancers.


Assuntos
5'-Nucleotidase/imunologia , Apirase/imunologia , Tolerância Imunológica , Proteínas de Neoplasias/imunologia , Neoplasias/imunologia , Transdução de Sinais/imunologia , Microambiente Tumoral/imunologia , Proteínas Ligadas por GPI/imunologia , Humanos
11.
Int J Mol Sci ; 22(14)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34299098

RESUMO

BACKGROUND: This study was designed to investigate the effect of cluster differentiation (CD)39 and CD73 inhibitors on the expresion of tumour-associated macrophages (TAMs), M1- versus M2-tumour phenotypes in mice with colon cancer. METHODS: An in vivo study of co-culture with colon cancer cells and immune cells from the bone marrow (BM) of mice was performed. After the confirmation of the effect of polyoxotungstate (POM-1) as an inhibitor of CD39 on TAMs, the mice were randomly divided into a control group without POM-1 and a study group with POM-1, respectively, after subcutaneous injection of CT26 cells. On day 14 after the injection, the mice were sacrificed, and TAMs were evaluated using fluorescence-activated cell sorting. RESULTS: In the in vivo study, the co-culture with POM-1 significantly increased the apoptosis of CT26 cells. The cell population from the co-culture with POM-1 showed significant increases in the expression of CD11b+ for myeloid cells, lymphocyte antigen 6 complex, locus C (Ly6C+) for monocytes, M1-tumour phenotypes from TAMs, and F4/80+ for macrophages. In the in vivo study, tumour growth in the study group with POM-1 was significantly limited, compared with the control group without POM-1. The expressions of Ly6C+ and major histocompatibility complex class II+ for M1-tumour phenotypes from TAMs on F4/80+ from the tumour tissue in the study group had significantly higher values compared with the control group. CONCLUSION: The inhibition of CD39 with POM-1 prevented the growth of colon cancer in mice, and it was associated with the increased expression of M1-tumour phenotypes from TAMs in the cancer tissue.


Assuntos
Apirase/antagonistas & inibidores , Neoplasias do Colo/prevenção & controle , Polímeros/farmacologia , Macrófagos Associados a Tumor/efeitos dos fármacos , Compostos de Tungstênio/farmacologia , Animais , Antígenos CD , Apoptose , Proliferação de Células , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Prognóstico , Células Tumorais Cultivadas , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
12.
FASEB J ; 35(7): e21684, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34159634

RESUMO

Adenine nucleotides represent crucial immunomodulators in the extracellular environment. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thus promoting an anti-inflammatory milieu induced by the "adenosine halo". AMPD2 intracellularly mediates AMP deamination to IMP, thereby both enhancing the degradation of inflammatory ATP and reducing the formation of anti-inflammatory adenosine. Here, we show that this enzyme is expressed on the surface of human immune cells and its predominance may modify inflammatory states by altering the extracellular milieu. Surface AMPD2 (eAMPD2) expression on monocytes was verified by immunoblot, surface biotinylation, mass spectrometry, and immunofluorescence microscopy. Flow cytometry revealed enhanced monocytic eAMPD2 expression after TLR stimulation. PBMCs from patients with rheumatoid arthritis displayed significantly higher levels of eAMPD2 expression compared with healthy controls. Furthermore, the product of AMPD2-IMP-exerted anti-inflammatory effects, while the levels of extracellular adenosine were not impaired by an increased eAMPD2 expression. In summary, our study identifies eAMPD2 as a novel regulator of the extracellular ATP-adenosine balance adding to the immunomodulatory CD39-CD73 system.


Assuntos
5'-Nucleotidase/metabolismo , AMP Desaminase/metabolismo , Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Leucócitos/metabolismo , Apirase , Células Cultivadas , Proteínas Ligadas por GPI/metabolismo , Humanos
13.
Nat Med ; 27(7): 1212-1222, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34183837

RESUMO

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.


Assuntos
Trifosfato de Adenosina/metabolismo , Apirase/metabolismo , Doenças Inflamatórias Intestinais/terapia , Probióticos/uso terapêutico , Receptores Purinérgicos P2Y2/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Apirase/genética , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Disbiose/prevenção & controle , Feminino , Fibrose/prevenção & controle , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Humanos , Doenças Inflamatórias Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Purinérgicos P2Y2/genética , Saccharomyces cerevisiae/genética
14.
Front Immunol ; 12: 687296, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34177939

RESUMO

Background: Targeting immune checkpoints for HIV treatment potentially provides a double benefit resulting from the ability to restore viral-specific CD8+ T-cell functions and enhance HIV production from reservoir cells. Despite promising pre-clinical data, PD-1 blockade alone in HIV-1-infected patients with advanced cancer has shown limited benefits in controlling HIV, suggesting the need for additional targets beyond PD-1. CD39 and PD-1 are highly co-expressed on CD8+ T cells in HIV-1 infection. However, the characteristics of CD39 and PD-1 dual-positive CD8+ T-cell subsets in chronic HIV-1 infection remain poorly understood. Methods: This study enrolled 72 HIV-1-infected patients, including 40 treatment naïve and 32 ART patients. A total of 11 healthy individuals were included as controls. Different subsets of CD8+ T cells defined by CD39 and/or PD-1 expression were studied by flow cytometry. The relationships between the frequencies of the different subsets and parameters indicating HIV-1 disease progression were analyzed. Functional (i.e., cytokine secretion, viral inhibition) assays were performed to evaluate the impact of the blockade of adenosine and/or PD-1 signaling on CD8+ T cells. Results: The proportions of PD-1+, CD39+, and PD-1+CD39+ CD8+ T cells were significantly increased in treatment naïve patients but were partially lowered in patients on antiretroviral therapy. In treatment naïve patients, the proportions of PD-1+CD39+ CD8+ T cells were negatively correlated with CD4+ T-cell counts and the CD4/CD8 ratio, and were positively correlated with viral load. CD39+CD8+ T cells expressed high levels of the A2A adenosine receptor and were more sensitive to 2-chloroadenosine-mediated functional inhibition than their CD39- counterparts. In vitro, a combination of blocking CD39/adenosine and PD-1 signaling showed a synergic effect in restoring CD8+ T-cell function, as evidenced by enhanced abilities to secrete functional cytokines and to kill autologous reservoir cells. Conclusion: In patients with chronic HIV-1 infection there are increased frequencies of PD-1+, CD39+, and PD-1+CD39+ CD8+ T cells. In treatment naïve patients, the frequencies of PD-1+CD39+ CD8+ T cells are negatively correlated with CD4+ T-cell counts and the CD4/CD8 ratio and positively correlated with viral load. Combined blockade of CD39/adenosine and PD-1 signaling in vitro may exert a synergistic effect in restoring CD8+ T-cell function in HIV-1-infected patients.


Assuntos
Adenosina/metabolismo , Fármacos Anti-HIV/farmacologia , Apirase/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Adulto , Apirase/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Estudos de Casos e Controles , Células Cultivadas , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/patogenicidade , Humanos , Masculino , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Carga Viral , Adulto Jovem
15.
Eur J Pharmacol ; 905: 174198, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34033815

RESUMO

CD39 is associated with diverse physiological and pathological processes, including cell proliferation and differentiation. Adenosine triphosphate (ATP) is hydrolysed to adenosine by different enzymes including ecto-nucleoside triphosphate diphosphohydrolase-1/ENTPD1 (CD39) and ecto-5'-nucleotidase (CD73), regulating many physiological and pathological processes in various diseases, but these changes and functions in alcoholic liver disease are generally unknown. In this study, an alcoholic liver disease model in vivo was induced by ethanol plus carbon tetrachloride(CCl4) administered to C57BL/6 mice, who were the intraperitoneally injected with the CD39 inhibitor sodium polyoxotungstate (POM1) or colchicine from the 5th week to the 8th week. Meanwhile, hepatic stellate cells were stimulated by acetaldehyde to replicate alcoholic liver fibrosis models in vitro. Exogenous ATP and POM1 were added in turn to the culture system. Pharmacological blockade of CD39 largely prevents liver damage and collagen deposition. We found that blockade or silencing of CD39 prevented acetaldehyde-induced proliferation of HSC-T6 cells and the expression of fibrogenic factors. Moreover, blockade or silencing of CD39 could block the activation of the adenosine A2A and adenosine A2B receptors and the TGF-ß/Smad3 pathway, which are essential events in HSC activation. Thus, blockade of CD39 to inhibit the transduction of ATP to adenosine may prevent HSC activation, alleviating alcoholic hepatic fibrosis. The findings from this study suggest ATP-adenosine signalling is a novel therapeutic and preventive target for alcoholic liver disease.


Assuntos
Trifosfato de Adenosina/metabolismo , Adenosina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Células Estreladas do Fígado/metabolismo , Hepatopatias Alcoólicas/etiologia , Hepatopatias Alcoólicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Acetaldeído/toxicidade , Animais , Antígenos CD/genética , Apirase/antagonistas & inibidores , Apirase/genética , Tetracloreto de Carbono/toxicidade , Colchicina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Etanol/toxicidade , Técnicas de Silenciamento de Genes , Humanos , Hepatopatias Alcoólicas/patologia , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Ratos , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Compostos de Tungstênio/farmacologia
16.
FASEB J ; 35(5): e21509, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33813781

RESUMO

Extracellular adenosine plays important roles in modulating the immune responses. We have previously demonstrated that infection of dendritic cells (DC) by Leishmania amazonensis leads to increased expression of CD39 and CD73 and to the selective activation of the low affinity A2B receptors (A2B R), which contributes to DC inhibition, without involvement of the high affinity A2A R. To understand this apparent paradox, we now characterized the alterations of both adenosine receptors in infected cells. With this aim, bone marrow-derived DC from C57BL/6J mice were infected with metacyclic promastigotes of L. amazonensis. Fluorescence microscopy revealed that L. amazonensis infection stimulates the recruitment of A2B R, but not of A2A R, to the surface of infected DC, without altering the amount of mRNA or the total A2B R density, an effect dependent on lipophosphoglycan (LPG). Log-phase promastigotes or axenic amastigotes of L. amazonensis do not stimulate A2B R recruitment. A2B R clusters are localized in caveolin-rich lipid rafts and the disruption of these membrane domains impairs A2B R recruitment and activation. More importantly, our results show that A2B R co-localize with CD39 and CD73 forming a "purinergic cluster" that allows for the production of extracellular adenosine in close proximity with these receptors. We conclude that A2B R activation by locally produced adenosine constitutes an elegant and powerful evasion mechanism used by L. amazonensis to down-modulate the DC activation.


Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Caveolina 1/metabolismo , Células Dendríticas/imunologia , Leishmaniose/imunologia , Microdomínios da Membrana/imunologia , Receptor A2B de Adenosina/metabolismo , Animais , Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Células Dendríticas/patologia , Imunidade , Imunomodulação , Leishmania/imunologia , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Leishmaniose/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Masculino , Microdomínios da Membrana/parasitologia , Microdomínios da Membrana/patologia , Camundongos , Camundongos Endogâmicos C57BL
17.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33807069

RESUMO

Studies implicating an important role for apyrase (NTPDase) enzymes in plant growth and development began appearing in the literature more than three decades ago. After early studies primarily in potato, Arabidopsis and legumes, especially important discoveries that advanced an understanding of the biochemistry, structure and function of these enzymes have been published in the last half-dozen years, revealing that they carry out key functions in diverse other plants. These recent discoveries about plant apyrases include, among others, novel findings on its crystal structures, its biochemistry, its roles in plant stress responses and its induction of major changes in gene expression when its expression is suppressed or enhanced. This review will describe and discuss these recent advances and the major questions about plant apyrases that remain unanswered.


Assuntos
Apirase/química , Apirase/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Apirase/antagonistas & inibidores , Apirase/genética , Domínio Catalítico , Fenômenos Químicos , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade
18.
Cancer Immunol Immunother ; 70(12): 3461-3475, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33885944

RESUMO

Mucosal-associated invariant T (MAIT) cells are unconventional T cells recognizing microbial metabolites, presented by the invariant MR1 protein. Upon activation, MAIT cells rapidly secrete cytokines and exert cytotoxic functions, and may thus be highly relevant also in tumor immunity. MAIT cells accumulate in colon tumors, but in contrast to other cytotoxic T cell subsets, their presence in tumors has been associated with worse patient outcome. Here we investigated if exhaustion may contribute to reduced anti-tumor immunity by MAIT cells. Freshly isolated lymphocytes from colon tumors, unaffected tissue and blood from the same patients were analyzed by flow cytometry to detect MAIT cells with effector functions that are relevant for tumor immunity, and their expression of inhibitory receptors and other exhaustion markers. Our studies show that MAIT cells with a PD-1highTim-3+CD39+ terminally exhausted phenotype and an increased proliferation accumulate in colon tumors. The exhausted MAIT cells have reduced polyfunctionality with regard to production of important anti-tumor effector molecules, and blocking antibodies to PD-1 partly improved activation of tumor-infiltrating MAIT cells in vitro. We conclude that the tumor microenvironment leads to exhaustion not only of conventional T cells, but also MAIT cells, and that checkpoint blockade therapy may be useful also to reinvigorate tumor-infiltrating MAIT cells.


Assuntos
Neoplasias do Colo/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/imunologia , Apirase/imunologia , Biomarcadores/metabolismo , Proliferação de Células/fisiologia , Citocinas/imunologia , Feminino , Receptor Celular 2 do Vírus da Hepatite A/imunologia , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptor de Morte Celular Programada 1/imunologia , Microambiente Tumoral/imunologia
19.
Int J Cancer ; 149(2): 327-336, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33675538

RESUMO

Gene expression profiling can be used for predicting survival in multiple myeloma (MM) and identifying patients who will benefit from particular types of therapy. Some germline single nucleotide polymorphisms (SNPs) act as expression quantitative trait loci (eQTLs) showing strong associations with gene expression levels. We performed an association study to test whether eQTLs of genes reported to be associated with prognosis of MM patients are directly associated with measures of adverse outcome. Using the genotype-tissue expression portal, we identified a total of 16 candidate genes with at least one eQTL SNP associated with their expression with P < 10-7 either in EBV-transformed B-lymphocytes or whole blood. We genotyped the resulting 22 SNPs in 1327 MM cases from the International Multiple Myeloma rESEarch (IMMEnSE) consortium and examined their association with overall survival (OS) and progression-free survival (PFS), adjusting for age, sex, country of origin and disease stage. Three polymorphisms in two genes (TBRG4-rs1992292, TBRG4-rs2287535 and ENTPD1-rs2153913) showed associations with OS at P < .05, with the former two also associated with PFS. The associations of two polymorphisms in TBRG4 with OS were replicated in 1277 MM cases from the International Lymphoma Epidemiology (InterLymph) Consortium. A meta-analysis of the data from IMMEnSE and InterLymph (2579 cases) showed that TBRG4-rs1992292 is associated with OS (hazard ratio = 1.14, 95% confidence interval 1.04-1.26, P = .007). In conclusion, we found biologically a plausible association between a SNP in TBRG4 and OS of MM patients.


Assuntos
Apirase/genética , Perfilação da Expressão Gênica/métodos , Proteínas Mitocondriais/genética , Mieloma Múltiplo/mortalidade , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Proteínas de Ligação a RNA/genética , Idoso , Feminino , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/genética , Análise de Sobrevida
20.
Life Sci ; 277: 119421, 2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-33785337

RESUMO

Diabetes mellitus (DM) is a metabolic disorder characterized by a chronic hyperglycemia state, increased oxidative stress parameters, and inflammatory processes. AIMS: To evaluate the effect of caffeic acid (CA) on ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and adenosine deaminase (ADA) enzymatic activity and expression of the A2A receptor of the purinergic system, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymatic activity and expression of the α7nAChR receptor of the cholinergic system as well as inflammatory and oxidative parameters in diabetic rats. METHODS: Diabetes was induced by a single dose intraperitoneally of streptozotocin (STZ, 55 mg/kg). Animals were divided into six groups (n = 10): control/oil; control/CA 10 mg/kg; control/CA 50 mg/kg; diabetic/oil; diabetic/CA 10 mg/kg; and diabetic/CA 50 mg/kg treated for thirty days by gavage. RESULTS: CA treatment reduced ATP and ADP hydrolysis (lymphocytes) and ATP levels (serum), and reversed the increase in ADA and AChE (lymphocytes), BuChE (serum), and myeloperoxidase (MPO, plasma) activities in diabetic rats. CA treatment did not attenuate the increase in IL-1ß and IL-6 gene expression (lymphocytes) in the diabetic state; however, it increased IL-10 and A2A gene expression, regardless of the animals' condition (healthy or diabetic), and α7nAChR gene expression. Additionally, CA attenuated the increase in oxidative stress markers and reversed the decrease in antioxidant parameters of diabetic animals. CONCLUSION: Overall, our findings indicated that CA treatment positively modulated purinergic and cholinergic enzyme activities and receptor expression, and improved oxi-inflammatory parameters, thus suggesting that this phenolic acid could improve redox homeostasis dysregulation and purinergic and cholinergic signaling in the diabetic state.


Assuntos
Acetilcolinesterase/metabolismo , Butirilcolinesterase/metabolismo , Ácidos Cafeicos/farmacologia , Diabetes Mellitus Experimental/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Acetilcolinesterase/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antioxidantes/farmacologia , Apirase/genética , Apirase/metabolismo , Butirilcolinesterase/genética , Citocinas/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Linfócitos/patologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peroxidase/metabolismo , Ratos , Ratos Wistar , Receptor Nicotínico de Acetilcolina alfa7/genética , Receptor Nicotínico de Acetilcolina alfa7/metabolismo
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