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1.
Sarcoidosis Vasc Diffuse Lung Dis ; 37(2): 225-230, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33093787

RESUMO

Objective: Immunoglobulin G4-related disease (IgG4-RD) is a recently described systemic disorder. Pleural effusion is considered an uncommon manifestation of the disease. We describe a case series of patients with IgG4-RD and clinically significant pleural effusions. Methods: A retrospective analysis of patients with histologically proven IgG4-RD treated for pleural effusion in our clinic. Results: We identified 4 male patients with pleural effusion caused by IgG4-RD. The effusions were lymphocytic exudates, with especially high protein concentrations. All patients had hyperglobulinemia, elevated serum immunoglobulin G (IgG) levels and elevated levels subclasses IgG1 and IgG4. In two patients, levels of adenosine deaminase (ADA) were measured in the effusion and were elevated (309 and 108 IU/L). Tuberculosis was excluded in both cases by pleural biopsy. Involvement of other organs by IgG4-RD was the rule, especially thoracic lymphadenopathy which was prominent in all patients. In all cases, effusion responded to corticosteroids therapy. One patient developed radiological findings compatible with rounded atelectasis during remission. Conclusions: IgG4-RD may cause an ADA-positive, lymphocytic exudate with a high protein concentration, characteristics resembling tuberculous effusion. Thoracic lymphadenopathy, hyperglobulinemia, and an increased total IgG, IgG1, IgG4 may suggest the diagnosis. Not previously described, IgG4-RD pleural inflammation may result in rounded atelectasis. (Sarcoidosis Vasc Diffuse Lung Dis 2020; 37 (2): 225-230).


Assuntos
Adenosina Desaminase/metabolismo , Doença Relacionada a Imunoglobulina G4/enzimologia , Linfócitos/enzimologia , Derrame Pleural/enzimologia , Corticosteroides/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Biópsia , Diagnóstico Diferencial , Humanos , Doença Relacionada a Imunoglobulina G4/diagnóstico , Doença Relacionada a Imunoglobulina G4/tratamento farmacológico , Doença Relacionada a Imunoglobulina G4/imunologia , Contagem de Linfócitos , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Masculino , Derrame Pleural/diagnóstico , Derrame Pleural/tratamento farmacológico , Derrame Pleural/imunologia , Valor Preditivo dos Testes , Estudos Retrospectivos , Tomografia Computadorizada por Raios X , Resultado do Tratamento
2.
Nat Chem Biol ; 16(7): 731-739, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32393898

RESUMO

Glucose is catabolized by two fundamental pathways, glycolysis to make ATP and the oxidative pentose phosphate pathway to make reduced nicotinamide adenine dinucleotide phosphate (NADPH). The first step of the oxidative pentose phosphate pathway is catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PD). Here we develop metabolite reporter and deuterium tracer assays to monitor cellular G6PD activity. Using these, we show that the most widely cited G6PD antagonist, dehydroepiandosterone, does not robustly inhibit G6PD in cells. We then identify a small molecule (G6PDi-1) that more effectively inhibits G6PD. Across a range of cultured cells, G6PDi-1 depletes NADPH most strongly in lymphocytes. In T cells but not macrophages, G6PDi-1 markedly decreases inflammatory cytokine production. In neutrophils, it suppresses respiratory burst. Thus, we provide a cell-active small molecule tool for oxidative pentose phosphate pathway inhibition, and use it to identify G6PD as a pharmacological target for modulating immune response.


Assuntos
Inibidores Enzimáticos/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Animais , Linhagem Celular , Desidroepiandrosterona/farmacologia , Relação Dose-Resposta a Droga , Ensaios Enzimáticos , Glucose/metabolismo , Glucosefosfato Desidrogenase/imunologia , Glucosefosfato Desidrogenase/metabolismo , Glicólise/imunologia , Células HCT116 , Células Hep G2 , Humanos , Imunidade Inata , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/citologia , Linfócitos/enzimologia , Linfócitos/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/enzimologia , Macrófagos/imunologia , NADP/antagonistas & inibidores , NADP/metabolismo , Neutrófilos/citologia , Neutrófilos/enzimologia , Neutrófilos/imunologia , Via de Pentose Fosfato/imunologia
3.
Biochimie ; 174: 34-43, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32315661

RESUMO

The nuclease activity of deoxyribonuclease 1 (DNase I) is regulated by alternative splicing (AS) of its mRNA. The aim of this study was to define the ability of a splice-switching oligonucleotide (SSO) that base-paired with DNase I pre-mRNA to induce AS and inhibit nuclease activity in human T, B and NK lymphocytes. The SSO for DNase I could significantly downregulate the expression of full-length active DNase I and upregulate a truncated splice variant with a deleted exon 4. Such an induction of AS resulted in inhibition of nuclease activity and slowed apoptosis progression in anti-CD95/FAS stimulated lymphocytes. These results should facilitate further investigations of apoptosis regulation in lymphocytes and demonstrate that SSOs for DNase I are promising cytoprotective agents.


Assuntos
Apoptose , Desoxirribonuclease I/antagonistas & inibidores , Linfócitos/citologia , Oligonucleotídeos/farmacologia , Adolescente , Adulto , Processamento Alternativo , Sobrevivência Celular , Desoxirribonuclease I/metabolismo , Voluntários Saudáveis , Humanos , Linfócitos/enzimologia , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Adulto Jovem
4.
Nature ; 580(7801): 130-135, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238926

RESUMO

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.


Assuntos
Apoptose/fisiologia , Microambiente Celular , Sistemas do Segundo Mensageiro/fisiologia , Animais , Artrite , Caspases/metabolismo , Linhagem Celular , Proliferação de Células/genética , Sobrevivência Celular/genética , Conexinas/metabolismo , Modelos Animais de Doenças , Rejeição de Enxerto , Humanos , Inflamação/genética , Transplante de Pulmão , Linfócitos/enzimologia , Linfócitos/metabolismo , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Fagócitos/metabolismo , Cicatrização/genética
5.
Sci Rep ; 10(1): 888, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31964936

RESUMO

To date, the effects of endurance exercise training on lymphocyte physiology at the kinome level are largely unknown. Therefore, the present study used a highly sensitive peptide-based kinase activity profiling approach to investigate if the basal activity of tyrosine (Tyr) and serine/threonine (Ser/Thr) kinases of human lymphocytes is affected by the aerobic endurance training status. Results revealed that the activity of various tyrosine kinases of the FGFR family and ZAP70 was increased, whereas the activity of multiple Ser/Thr kinases such as IKKα, CaMK4, PKAα, PKCα+δ (among others) was decreased in lymphocytes of endurance trained athletes (ET). Moreover, functional associations between several differentially regulated kinases in ET-derived lymphocytes were demonstrated by phylogenetic mapping and network analysis. Especially, Ser/Thr kinases of the AGC-kinase (protein kinase A, G, and C) family represent exercise-sensitive key components within the lymphocytes kinase network that may mediate the long-term effects of endurance training. Furthermore, KEGG (Kyoto Encyclopedia of Genes and Genomes) and Reactome pathway analysis indicate that Ras as well as intracellular signaling by second messengers were found to be enriched in the ET individuals. Overall, our data suggest that endurance exercise training improves the adaptive immune competence by modulating the activity of multiple protein kinases in human lymphocytes.


Assuntos
Treino Aeróbico , Linfócitos/enzimologia , Proteínas Quinases/metabolismo , Adulto , Atletas , Teste de Esforço , Humanos , Linfócitos/fisiologia , Fosforilação , Filogenia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Corrida , Tirosina/metabolismo
6.
Med Hypotheses ; 134: 109419, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31622925

RESUMO

To remedy carotid artery stenosis and prevent stroke surgical intervention is commonly used, and the gold standard being carotid endarterectomy (CEA). During CEA cerebrovascular hemoglobin oxygen saturation decreases and when this decrease reaches critical levels it leads to cerebral hypoxia that causes neuronal damage. One of the proposed mechanism that affects changes during CEA and contribute to acute brain ischemia (ABI) is oxidative stress. The increased production of reactive oxygen species and reactive nitrogen species during ABI may cause an unregulated inflammatory response and further lead to structural and functional injury of neurons. Antioxidant activity are involved in the protection against neuronal damage after cerebral ischemia. We hypothesized that neuronal injury and poor outcomes in patients undergoing CEA may be results of oxidative stress that disturbed function of antioxidant enzymes and contributed to the DNA damage in lymphocytes.


Assuntos
Isquemia Encefálica/enzimologia , Catalase/biossíntese , Endarterectomia das Carótidas/efeitos adversos , Hipóxia Encefálica/enzimologia , Complicações Intraoperatórias/enzimologia , Linfócitos/enzimologia , Superóxido Dismutase-1/biossíntese , Superóxido Dismutase/biossíntese , Isquemia Encefálica/etiologia , Estenose das Carótidas/enzimologia , Estenose das Carótidas/cirurgia , Catalase/sangue , Catalase/genética , Dano ao DNA , Radicais Livres , Regulação Enzimológica da Expressão Gênica , Humanos , Hipóxia Encefálica/etiologia , Complicações Intraoperatórias/etiologia , Mitocôndrias/metabolismo , Modelos Biológicos , Estresse Oxidativo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/etiologia , Superóxido Dismutase/sangue , Superóxido Dismutase/genética , Superóxido Dismutase-1/sangue , Superóxido Dismutase-1/genética
7.
Ecotoxicol Environ Saf ; 190: 110064, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31838230

RESUMO

Astilbin (ASB), a dihydroflavonol glycoside, is widely found in a variety of plants and in functional foods and acts as a powerful antioxidant. The aim of this study was to investigate the underlying mechanisms involved in the antagonistic effects of ASB on cadmium (Cd)-induced necroptosis in chicken peripheral blood lymphocytes. Peripheral blood lymphocytes were aseptically collected from Roman white hens and then randomly divided into five groups: the control group was incubated without additional reagents, while the other groups were incubated with Cd, ASB, a combination of Cd and ASB, and 0.1% DMSO. After a 24 h treatment, cell samples were collected. The results showed that some morphological changes consistent with necroptosis were observed in the Cd-treated groups, suggesting the occurrence of necroptosis. Simultaneously, antioxidant activity markers (CAT, SOD, GSH, GSH-px, and T-AOC) decreased and indicators of oxidative stress (MDA, iNOS, NO, H2O2, ·OH and ROS) increased. The production of ROS induced the activation of the PI3K/Akt signaling pathway, as the expression levels of PI3K, Akt and PDK1 were significantly elevated. Additionally, the expression levels of RIPK3, RIPK1, MLKL, TAK1, TAB2 and TAB3 were increased and that of Caspase-8 was decreased, which could cause the necroptosis. However, the most important our results was that ASB supplements remarkably attenuated the Cd-induced effects. We conclude that the Cd treatment promoted an imbalance of the antioxidant status and activated the PI3K/Akt pathway, leading to necroptosis in chicken peripheral blood lymphocytes, and that ASB was able to partially ameliorate the effect of Cd-induced necroptosis.


Assuntos
Cádmio/toxicidade , Flavonóis/farmacologia , Necroptose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Cádmio/metabolismo , Galinhas/metabolismo , Feminino , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Substâncias Protetoras/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo
8.
BMC Pharmacol Toxicol ; 20(1): 74, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31791417

RESUMO

BACKGROUND: Anti-neoplastic agents are widely used in the treatment of cancer and some non-neoplastic diseases. These drugs have been proved to be carcinogens, teratogens, and mutagens. Concern exists regarding the possible dangers of the staff handling anti-cancer drugs. The long-term exposure of nurses to anti-neoplastic drugs is still a controversial issue. The purpose of this study was to monitor cellular toxicity parameters and gene expression in nurses who work in chemotherapy wards and compare them to nurses who work in other wards. METHODS: To analyze the apoptosis-related genes overexpression and cytotoxicity effects, peripheral blood lymphocytes obtained from oncology nurses and the control group. THE RESULTS: Significant alterations in four analyzed apoptosis-related genes were observed in oncology nurses. In most individual samples being excavated, Bcl-2 overexpression is superior to that of Bax. Prominent P53 and Hif-1α up-regulation were observed in oncology nurses. Moreover, all cytotoxicity parameters (cell viability, ROS formation, MMP collapse, Lysosomal membrane damage, Lipid peroxidation, Caspase 3 activity and Apoptosis phenotype) in exposed oncology nurses were significantly (p < 0.001) higher than those of unexposed control nurses. Up-regulation of three analyzed apoptosis-related genes were observed in nurses occupationally exposed to anti-cancer drugs. CONCLUSION: Our data show that oxidative stress and mitochondrial toxicity induced by anti-neoplastic drugs lead to overexpression of apoptosis-related genes in oncology nurses.


Assuntos
Antineoplásicos/efeitos adversos , Proteínas Reguladoras de Apoptose/genética , Expressão Gênica/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Enfermeiras e Enfermeiros , Exposição Ocupacional/efeitos adversos , Adulto , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Apoptose/efeitos dos fármacos , Apoptose/genética , Estudos de Casos e Controles , Caspase 3/sangue , Feminino , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/fisiologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Exposição Ocupacional/análise , Espécies Reativas de Oxigênio/sangue
9.
Free Radic Biol Med ; 143: 560-572, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31493505

RESUMO

Radiation induced damage to normal cells is a major shortcoming of conventional radiotherapy, which necessitates the development of novel radio-protective drugs. An ideal radio-modulator would protect normal cells while having cytotoxic effects on cancer cells. Plumbagin is a potent anti-tumour agent and has been shown to sensitize tumour cells to radiation-induced damage. In the present study, we have evaluated the radio-protective potential of plumbagin and found that it protected normal lymphocytes against radiation-induced apoptosis, but did not protect cancer cells against radiation. Plumbagin offered radioprotection even when it was added to cells after irradiation. The ability of only thiol based antioxidants to abrogate the radio-protective effects of plumbagin suggested a pivotal role of thiol groups in the radio-protective activity of plumbagin. Further, protein interaction network (PIN) analysis was used to predict the molecular targets of plumbagin. Based on the inputs from plumbagin's PIN and in light of its well-documented ability to modulate thiol groups, we proposed that plumbagin may act via modulation of caspase enzyme which harbours a critical catalytic cysteine. Indeed, plumbagin suppressed radiation-induced increase in homogenous caspase and caspase-3 activity in lymphocytes. Plumbagin also inhibited the activity of recombinant caspase-3 and mass spectrometric analysis revealed that plumbagin covalently interacts with caspase-3. Further, the in vivo radioprotective efficacy of plumbagin (single dose of 2mg/kg body weight) was demonstrated by its ability to rescue mice against radiation (7.5 Gy; Whole Body Irradiation) induced mortality. These results indicate that plumbagin prevents radiation induced apoptosis specifically in normal cells by inhibition of caspase-3 activity.


Assuntos
Caspase 3/metabolismo , Raios gama/efeitos adversos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pulmonares/enzimologia , Linfócitos/enzimologia , Naftoquinonas/farmacologia , Protetores contra Radiação/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Caspase 3/genética , Proliferação de Células , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/radioterapia , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Camundongos , Oxirredução , Fosforilação
10.
Med Oncol ; 36(9): 78, 2019 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-31375946

RESUMO

Cigarette smoking is directly associated with lung cancer. Non-small cell lung carcinoma (NSCLC) represents approximately 80% from all types of lung cancer. This latter is hard to diagnose and to treat due to the lack of symptoms in early stages of the disease. The aim of this study was to evaluate ADA activity and the expression of P2X7, A1, and A2A receptors and in lymphocytes. In addition, the profile of pro-inflammatory and anti-inflammatory cytokines serum levels of patients with lung cancer in advanced stage was evaluated. Patients (n = 13) previously treated for lung cancer at stage IV (UICC) with chemotherapy had their blood collected. Cancer patients showed a decrease in ADA activity and an increase in A1 receptor expression in lymphocytes when compared to the control group. Moreover, patients exhibited an increase in IL-6 and TNF-α, while IL-17 and INF-ϒ serum levels were lower in patients with lung cancer. The decreased ADA activity and the increase in A1 receptor expression may contribute to adenosine pro-tumor effects by increasing IL-6 and TNF-α and decreasing IL-17 and INF-γ serum levels. Our data show an indirect evidence that purinergic signaling may have a role in promoting a profile of cytokines levels that favors tumor progression.


Assuntos
Adenosina Desaminase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Linfócitos/enzimologia , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Citocinas/sangue , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/metabolismo , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Receptores Purinérgicos/metabolismo , Transdução de Sinais
11.
Commun Biol ; 2: 152, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044177

RESUMO

Metabolic reprogramming is an important feature of host-pathogen interactions and a hallmark of tumorigenesis. The intracellular apicomplexa parasite Theileria induces a Warburg-like effect in host leukocytes by hijacking signaling machineries, epigenetic regulators and transcriptional programs to create a transformed cell state. The molecular mechanisms underlying host cell transformation are unclear. Here we show that a parasite-encoded prolyl-isomerase, TaPin1, stabilizes host pyruvate kinase isoform M2 (PKM2) leading to HIF-1α-dependent regulation of metabolic enzymes, glucose uptake and transformed phenotypes in parasite-infected cells. Our results provide a direct molecular link between the secreted parasite TaPin1 protein and host gene expression programs. This study demonstrates the importance of prolyl isomerization in the parasite manipulation of host metabolism.


Assuntos
Proteínas de Transporte/genética , Transformação Celular Neoplásica/genética , Interações Hospedeiro-Patógeno/genética , Proteínas de Membrana/genética , Peptidilprolil Isomerase de Interação com NIMA/genética , Proteínas de Protozoários/genética , Theileria/genética , Hormônios Tireóideos/genética , Animais , Antiprotozoários/farmacologia , Transporte Biológico , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Bovinos , Linhagem Celular Transformada , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Glucose/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/parasitologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas/genética , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Naftoquinonas/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Theileria/efeitos dos fármacos , Theileria/enzimologia , Theileria/crescimento & desenvolvimento , Hormônios Tireóideos/metabolismo
12.
Cell Immunol ; 340: 103915, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31054776

RESUMO

During viral infection, the innate immune system represents the first defense line of the human body. The pathogen associated molecular patterns (PAMPs) from the viruses are recognized by pattern recognition receptors (PRRs) of the host cell, especially from those of the immune cells. Sensing of PAMPs by PRRs elicits an elegant signal transduction system, ultimately leading to the production of type I interferons (IFNs) and proinflammatory cytokines. Ubiquitination, with its versatile functions, plays a central role in modulating almost every single step of this signaling cascade. Ubiquitin ligases, which catalyze different types of ubiquitination correlating with multiple functions, are the key participant in fine-tuning antiviral signal transduction. In this review, we focus on summarizing the ubiquitin ligases that regulate the key signaling molecules in antiviral innate immunity.


Assuntos
Imunidade Inata , Isoenzimas/genética , Processamento de Proteína Pós-Traducional , Receptores de Reconhecimento de Padrão/genética , Ubiquitina-Proteína Ligases/genética , Viroses/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Isoenzimas/imunologia , Linfócitos/enzimologia , Linfócitos/imunologia , Linfócitos/virologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Transdução de Sinais , Ubiquitina-Proteína Ligases/imunologia , Ubiquitinação , Viroses/enzimologia , Viroses/genética , Viroses/virologia
14.
J Neuropathol Exp Neurol ; 78(6): 492-500, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31058279

RESUMO

Leukocyte infiltration is an important pathological hallmark of multiple sclerosis (MS) and is therefore targeted by current MS therapies. The enzyme tissue transglutaminase (TG2) contributes to monocyte/macrophage migration and is present in MS lesions and could be a potential therapeutic target. We examined the cellular identity of TG2-expressing cells by immunohistochemistry in white matter lesions of 13 MS patients; 9 active and chronic active lesions from 4 patients were analyzed in detail. In these active MS lesions, TG2 is predominantly expressed in leukocytes (CD45+) but not in cells of the lymphocyte lineage, that is, T cells (CD3+) and B cells (CD20+). In general, cells of the monocyte/macrophage lineage (CD11b+ or CD68+) are TG2+ but no further distinction could be made regarding pro- or anti-inflammatory macrophage subtypes. In conclusion, TG2 is abundantly present in cells of the monocyte/macrophage lineage in active white matter MS lesions. We consider that TG2 can play a role in MS as it is associated with macrophage infiltration into the CNS. As such, TG2 potentially presents a novel target for therapeutic intervention that can support available MS therapies targeting lymphocyte infiltration.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Linfócitos/enzimologia , Macrófagos/enzimologia , Monócitos/enzimologia , Esclerose Múltipla/enzimologia , Transglutaminases/metabolismo , Substância Branca/enzimologia , Adulto , Idoso , Linhagem da Célula , Feminino , Humanos , Imuno-Histoquímica , Linfócitos/patologia , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/patologia , Esclerose Múltipla/patologia , Bancos de Tecidos , Substância Branca/patologia
15.
Nucleic Acids Res ; 47(8): 4240-4254, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30809670

RESUMO

Enzymes of intermediary metabolism are often reported to have moonlighting functions as RNA-binding proteins and have regulatory roles beyond their primary activities. Human serine hydroxymethyltransferase (SHMT) is essential for the one-carbon metabolism, which sustains growth and proliferation in normal and tumour cells. Here, we characterize the RNA-binding function of cytosolic SHMT (SHMT1) in vitro and using cancer cell models. We show that SHMT1 controls the expression of its mitochondrial counterpart (SHMT2) by binding to the 5'untranslated region of the SHMT2 transcript (UTR2). Importantly, binding to RNA is modulated by metabolites in vitro and the formation of the SHMT1-UTR2 complex inhibits the serine cleavage activity of the SHMT1, without affecting the reverse reaction. Transfection of UTR2 in cancer cells controls SHMT1 activity and reduces cell viability. We propose a novel mechanism of SHMT regulation, which interconnects RNA and metabolites levels to control the cross-talk between cytosolic and mitochondrial compartments of serine metabolism.


Assuntos
Citosol/enzimologia , Glicina Hidroximetiltransferase/genética , Mitocôndrias/enzimologia , Proteínas de Ligação a RNA/genética , Serina/metabolismo , Regiões 5' não Traduzidas , Compartimento Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Fibroblastos/citologia , Fibroblastos/enzimologia , Regulação da Expressão Gênica , Glicina Hidroximetiltransferase/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/enzimologia , Mitocôndrias/genética , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo
16.
Cells ; 8(2)2019 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-30744056

RESUMO

Increased DNA damage and the propension to cancer development, depend on the modulation of the mechanisms to control and maintain genomic integrity. Poly(ADP-Ribose)Polymerase activation and automodification are early responses to genotoxic stress. Upon binding to DNA strand breaks, the enzyme, a molecular DNA nick sensor, is hyperactivated: this is the first step in a series of events leading to either DNA repair or apoptosis. Enzyme hyperactivation and automodification can be easily measured and are widely used to look at DNA damage extent in the cell. We investigated whether these two markers (increased catalytic activity and auto modification), could help to monitor DNA damage in lymphocytes of flower growers from Southern Italy, occupationally exposed to pesticides. Peripheral lymphocyte lysates were analyzed for Poly(ADP-Ribose)Polymerase activity, and by SDS-PAGE and anti-Poly(ADP-Ribose)Polymerase 1-antibodyto measure automodified Poly(ADP-Ribose)Polymerase levels bydensitometry. Poly(ADP-Ribose)Polymerase activity and PARP automodification followed the same trend. Growers daily exposed to pesticides, showed both biomarkers very high, either in the presence or in the absence of pathologies. PARP activity and auto-modification in peripheral blood lymphocytes are possible, non-invasive, androutinartools to monitor the healthy conditions of floricoltorists.


Assuntos
Agricultura , Dano ao DNA , Flores/crescimento & desenvolvimento , Linfócitos/enzimologia , Linfócitos/patologia , Exposição Ocupacional , Praguicidas/efeitos adversos , Poli(ADP-Ribose) Polimerases/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Masculino , Poli Adenosina Difosfato Ribose/sangue , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/sangue
17.
Clin Sci (Lond) ; 133(2): 253-267, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30606816

RESUMO

Objective: Currently, no guidelines are established for pharmacogenomic testing involving folate metabolic genes in long-term disease-modifying antirheumatic drugs' (DMARD) therapies. We carefully investigated how common genetic variations in methylenetetrahydrofolate reductase (MTHFR) influence cellular metabolic kinetics in response to methotrexate (MTX). Designs: Two distinct cell models: HepG2 with stabilized MTHFR inhibition using shRNA delivered by a Lentiviral vector; and Epstein-Barr virus transformed human lymphoblasts expressing MTHFR polymorphic allele 677C and 677T were used. Disease activity and DMARD use were compared between MTHFR-677CC, CT and TT rheumatoid arthritis (RA) patients in a cross-sectional study (n=120). Results: Compared with MTHFR-CC, MTHFR-TT carriers had lower mean weakly MTX dose (9.8 ± 3.3 compared with 12.1 ± 3.5, P<0.05). More MTHFR-TT carriers (8/11, 73%) reported MTX-related side effects compared with MTHFR-677CC (32/57, 56%) and MTHFR-677CT (30/51, 59%). No genotypic difference was found in other DMARDs. At the same dose of MTX, lymphoblasts were more sensitive in cell survival, protein and thymidine syntheses whereas HepG2 models were more susceptible to the inhibition of S-adenosylmethionine (adoMet) synthesis. MTHFR-C677T altered protein turnover and folate mediated 1-carbon metabolic fluxes in lymphoblasts with and without MTX. MTHFR function significantly affected transmethylation fluxes and adoMet homeostasis but not nucleotide biosyntheses in MTX-treated HepG2 cell-lines. Conclusion: Combining cell models, kinetic studies, and genetic tests in humans, the present study gives insight on how MTHFR effects hepatic transmethylation homeostasis during MTX therapy. We provide platforms that help predict the genetic impact on antifolate drugs, and further delineate tissue-specific target pathway in DMARD therapies. We suggest that genetic factors should be taken into account in clinical practice.


Assuntos
Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Fígado/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Metotrexato/uso terapêutico , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Variantes Farmacogenômicos , S-Adenosilmetionina/metabolismo , Adulto , Idoso , Antirreumáticos/metabolismo , Artrite Reumatoide/enzimologia , Artrite Reumatoide/genética , Linhagem Celular Transformada , Feminino , Células Hep G2 , Heterozigoto , Homozigoto , Humanos , Cinética , Fígado/enzimologia , Linfócitos/enzimologia , Masculino , Metotrexato/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Pessoa de Meia-Idade , Fenótipo , Estudos Prospectivos
18.
Proteomics Clin Appl ; 13(4): e1800119, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30648813

RESUMO

PURPOSE: Psoriatic skin lesions are associated with chronic inflammation related to immune cell activity. Therefore, the aim of this study is to compare changes in the proteome of psoriatic keratinocytes and lymphocytes. EXPERIMENTAL DESIGN: A proteomics approach is used to analyze the expression of proteins in keratinocytes and lymphocytes from psoriatic patients and healthy controls. RESULTS: As a result 2119 proteins for keratinocytes and 1235 proteins for lymphocytes are identified. Psoriatic keratinocytes has 68 downregulated and 7 upregulated proteins and psoriatic lymphocytes has 106 downregulated and 67 upregulated proteins compared to healthy individuals. The list of downregulated proteins includes proteins involved in antioxidant homeostasis and, transcription regulation; upregulated proteins are involved in glycolytic processes and translation. These changes are accompanied by an increased level of 4-Hydroxynonenal-protein adducts; control cells are characterized by 4-Hydroxynonenal-Lysine adducts formed with structural and binding proteins, while in psoriatic cells 4-Hydroxynonenal-Lysine, 4-Hydroxynonenal-Histidine, and 4-Hydroxynonenal-Cysteine adducts with various molecular function proteins occur. CONCLUSIONS AND CLINICAL RELEVANCE: This study highlights the changes in psoriatic keratinocytes and lymphocytes that can be directly involved in the development of psoriasis. In both cell types the most significant changes are associated with upregulation of phosphoglycerate mutase 1 and downregulation of thioredoxin reductase.


Assuntos
Regulação Enzimológica da Expressão Gênica , Queratinócitos/enzimologia , Linfócitos/enzimologia , Fosfoglicerato Mutase/biossíntese , Proteoma/biossíntese , Psoríase/enzimologia , Tiorredoxina Dissulfeto Redutase/biossíntese , Adulto , Feminino , Humanos , Queratinócitos/patologia , Linfócitos/patologia , Masculino , Pessoa de Meia-Idade , Psoríase/patologia
19.
Matrix Biol ; 77: 58-72, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30096360

RESUMO

It is now well recognized that heparanase, an endo-ß-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, promotes tumorigenesis by diverse mechanisms. Compelling evidence strongly implies that heparanase is a viable target for cancer therapy, thus encouraging the development of heparanase inhibitors as anti-cancer therapeutics. Here, we examined the efficacy and mode of action of PG545, an HS-mimetic heparanase inhibitor, in human lymphoma. We found that PG545 exhibits a strong anti-lymphoma effect, eliciting lymphoma cell apoptosis. Notably, this anti-lymphoma effect involves ER stress response that was accompanied by increased autophagy. The persistent ER stress evoked by PG545 is held responsible for cell apoptosis because apoptotic cell death was attenuated by an inhibitor of PERK, a molecular effector of ER stress. Importantly, PG545 had no such apoptotic effect on naïve splenocytes, further encouraging the development of this compound as anti-lymphoma drug. Surprisingly, we found that PG545 also elicits apoptosis in lymphoma cells that are devoid of heparanase activity (i.e., Raji), indicating that the drug also exerts heparanase-independent function(s) that together underlie the high potency of PG545 in preclinical cancer models.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Glucuronidase/genética , Linfoma/tratamento farmacológico , Saponinas/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Regulação da Expressão Gênica , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Heparina/análogos & derivados , Heparina/farmacologia , Heparitina Sulfato , Humanos , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfoma/enzimologia , Linfoma/genética , Linfoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Cultura Primária de Células , Baço/citologia , Baço/efeitos dos fármacos , Baço/enzimologia , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Clin Chim Acta ; 488: 90-97, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30409763

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease, where there is irreversible breakdown of immunological self-tolerance. Extracellular adenosine triphosphate (ATP) and adenosine are signaling molecules that play an important part in the immune response. During inflammation and the immune response, a group of enzymes control these molecules, including ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5'-nucleotidase, and ecto-adenosine deaminase (E-ADA). We determined the activity and expression of E-NTPDase, the expression of E-5'-nucleotidase, the activity of E-ADA in lymphocytes and serum of SLE patients. METHODS: This study involved 35 patients with SLE and 30 healthy subjects as a control group. E-NTPDase activity and expression were increased in lymphocytes from SLE patients (31% and 37% for activity and expression, respectively) compared with the control group. RESULTS: An approximately 42% increase in E-ADA activity in lymphocytes was observed in SLE patients compared with the control group, in serum the ADA activity was decreased by 57% in SLE patients. Expression of E-5'-nucleotidase was not changed in SLE patients. CONCLUSIONS: E-NTPDase and E-ADA perform key functions in the modulation of the immune and inflammatory response in SLE.


Assuntos
5'-Nucleotidase/metabolismo , Apirase/metabolismo , Lúpus Eritematoso Sistêmico/enzimologia , Linfócitos/enzimologia , 5'-Nucleotidase/biossíntese , Adulto , Apirase/biossíntese , Feminino , Proteínas Ligadas por GPI/biossíntese , Proteínas Ligadas por GPI/metabolismo , Humanos , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Linfócitos/metabolismo , Masculino
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