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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
Artigo em Inglês | MEDLINE | ID: mdl-30285552

RESUMO

The enzyme thymidine phosphorylase (TP) is important for activation of capecitabine and 5-fluorouracil. Assessment of TP phenotype might be suitable for identification of patients at risk of fluoropyrimidine-induced toxicity. In this paper, we describe the development and validation an assay for TP activity in peripheral blood mononuclear cells (PBMCs). The assay was based on ex vivo conversion of the TP substrate thymidine to thymine. The amount of thymine formed was determined by high-performance liquid chromatography - ultraviolet detection (HPLC-UV) with 5-bromouracil as internal standard. Lymphocytes and monocytes were purified from isolated PBMCs to examine cell-specific TP activity. TP activity in PBMCs demonstrated Michaelis-Menten kinetics. The lower limit of quantification was 2.3 µg PBMC protein and assay linearity was demonstrated up to 22.7 µg PBMC protein. Within-day and between-day precisions were ≤9.2% and ≤6.0%, respectively. Adequate stability TP activity was demonstrated after long-term storage of PBMC dry pellets and lysates at -80 °C. In monocytes, TP activity was approximately 3 times higher than in lymphocytes. Clinical applicability was demonstrated in samples that were collected from five cancer patients. A simple, precise and sensitive HPLC-UV assay for quantification of TP activity in PBMCs was developed that can be applied for clinical research.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Leucócitos Mononucleares/enzimologia , Timidina Fosforilase/sangue , Antimetabólitos Antineoplásicos/uso terapêutico , Capecitabina/uso terapêutico , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Limite de Detecção , Linfócitos/enzimologia , Monócitos/enzimologia , Neoplasias/sangue , Sensibilidade e Especificidade , Timidina/metabolismo , Timidina Fosforilase/metabolismo , Timina/metabolismo
9.
Sci Rep ; 8(1): 15446, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30337601

RESUMO

Missense mutations in glucocerebrosidase (GBA1) that impair the activity of the encoded lysosomal lipid metabolism enzyme (GCase) are linked to an increased risk of Parkinson's disease. However, reduced GCase activity is also found in brain tissue from Parkinson's disease patients without GBA1 mutations, implicating GCase dysfunction in the more common idiopathic form of Parkinson's disease. GCase is very highly expressed in monocytes, and thus we measured GCase activity in blood samples from recently diagnosed Parkinson's disease patients. Flow cytometry and immunoblotting assays were used to measure levels of GCase activity and protein in monocytes and lymphocytes from patients with Parkinson's disease (n = 48) and matched controls (n = 44). Gene sequencing was performed to screen participants for GBA1 missense mutations. In the Parkinson's disease patients, GCase activity was significantly reduced in monocytes, but not lymphocytes, compared to controls, even when GBA1 mutation carriers were excluded. Monocyte GCase activity correlated with plasma ceramide levels in the Parkinson's disease patients. Our results add to evidence for GCase dysfunction in idiopathic Parkinson's disease and warrant further work to determine if monocyte GCase activity associates with Parkinson's disease progression.


Assuntos
Ceramidas/sangue , Glucosilceramidase/deficiência , Monócitos/enzimologia , Doença de Parkinson/enzimologia , Idoso , Progressão da Doença , Feminino , Citometria de Fluxo , Genótipo , Glucosilceramidase/análise , Glucosilceramidase/genética , Humanos , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Doença de Parkinson/sangue , Interferência de RNA , RNA Interferente Pequeno/genética
10.
Biomed Pharmacother ; 107: 1259-1267, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30257340

RESUMO

Meloxicam is an anti-inflammatory drug that has a potential protective effect in many common diseases. However, this molecule is quickly eliminated from the body due to it short half-life. One way to overcome this problem is to incorporate meloxicam into lipid-core nanocapsules which may increase it anti-inflammatory effects. In view of this, the objective of this work was to evaluate the potential toxicity and safety of these novel nanomaterials both in vitro and in vivo. Here, we evaluated the effects of uncoated meloxicam-loaded nanocapsules (M-NC), uncoated and not loaded with meloxicam or blank (B-NC), PEGylated meloxicam-loaded lipid-core nanocapsules (M-NCPEG), blank PEGylated lipid-core nanocapsules (B-NCPEG) and free meloxicam (M-F) in vitro through the analysis of cell viability, caspase activity assays and gene expression of perforin and granzyme B. Meanwhile, the in vivo safety was assessed using C57BL/6 mice that received nanocapsules for seven days. Thus, no change in cell viability was observed after treatments. Furthermore, M-NC, M-NCPEG and M-F groups reversed the damage caused by H2O2 on caspase-1, 3 and 8 activities. Overall, in vivo results showed a safe profile of these nanocapsules including hematological, biochemical, histological and genotoxicity analysis. In conclusion, we observed that meloxicam nanocapsules present a safe profile to use in future studies with this experimental protocol and partially reverse in vitro damage caused by H2O2.


Assuntos
Anti-Inflamatórios não Esteroides , Caspases/metabolismo , Linfócitos/efeitos dos fármacos , Meloxicam , Nanocápsulas/química , Polietilenoglicóis/química , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/toxicidade , Peso Corporal/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Ingestão de Alimentos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/toxicidade , Linfócitos/enzimologia , Linfócitos/patologia , Masculino , Meloxicam/farmacologia , Meloxicam/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Baço/efeitos dos fármacos , Baço/patologia , Testes de Toxicidade
11.
J Biochem Mol Toxicol ; 32(8): e22171, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30019796

RESUMO

The present study examined the impacts of sodium acetate (SA), sodium acid pyrophosphate (SAPP), and citric acid (CA) on the viability, proliferation, and DNA damage of isolated lymphocytes in vitro. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release assays were adopted to evaluate cell viability, while comet assay was employed to assess the genotoxic effects. The cells were incubated with different levels of SA (50, 100, and 200 mM), SAPP (25, 50, and 100 mM/L), or CA (100, 200, and 300 µg/mL). The lymphocytes treated with the tested food additives showed concentration-dependent decreases in both cell viability and proliferation. A concentration-dependent increase in LDH release was also observed. The comet assay results indicated that SA, SAPP, and CA increased DNA damage percentage, tail DNA percentage, tail length, and tail moment in a concentration-dependent manner. The current results showed that SA, SAPP, and CA are cytotoxic and genotoxic to isolated lymphocytes in vitro.


Assuntos
Proliferação de Células/efeitos dos fármacos , Ácido Cítrico/toxicidade , Dano ao DNA , Difosfatos/toxicidade , Linfócitos/efeitos dos fármacos , Acetato de Sódio/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Ensaio Cometa , Relação Dose-Resposta a Droga , Aditivos Alimentares/toxicidade , L-Lactato Desidrogenase/metabolismo , Limite de Detecção , Linfócitos/citologia , Linfócitos/enzimologia , Linfócitos/metabolismo , Masculino , Ratos Sprague-Dawley
12.
Hum Pathol ; 81: 131-137, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29969607

RESUMO

Benign terminal deoxynucleotidyl transferase (TdT)-positive cells have been documented in a variety of nonhematopoietic tissues. Scant data are, however, available on their presence in nonneoplastic lymph nodes. This study is aimed to (1) characterize the presence/distribution of benign TdT-positive cells in pediatric and adult reactive lymph nodes and (2) define the phenotype and nature of such elements. This retrospective study considered 141 reactive lymph nodes from pediatric and adult patients without history of neoplastic disease. TdT-positive cells were characterized by immunohistochemical and morphometric analyses, and their presence was correlated with the clinical-pathological features. The nature of TdT-positive cells was investigated by (1) double immunostaining for early lymphoid cell markers and (2) assessment of TdT expression in fetal lymph nodes. Sparse TdT-positive cells were documented in all pediatric cases and in most (76%) adult lymph nodes. TdT-positive cell density was higher in children than adults (15.9/mm2 versus 8.6/mm2; P < .05). TdT positivity did not correlate with any clinical or histological parameter, and double immunostaining disclosed a phenotype compatible with early lymphoid precursors (positivity for CD34 and CD10, and variable expression of CD7). A very high TdT-positive cell density (802.4/mm2) was reported in all fetal lymph nodes. In conclusion, TdT-positive cells are a common finding in pediatric and adult lymph nodes. The interstitial distribution and low number of such cells allow for the differential diagnosis with precursor lymphoid neoplasms. The high density in fetal lymph nodes and the phenotype of such cells suggest their belonging to an immature lymphoid subset gradually decreasing with age.


Assuntos
DNA Nucleotidilexotransferase/análise , Linfonodos/enzimologia , Doenças Linfáticas/enzimologia , Linfócitos/enzimologia , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/análise , Linhagem da Célula , Proliferação de Células , Criança , Pré-Escolar , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica , Lactente , Itália , Linfonodos/patologia , Doenças Linfáticas/patologia , Linfócitos/patologia , Pessoa de Meia-Idade , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Adulto Jovem
13.
Immunogenetics ; 70(9): 585-597, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29947943

RESUMO

Granzymes are a family of serine proteases found in the lytic granules of cytotoxic T lymphocytes and natural killer (NK) cells, which are involved in killing of susceptible target cells. Most information on granzymes and their enzymatic specificities derive from studies in humans and mice. Although granzymes shared by both species show a high level of conservation, the complement of granzyme genes differs between the species. The aim of this study was to identify granzyme genes expressed in cattle, determine their genomic locations and analyse their sequences to predict likely functional specificities. Orthologues of the five granzyme genes found in humans (A, B, H, K and M) were identified, as well a novel gene designated granzyme O, most closely related to granzyme A. An orthologue of granzyme O was found in pigs and a non-function version was detected in the human genome. Use of specific PCRs demonstrated that all of these genes, including granzyme O, are expressed in activated subsets of bovine lymphocytes, with particularly high levels in CD8 T cells. Consistent with findings in humans and mice, the granzyme-encoding genes were located on three distinct genomic loci, which correspond to different proteolytic enzymatic activities, namely trypsin-like, chymotrypsin-like and metase-like. Analysis of amino acid sequences indicated that the granzyme proteins have broadly similar enzymatic specificities to their human and murine counterparts but indicated that granzyme B has a different secondary specificity. These findings provide the basis for further work to examine their role in the cytotoxic activity of bovine CD8 T cells.


Assuntos
Granzimas/genética , Linfócitos/enzimologia , Filogenia , Animais , Bovinos , Mapeamento Cromossômico , Granzimas/química , Granzimas/metabolismo , Ativação Linfocitária , Anotação de Sequência Molecular , Perforina/genética , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tripsina/genética
14.
Science ; 360(6387): 449-453, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29599194

RESUMO

Activated immune cells undergo a metabolic switch to aerobic glycolysis akin to the Warburg effect, thereby presenting a potential therapeutic target in autoimmune disease. Dimethyl fumarate (DMF), a derivative of the Krebs cycle intermediate fumarate, is an immunomodulatory drug used to treat multiple sclerosis and psoriasis. Although its therapeutic mechanism remains uncertain, DMF covalently modifies cysteine residues in a process termed succination. We found that DMF succinates and inactivates the catalytic cysteine of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in mice and humans, both in vitro and in vivo. It thereby down-regulates aerobic glycolysis in activated myeloid and lymphoid cells, which mediates its anti-inflammatory effects. Our results provide mechanistic insight into immune modulation by DMF and represent a proof of concept that aerobic glycolysis is a therapeutic target in autoimmunity.


Assuntos
Autoimunidade/efeitos dos fármacos , Fumarato de Dimetilo/farmacologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/efeitos dos fármacos , Imunossupressores/farmacologia , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/enzimologia , Ciclo do Ácido Cítrico , Cisteína/metabolismo , Fumarato de Dimetilo/uso terapêutico , Humanos , Imunossupressores/uso terapêutico , Linfócitos/efeitos dos fármacos , Linfócitos/enzimologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/enzimologia , Células Mieloides/imunologia , Succinatos/química
15.
J Biol Chem ; 293(20): 7564-7577, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29563154

RESUMO

The mitochondrial polyglycerophospholipid cardiolipin (CL) is remodeled to obtain specific fatty acyl chains. This is predominantly accomplished by the transacylase enzyme tafazzin (TAZ). Barth syndrome (BTHS) patients with TAZ gene mutations exhibit impaired TAZ activity and loss in mitochondrial respiratory function. Previous studies identified monolysocardiolipin acyltransferase-1 (MLCL AT-1) as a mitochondrial enzyme capable of remodeling CL with fatty acid. In this study, we analyzed what relationship, if any, exists between TAZ and MLCL AT-1 with regard to CL remodeling and whether transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct improves mitochondrial respiratory function. In healthy lymphoblasts, reduction in TAZ expression through TAZ RNAi transfection resulted in a compensatory increase in MLCL AT-1 mRNA, protein, and enzyme activity, but CL mass was unaltered. In contrast, BTHS lymphoblasts exhibited decreased TAZ gene and protein expression but in addition decreased MLCL AT-1 expression and CL mass. Transfection of BTHS lymphoblasts with MLCL AT-1 expression construct increased CL, improved mitochondrial basal respiration and protein leak, and decreased the proportion of cells producing superoxide but did not restore CL molecular species composition to control levels. In addition, BTHS lymphoblasts exhibited higher rates of glycolysis compared with healthy controls to compensate for reduced mitochondrial respiratory function. Mitochondrial supercomplex assembly was significantly impaired in BTHS lymphoblasts, and transfection of BTHS lymphoblasts with MLCL AT-1 expression construct did not restore supercomplex assembly. The results suggest that expression of MLCL AT-1 depends on functional TAZ in healthy cells. In addition, transfection of BTHS lymphoblasts with an MLCL AT-1 expression construct compensates, but not completely, for loss of mitochondrial respiratory function.


Assuntos
Aciltransferases/metabolismo , Síndrome de Barth/prevenção & controle , Cardiolipinas/metabolismo , Linfócitos/enzimologia , Lisofosfolipídeos/metabolismo , Mitocôndrias/metabolismo , Aciltransferases/genética , Síndrome de Barth/enzimologia , Síndrome de Barth/patologia , Estudos de Casos e Controles , Células Cultivadas , Ácidos Graxos/metabolismo , Humanos , Mitocôndrias/patologia , Mutação
16.
Mol Cell Biochem ; 448(1-2): 9-15, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29435869

RESUMO

The aim of this study was to evaluate the therapeutic efficacy of specific avian polyclonal antibodies (IgY) against Trypanosoma cruzi and their interaction with ecto-enzymes of the purinergic system (NTPDase and adenosine deaminase (ADA) activities) in splenic lymphocytes. For this, mice were divided into six groups: three non-infected (A, B, and C) and three infected (D, E, and F). The groups A and D were composed by negative and positive controls, respectively; while the groups B and E were treated prophylactically with IgY (50 mg/kg), and the groups C and F were treated therapeutically with IgY (50 mg/kg). Treatment with IgY reduced parasitemia on day 6 post-infection (PI) compared to the infected control group, but it was similar on day 8 PI. Moreover, infected and treated animals (the groups E and F) did not show neither amastigotes in the cardiac tissue nor cardiac lesions when compared to the positive control group (the group D). The E-NTPDase (ATP and ADP as substrate) and ADA activities in splenic lymphocytes increased significantly in the positive control group (the group D) compared to the negative control group (the group A). The therapeutic treatment of IgY (the group F) was able to prevent the increase of E-NTPDase and E-ADA activities compared to the positive control group (the group D), but this finding was not observed in animals that received the prophylactic treatment (the group E). The therapeutic treatment of IgY may be considered an interesting approach to improve the immune response of mice experimentally infected by T. cruzi.


Assuntos
Adenosina Desaminase , Anticorpos Antiprotozoários/farmacologia , Proteínas Aviárias/farmacologia , Doença de Chagas , Imunoglobulinas/farmacologia , Proteínas de Protozoários , Baço , Trypanosoma cruzi , Adenosina Desaminase/imunologia , Adenosina Desaminase/metabolismo , Animais , Doença de Chagas/tratamento farmacológico , Doença de Chagas/enzimologia , Doença de Chagas/imunologia , Galinhas , Feminino , Linfócitos/enzimologia , Linfócitos/imunologia , Linfócitos/patologia , Camundongos , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Baço/enzimologia , Baço/imunologia , Baço/parasitologia , Baço/fisiologia , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologia
17.
Arterioscler Thromb Vasc Biol ; 38(3): 592-598, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29284604

RESUMO

OBJECTIVE: Evolocumab, a PCSK9 (proprotein convertase subtilisin kexin type 9)-neutralizing antibody, lowers low-density lipoprotein cholesterol (LDL-C) in homozygous familial hypercholesterolemic (HoFH) patients with reduced LDLR (low-density lipoprotein receptor) function. However, their individual responses are highly variable, even among carriers of identical LDLR genetic defects. We aimed to elucidate why HoFH patients variably respond to PCSK9 inhibition. APPROACH AND RESULTS: Lymphocytes were isolated from 22 HoFH patients enrolled in the TAUSSIG trial (Trial Assessing Long Term Use of PCSK9 Inhibition in Subjects With Genetic LDL Disorders). Ten patients were true homozygotes (FH1/FH1) and 5 identical compound heterozygotes (FH1/FH2). Lymphocytes were plated with or without mevastatin, recombinant PCSK9 (rPCSK9), or a PCSK9-neutralizing antibody. Cell surface LDLR expression was analyzed by flow cytometry. All HoFH lymphocytes had reduced cell surface LDLR expression compared with non-FH lymphocytes, for each treatment modality. Lymphocytes from FH1/FH2 patients (LDLR defective/negative) displayed the lowest LDLR expression levels followed by lymphocytes from FH1/FH1 patients (defective/defective). Mevastatin increased, whereas rPCSK9 reduced LDLR expression. The PCSK9-neutralizing antibody restored LDLR expression. Lymphocytes displaying higher LDLR expression levels were those isolated from patients presenting with lowest levels of LDL-C and apolipoprotein B, before and after 24 weeks of evolocumab treatment. These negative correlations remained significant in FH1/FH1 patients and appeared more pronounced when patients with apolipoprotein E3/E3 genotypes were analyzed separately. Significant positive correlations were found between the levels of LDLR expression and the percentage reduction in LDL-C on evolocumab treatment. CONCLUSIONS: Residual LDLR expression in HoFH is a major determinant of LDL-C levels and seems to drive their individual response to evolocumab.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticolesterolemiantes/uso terapêutico , Homozigoto , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Linfócitos/efeitos dos fármacos , Mutação , Pró-Proteína Convertase 9/antagonistas & inibidores , Receptores de LDL/genética , Inibidores de Serino Proteinase/uso terapêutico , Adolescente , Adulto , Anticorpos Monoclonais Humanizados , Apolipoproteína B-100/sangue , Células Cultivadas , LDL-Colesterol/sangue , Quimioterapia Combinada , Ezetimiba/uso terapêutico , Feminino , Predisposição Genética para Doença , Heterozigoto , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Lovastatina/análogos & derivados , Lovastatina/uso terapêutico , Linfócitos/enzimologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Receptores de LDL/metabolismo , Resultado do Tratamento , Adulto Jovem
18.
Toxins (Basel) ; 9(12)2017 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-29232860

RESUMO

Innate lymphoid cells (ILCs) are an emerging group of immune cells that provide the first line of defense against various pathogens as well as contributing to tissue repair and inflammation. ILCs have been classically divided into three subgroups based on their cytokine secretion and transcription factor profiles. ILC nomenclature is analogous to that of T helper cells. Group 1 ILCs composed of natural killer (NK) cells as well as IFN-γ secreting ILC1s. ILC2s have the capability to produce TH2 cytokines while ILC3s and lymphoid tissue inducer (LTis) are subsets of cells that are able to secrete IL-17 and/or IL-22. A recent subset of ILC known as ILC4 was discovered, and the cells of this subset were designated as NK17/NK1 due to their release of IL-17 and IFN-γ. In this review, we sought to explain the subclasses of ILCs and their roles as mediators of lytic enzymes and inflammation.


Assuntos
Citocinas/metabolismo , Imunidade Inata , Linfócitos , Animais , Humanos , Inflamação , Linfócitos/classificação , Linfócitos/enzimologia , Linfócitos/imunologia
19.
Blood ; 130(25): 2750-2761, 2017 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-29101238

RESUMO

Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glucocorticoides/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Dasatinibe/farmacologia , Dexametasona/farmacologia , Xenoenxertos , Humanos , Interleucina-4/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/enzimologia , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prednisona/farmacologia
20.
Viruses ; 9(11)2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29099045

RESUMO

Programmable endonucleases introduce DNA breaks at specific sites, which are repaired by non-homologous end joining (NHEJ) or homology recombination (HDR). Genome editing in human lymphoid cells is challenging as these difficult-to-transfect cells may also inefficiently repair DNA by HDR. Here, we estimated efficiencies and dynamics of knockout (KO) and knockin (KI) generation in human T and B cell lines depending on repair template, target loci and types of genomic endonucleases. Using zinc finger nuclease (ZFN), we have engineered Jurkat and CEM cells with the 8.2 kb human immunodeficiency virus type 1 (HIV-1) ∆Env genome integrated at the adeno-associated virus integration site 1 (AAVS1) locus that stably produce virus particles and mediate infection upon transfection with helper vectors. Knockouts generated by ZFN or clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) double nicking techniques were comparably efficient in lymphoid cells. However, unlike polyclonal sorted cells, gene-edited cells selected by cloning exerted tremendous deviations in functionality as estimated by replication of HIV-1 and human T cell leukemia virus type 1 (HTLV-1) in these cells. Notably, the recently reported high-fidelity eCas9 1.1 when combined to the nickase mutation displayed gene-dependent decrease in on-target activity. Thus, the balance between off-target effects and on-target efficiency of nucleases, as well as choice of the optimal method of edited cell selection should be taken into account for proper gene function validation in lymphoid cells.


Assuntos
Edição de Genes , HIV-1/genética , Linfócitos/fisiologia , Linfócitos/virologia , Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Técnicas de Inativação de Genes , Genoma Viral , Genômica/métodos , HIV-1/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Células Jurkat , Linfócitos/enzimologia , Transfecção , Nucleases de Dedos de Zinco/genética
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