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1.
Front Immunol ; 10: 1732, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428087

RESUMO

Previous studies indicate that B-lymphocytes play a key role activating diabetogenic T-lymphocytes during the development of autoimmune diabetes. Recently, two transgenic NOD mouse models were generated: the NOD-PerIg and the 116C-NOD mice. In NOD-PerIg mice, B-lymphocytes acquire an activated proliferative phenotype and support accelerated autoimmune diabetes development. In contrast, in 116C-NOD mice, B-lymphocytes display an anergic-like phenotype delaying autoimmune diabetes onset and decreasing disease incidence. The present study further evaluates the T- and B-lymphocyte phenotype in both models. In islet-infiltrating B-lymphocytes (IIBLs) from 116C-NOD mice, the expression of H2-Kd and H2-Ag7 is decreased, whereas that of BAFF, BAFF-R, and TACI is increased. In contrast, IIBLs from NOD-PerIg show an increase in CD86 and FAS expression. In addition, islet-infiltrating T-lymphocytes (IITLs) from NOD-PerIg mice exhibit an increase in PD-1 expression. Moreover, proliferation assays indicate a high capacity of B-lymphocytes from NOD-PerIg mice to secrete high amounts of cytokines and induce T-lymphocyte activation compared to 116C B-lymphocytes. This functional variability between 116C and PerIg B-lymphocytes ultimately results in differences in the ability to shape T-lymphocyte phenotype. These results support the role of B-lymphocytes as key regulators of T-lymphocytes in autoimmune diabetes and provide essential information on the phenotypic characteristics of the T- and B-lymphocytes involved in the autoimmune response in autoimmune diabetes.

2.
Front Cell Dev Biol ; 7: 107, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31259172

RESUMO

Cell migration is a key procedure involved in many biological processes including embryological development, tissue formation, immune defense or inflammation, and cancer progression. How physical, chemical, and molecular aspects can affect cell motility is a challenge to understand migratory cells behavior. In vitro assays are excellent approaches to extrapolate to in vivo situations and study live cells behavior. Here we present four in vitro protocols that describe step-by-step cell migration, invasion and adhesion strategies and their corresponding image data quantification. These current protocols are based on two-dimensional wound healing assays (comparing traditional pipette tip-scratch assay vs. culture insert assay), 2D individual cell-tracking experiments by live cell imaging and three-dimensional spreading and transwell assays. All together, they cover different phenotypes and hallmarks of cell motility and adhesion, providing orthogonal information that can be used either individually or collectively in many different experimental setups. These optimized protocols will facilitate physiological and cellular characterization of these processes, which may be used for fast screening of specific therapeutic cancer drugs for migratory function, novel strategies in cancer diagnosis, and for assaying new molecules involved in adhesion and invasion metastatic properties of cancer cells.

3.
Pigment Cell Melanoma Res ; 31(4): 484-495, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29385656

RESUMO

Melanoma is a malignant tumor derived from melanocytes. Once disseminated, it is usually highly resistant to chemotherapy and is associated with poor prognosis. We have recently reported that T-type calcium channels (TTCCs) are overexpressed in melanoma cells and play an important role in melanoma progression. Importantly, TTCC pharmacological blockers reduce proliferation and deregulate autophagy leading to apoptosis. Here, we analyze the role of autophagy during migration/invasion of melanoma cells. TTCC Cav3.1 and LC3-II proteins are highly expressed in BRAFV600E compared with NRAS mutant melanomas, both in cell lines and biopsies. Chloroquine, pharmacological blockade, or gene silencing of TTCCs inhibit the autophagic flux and impair the migration and invasion capabilities, specifically in BRAFV600E melanoma cells. Snail1 plays an important role in motility and invasion of melanoma cells. We show that Snail1 is strongly expressed in BRAFV600E melanoma cells and patient biopsies, and its expression decreases when autophagy is blocked. These results demonstrate a role of Snail1 during BRAFV600E melanoma progression and strongly suggest that targeting macroautophagy and, particularly TTCCs, might be a good therapeutic strategy to inhibit metastasis of the most common melanoma type (BRAFV600E).


Assuntos
Canais de Cálcio Tipo T/metabolismo , Movimento Celular , Melanoma/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação de Sentido Incorreto , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Substituição de Aminoácidos , Canais de Cálcio Tipo T/genética , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas Associadas aos Microtúbulos/genética , Invasividade Neoplásica , Proteínas Proto-Oncogênicas B-raf/genética , Fatores de Transcrição da Família Snail/genética
4.
Eur J Immunol ; 41(5): 1344-51, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21469125

RESUMO

CD4(+) T lymphocytes are required to induce spontaneous autoimmune diabetes in the NOD mouse. Since pancreatic ß cells upregulate Fas expression upon exposure to pro-inflammatory cytokines, we studied whether the diabetogenic action of CD4(+) T lymphocytes depends on Fas expression on target cells. We assayed the diabetogenic capacity of NOD spleen CD4(+) T lymphocytes when adoptively transferred into a NOD mouse model combining: (i) Fas-deficiency, (ii) FasL-deficiency, and (iii) SCID mutation. We found that CD4(+) T lymphocytes require Fas expression in the recipients' target cells to induce diabetes. IL-1ß has been described as a key cytokine involved in Fas upregulation on mouse ß cells. We addressed whether CD4(+) T cells require IL-1ß to induce diabetes. We also studied spontaneous diabetes onset in NOD/IL-1 converting enzyme-deficient mice, in NOD/IL-1ß-deficient mice, and CD4(+) T-cell adoptively transferred diabetes into NOD/SCID IL-1ß-deficient mice. Neither IL-1ß nor IL-18 are required for either spontaneous or CD4(+) T-cell adoptively transferred diabetes. We conclude that CD4(+) T-cell-mediated ß-cell damage in autoimmune diabetes depends on Fas expression, but not on IL-1ß unveiling the existing redundancy regarding the cytokines involved in Fas upregulation on NOD ß cells in vivo.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Receptor fas/metabolismo , Transferência Adotiva , Animais , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Proteína Ligante Fas/deficiência , Proteína Ligante Fas/metabolismo , Genótipo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/patologia , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Receptor fas/deficiência , Receptor fas/genética
5.
PLoS One ; 5(6): e11328, 2010 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-20593029

RESUMO

Ribonucleotide reductases (RNRs) are essential enzymes that carry out the de novo synthesis of deoxyribonucleotides by reducing ribonucleotides. There are three different classes of RNRs (I, II and III), all having different oxygen dependency and biochemical characteristics. Salmonella enterica serovar Typhimurium (S. Typhimurium) harbors class Ia, class Ib and class III RNRs in its genome. We have studied the transcriptional regulation of these three RNR classes in S. Typhimurium as well as their differential function during infection of macrophage and epithelial cells. Deletion of both NrdR and Fur, two main transcriptional regulators, indicates that Fur specifically represses the class Ib enzyme and that NrdR acts as a global repressor of all three classes. A Fur recognition sequence within the nrdHIEF promoter has also been described and confirmed by electrophoretic mobility shift assays (EMSA). In order to elucidate the role of each RNR class during infection, S. Typhimurium single and double RNR mutants (as well as Fur and NrdR mutants) were used in infection assays with macrophage and epithelial cell lines. Our results indicate class Ia to be mainly responsible for deoxyribonucleotide production during invasion and proliferation inside macrophages and epithelial cells. Neither class Ib nor class III seem to be essential for growth under these conditions. However, class Ib is able to maintain certain growth in an nrdAB mutant during the first hours of macrophage infection. Our results suggest that, during the early stages of macrophage infection, class Ib may contribute to deoxyribonucleotide synthesis by means of both an NrdR and a Fur-dependent derepression of nrdHIEF due to hydrogen peroxide production and DNA damage associated with the oxidative burst, thus helping to overcome the host defenses.


Assuntos
Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Ribonucleotídeo Redutases/metabolismo , Salmonella typhimurium/enzimologia , Transcrição Genética , Sequência de Bases , DNA Bacteriano , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes Bacterianos , Hidroxiureia/farmacologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Ribonucleotídeo Redutases/genética , Salmonella typhimurium/patogenicidade , Transcrição Genética/efeitos dos fármacos
6.
Int Microbiol ; 11(1): 49-56, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18683632

RESUMO

The nrdDG promoter regulates transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli, an essential enzyme required to supply the building blocks for DNA synthesis. In this work, binding of the pleiotropic FNR (fumarate and nitrate reduction) transcriptional regulator to the nrdDG promoter region and the effects of binding on transcription were investigated. Gel retardation analysis with purified FNR* demonstrated FNR interaction at two FNR sites, termed FNR-2 and FNR-1, while studies with altered FNR boxes indicated that the upstream FNR-2 site was essential for anaerobic activation of the nrdDG promoter. Although the FNR-1 site was not absolutely required, it allowed maximal expression of this promoter. These results suggest that the two sites have an additive effect in coordinating nrdDG expression in response to shifting oxygen concentrations.


Assuntos
Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas com Ferro-Enxofre/genética , Ribonucleotídeo Redutases/genética , Anaerobiose , Sequência de Bases , Proteínas de Ligação a DNA , Escherichia coli K12/enzimologia , Proteínas de Escherichia coli/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ribonucleotídeo Redutases/metabolismo , Fatores de Transcrição/metabolismo
7.
Int. microbiol ; 11(1): 49-56, mar. 2008. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-67266

RESUMO

The nrdDG promoter regulates transcriptional expression of the anaerobic ribonucleotide reductase of Escherichia coli, an essential enzyme required to supply the building blocks for DNA synthesis. In this work, binding of the pleiotropic FNR (fumarate and nitrate reduction) transcriptional regulator to the nrdDG promoter region and the effects of binding on transcription were investigated. Gel retardation analysis with purified FNR demonstrated FNR interaction at two FNR sites, termed FNR-2 and FNR-1, while studies with altered FNR boxes indicated that the upstream FNR-2 site was essential for anaerobic activation of the nrdDG promoter. Although the FNR-1 site was not absolutely required, it allowed maximal expression of this promoter. These results suggest that the two sites have an additive effect in coordinating nrdDG expression in response to shifting oxygen concentrations (AU)


No disponible


Assuntos
Fumaratos/análise , Nitratos/análise , Escherichia coli , Ribonucleotídeo Redutases/genética , Regulação Bacteriana da Expressão Gênica , Proteínas com Ferro-Enxofre/genética , Oxirredutases/análise , Fatores de Transcrição/metabolismo , Anaerobiose
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