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1.
Nat Commun ; 12(1): 4878, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385447

RESUMO

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPß, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Clofazimina/farmacologia , Dieta Hiperlipídica/efeitos adversos , Progressão da Doença , Inativação Gênica , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo
2.
Int J Mol Sci ; 22(11)2021 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-34070750

RESUMO

The immune system is a fine modulator of the tumor biology supporting or inhibiting its progression, growth, invasion and conveys the pharmacological treatment effect. Tumors, on their side, have developed escaping mechanisms from the immune system action ranging from the direct secretion of biochemical signals to an indirect reaction, in which the cellular actors of the tumor microenvironment (TME) collaborate to mechanically condition the extracellular matrix (ECM) making it inhospitable to immune cells. TME is composed of several cell lines besides cancer cells, including tumor-associated macrophages, cancer-associated fibroblasts, CD4+ and CD8+ lymphocytes, and innate immunity cells. These populations interface with each other to prepare a conservative response, capable of evading the defense mechanisms implemented by the host's immune system. The presence or absence, in particular, of cytotoxic CD8+ cells in the vicinity of the main tumor mass, is able to predict, respectively, the success or failure of drug therapy. Among various mechanisms of immunescaping, in this study, we characterized the modulation of the phenotypic profile of CD4+ and CD8+ cells in resting and activated states, in response to the mechanical pressure exerted by a three-dimensional in vitro system, able to recapitulate the rheological and stiffness properties of the tumor ECM.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Matriz Extracelular/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Evasão Tumoral , Microambiente Tumoral/imunologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Fibroblastos Associados a Câncer/imunologia , Fibroblastos Associados a Câncer/patologia , Técnicas de Cultura de Células , Módulo de Elasticidade , Matriz Extracelular/química , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Hidrogéis/química , Interferon gama/genética , Interferon gama/imunologia , Ativação Linfocitária , Mecanotransdução Celular , Modelos Biológicos , NF-kappa B/genética , NF-kappa B/imunologia , Fenótipo , Cultura Primária de Células , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologia , Reologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/imunologia , Neoplasias de Mama Triplo Negativas/patologia , Microambiente Tumoral/genética , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/patologia
3.
Brain ; 2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-33974044

RESUMO

Brain cholesterol is produced mainly by astrocytes and is important for neuronal function. Its biosynthesis is severely reduced in mouse models of Huntington's disease. One possible mechanism is a diminished nuclear translocation of the transcription factor sterol regulatory element binding protein 2 (SREBP2) and, consequently, reduced activation of SREBP-controlled genes in the cholesterol biosynthesis pathway. Here we evaluated the efficacy of a gene therapy based on the unilateral intra-striatal injection of a recombinant adeno-associated virus 2/5 (AAV2/5) targeting astrocytes specifically and carrying the transcriptionally active N-terminal fragment of human SREBP2. Robust hSREBP2 expression in striatal glial cells in R6/2 Huntington's disease mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared mutant Huntingtin aggregates and attenuated behavioral deficits. We conclude that glial SREBP2 participates in Huntington's disease brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of the disease.

4.
Nat Immunol ; 22(6): 735-745, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34017124

RESUMO

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Hematopoiese Clonal/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Carcinoma Pulmonar de Células não Pequenas/terapia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células/genética , Quimioterapia Adjuvante/métodos , Quitinases/metabolismo , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Conjuntos de Dados como Assunto , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Granzimas/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA-Seq , Análise de Célula Única , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismo
5.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670450

RESUMO

Hemophilia is an X-linked recessive bleeding disorder. In pregnant women carrier of hemophilia, the fetal sex can be determined by non-invasive analysis of fetal DNA circulating in the maternal blood. However, in case of a male fetus, conventional invasive procedures are required for the diagnosis of hemophilia. Fetal cells, circulating in the maternal bloodstream, are an ideal target for a safe non-invasive prenatal diagnosis. Nevertheless, the small number of cells and the lack of specific fetal markers have been the most limiting factors for their isolation. We aimed to develop monoclonal antibodies (mAbs) against the ribosomal protein RPS4Y1 expressed in male cells. By Western blotting, immunoprecipitation and immunofluorescence analyses performed on cell lysates from male human hepatoma (HepG2) and female human embryonic kidney (HEK293) we developed and characterized a specific monoclonal antibody against the native form of the male RPS4Y1 protein that can distinguish male from female cells. The availability of the RPS4Y1-targeting monoclonal antibody should facilitate the development of novel methods for the reliable isolation of male fetal cells from the maternal blood and their future use for non-invasive prenatal diagnosis of X-linked inherited disease such as hemophilia.


Assuntos
Anticorpos Monoclonais/imunologia , Ácidos Nucleicos Livres/imunologia , Doenças Fetais/imunologia , Hemofilia A/imunologia , Diagnóstico Pré-Natal/métodos , Proteínas Ribossômicas/imunologia , Especificidade de Anticorpos/imunologia , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/metabolismo , Feminino , Doenças Fetais/sangue , Doenças Fetais/diagnóstico , Células HEK293 , Hemofilia A/sangue , Hemofilia A/diagnóstico , Células Hep G2 , Humanos , Masculino , Gravidez , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Sensibilidade e Especificidade
6.
Nat Commun ; 11(1): 4178, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826895

RESUMO

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.


Assuntos
Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Gânglios Espinais/metabolismo , Edição de Genes/métodos , Proteínas de Ligação ao Ferro/genética , Organoides/metabolismo , Células Receptoras Sensoriais/metabolismo , Antioxidantes/farmacologia , Sistemas CRISPR-Cas , Diferenciação Celular , Cromatina/metabolismo , Ataxia de Friedreich/tratamento farmacológico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/patologia , Predisposição Genética para Doença/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Íntrons , Mitocôndrias/metabolismo , Organoides/efeitos dos fármacos , Organoides/patologia , Células Receptoras Sensoriais/patologia , Análise de Sequência de RNA , Transcriptoma
7.
J Orthop Res ; 2020 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-32779775

RESUMO

The etiology of intervertebral disc degeneration is largely unknown, but local neuroinflammation may exert a crucial role through activation of cells as microglia and pro-inflammatory cytokines production. We aimed to compare the effect of degenerated and normal intervertebral disc microenvironment on microglial cells and the potential role of sphingosine-1-phosphate, a pro-inflammatory sphingolipid, in their crosstalk. Human degenerated intervertebral discs (Pfirrmann grade IV) were obtained at surgery for spondylolisthesis. Normal intervertebral discs were collected from cadaveric normal lumbar spines. Normal and degenerated-intervertebral discs were kept in culture to obtain media conditioning. Then, microglial cells were cocultured with conditioned media and viability, proliferation, migration, chemotaxis, and inflammatory gene expression were evaluated. The results demonstrate that conditioned media from degenerated intervertebral discs activate microglial cells, increasing chemotaxis, migration, and pro-inflammatory mediators release to a great extent than normal discs. In addition, we show that the administration of sphingosine-1-phosphate to normal intervertebral disc/microglia coculture mimicked degenerative effects. Interestingly, sphingosine-1-phosphate content in conditioned media from degenerated discs was significantly higher than that from normal ones. In addition, FTY720, a functional antagonist of sphingosine-1-phosphate, potently inhibited the effect of degenerated intervertebral discs on microglial inflammatory factor transcription and migration. Our data report, for the first time, that sphingosine-1-phosphate is involved as signal in the microenvironment of human degenerated intervertebral discs. Sphingosine-1-phosphate signaling modulation by FTY720 may induce beneficial effects in counteracting microglial activation during intervertebral disc degeneration.

8.
Cells ; 9(2)2020 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-31991805

RESUMO

: Circulating platelets (PLTs) are able to affect glioblastoma (GBM) microenvironment by supplying oncopromoter and pro-angiogenic factors. Among these mediators, sphingosine-1-phophate (S1P) has emerged as a potent bioactive lipid enhancing cell proliferation and survival. Here, we investigated the effect of "tumor education", characterizing PLTs from GBM patients in terms of activation state, protein content, and pro-angiogenic potential. PLTs from healthy donors (HD-PLTs) and GBM patients (GBM-PLTs) were collected, activated, and analyzed by flow cytometry, immunofluorescence, and Western blotting. To assess the pro-angiogenic contribution of GBM-PLTs, a functional cord formation assay was performed on GBM endothelial cells (GECs) with PLT-releasate. GBM-PLTs expressed higher positivity for P-selectin compared to HD-PLTs, both in basal conditions and after stimulation with adenosine triphosphate (ADP) and thrombin receptor activating peptide (TRAP). PLTs showed higher expression of VEGFR-1, VEGFR-2, VWF, S1P, S1PR1, SphK1, and SPNS. Interestingly, increased concentrations of VEGF and its receptors VEGFR1 and VEGFR2, VWF, and S1P were found in GBM-PLT-releasate with respect to HD-PLTs. Finally, GBM-PLT-releasate showed a pro-angiogenic effect on GECs, increasing the vascular network's complexity. Overall, our results demonstrated the contribution of PLTs to GBM angiogenesis and aggressiveness, advancing the potential of an anti-PLT therapy and the usefulness of PLT cargo as predictive and monitoring biomarkers.


Assuntos
Biomarcadores Tumorais/metabolismo , Plaquetas/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Neovascularização Patológica/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Difosfato de Adenosina/farmacologia , Adulto , Idoso , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Neoplasias Encefálicas/patologia , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Prognóstico , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
9.
Cell Rep ; 29(13): 4646-4656.e4, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875567

RESUMO

Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson's disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity.


Assuntos
Neurônios Dopaminérgicos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Dispositivos Lab-On-A-Chip , Mitocôndrias/metabolismo , Neostriado/metabolismo , Substância Negra/metabolismo , Sinapses/metabolismo , Axônios/metabolismo , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Rede Nervosa/metabolismo , Neuritos/metabolismo , Doença de Parkinson/metabolismo
10.
Liver Int ; 39(11): 2124-2135, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31033124

RESUMO

BACKGROUND AND AIMS: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis. METHODS: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker. RESULTS: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients. CONCLUSIONS: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.


Assuntos
Autoanticorpos/sangue , Autoantígenos/imunologia , Cirrose Hepática Biliar/diagnóstico , Proteínas de Membrana/imunologia , Mitocôndrias/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Feminino , Humanos , Cirrose Hepática Biliar/sangue , Cirrose Hepática Biliar/imunologia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
11.
Front Immunol ; 10: 2949, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31956324

RESUMO

Beneficial effects of probiotics on gut microbiota homeostasis and inflammatory immune responses suggested the investigation of their potential clinical efficacy in experimental models of autoimmune diseases. Indeed, administration of two bifidobacteria and lactobacilli probiotic strains prevented disease manifestations in the Lewis rat model of Myasthenia Gravis (EAMG). Here, we demonstrate the clinical efficacy of therapeutic administration of vital bifidobacteria (i.e., from EAMG onset). The mechanisms involved in immunomodulation were investigated with ex vivo and in vitro experiments. Improvement of EAMG symptoms was associated to decreased anti-rat AChR antibody levels, and differential expression of TGFß and FoxP3 immunoregulatory transcripts in draining lymph nodes and spleen of treated-EAMG rats. Exposure of rat bone marrow-derived dendritic cells to bifidobacteria or lactobacilli strains upregulated toll-like receptor 2 mRNA expression, a key molecule involved in bacterium recognition via lipotheicoic acid. Live imaging experiments of AChR-specific effector T cells, co-cultured with BMDCs pre-exposed to bifidobacteria, demonstrated increased percentages of motile effector T cells, suggesting a hindered formation of TCR-peptide-MHC complex. Composition of gut microbiota was studied by 16S rRNA gene sequencing, and α and ß diversity were determined in probiotic treated EAMG rats, with altered ratios between Tenericutes and Verrucomicrobia (phylum level), and Ruminococcaceae and Lachnospiraceae (family level). Moreover, the relative abundance of Akkermansia genus was found increased compared to healthy and probiotic treated EAMG rats. In conclusion, our findings confirms that the administration of vital bifidobacteria at EAMG onset has beneficial effects on disease progression; this study further supports preclinical research in human MG to evaluate probiotic efficacy as supplementary therapy in MG.


Assuntos
Bifidobacterium , Miastenia Gravis Autoimune Experimental/etiologia , Probióticos/administração & dosagem , Animais , Autoimunidade , Movimento Celular , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal , Sequenciamento de Nucleotídeos em Larga Escala , Metagenoma , Metagenômica/métodos , RNA Ribossômico 16S , Ratos , Ratos Endogâmicos Lew
12.
Sci Rep ; 8(1): 16775, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425261

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date, but the precise NAADP/Ca2+ signalling mechanisms are still controversial. We report the synthesis of small-molecule inhibitors of NAADP-induced Ca2+ release based upon the nicotinic acid motif. Alkylation of nicotinic acid with a series of bromoacetamides generated a diverse compound library. However, many members were only weakly active or had poor physicochemical properties. Structural optimisation produced the best inhibitors that interact specifically with the NAADP/Ca2+ release mechanism, having no effect on Ca2+ mobilized by the other well-known second messengers D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] or cyclic adenosine 5'-diphospho-ribose (cADPR). Lead compound (2) was an efficient antagonist of NAADP-evoked Ca2+ release in vitro in intact T lymphocytes and ameliorated clinical disease in vivo in a rat experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis. Compound (3) (also known as BZ194) was synthesized as its bromide salt, confirmed by crystallography, and was more membrane permeant than 2. The corresponding zwitterion (3a), was also prepared and studied by crystallography, but 3 had more desirable physicochemical properties. 3 Is potent in vitro and in vivo and has found widespread use as a tool to modulate NAADP effects in autoimmunity and cardiovascular applications. Taken together, data suggest that the NAADP/Ca2+ signalling mechanism may serve as a potential target for T cell- or cardiomyocyte-related diseases such as multiple sclerosis or arrhythmia. Further modification of these lead compounds may potentially result in drug candidates of clinical use.


Assuntos
Cálcio/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , NADP/análogos & derivados , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Modelos Moleculares , Conformação Molecular , NADP/farmacologia , Ratos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade
13.
World Neurosurg ; 120: e380-e391, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30144594

RESUMO

BACKGROUND: Glioblastoma (GBM) is the most common and fatal human brain tumor, with the worst prognosis. The aberrant microenvironment, enhanced by the activation of proangiogenic mediators such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), and their downstream effectors, sustain GBM malignancy. Proangiogenic signaling represents an attractive chemotherapeutic target. Recent evidence suggests a therapeutic benefit from aspirin (acetylsalicylic acid, or ASA) intake in reducing risk and cancer progression. METHODS: In the present study, human primary GBM-endothelial cells (ECs) were used to ascertain whether ASA could inhibit angiogenesis and improve cell sensitivity to drugs. The impact of ASA was observed by measuring cell viability, tube-like structure formation, migration, VEGF production, and proliferative, proangiogenic, and apoptotic modulators expression, such as HIF-1α/VEGF/vascular endothelial growth factor receptor/(VEGFR)-1/VEGFR-2, Ras/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase, phosphoinositide 3-kinase/AKT signaling axis, and Bcl-2-associated X protein/B-cell lymphoma 2 (BCL-2) ratio. Furthermore, we evaluated the effect of ASA alone or in combination with temozolomide (TMZ), bevacizumab (BEV), and sunitinib (SUN). RESULTS: Our data reported that ASA affected GBM-EC viability, tube-like structure formation, cell migration, and VEGF releasing in a dose-dependent manner and that combined treatments with TMZ, BEV, and SUN synergized to counteract proangiogenic cell ability. mRNA expression analysis displayed a marked effect of ASA in reducing VEGF, VEGFR-1, HIF-1α, RAS, mitogen-activated protein kinase kinase, AKT, and BCL-2, as well a combined anticancer effect of ASA together with TMZ, BEV, and SUN. Levels of HIF-1α, VEGFR-2, Bcl-2-associated X protein, and BCL-2 protein expression confirmed a positive trend. CONCLUSIONS: ASA and antiangiogenic therapies showed synergetic anticancer efficacy in human primary GBM-ECs. Thus, the combination of conventional chemotherapy with ASA may offer a new strategy to counteract tumor malignancy.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Aspirina/farmacologia , Bevacizumab/farmacologia , Neoplasias Encefálicas/irrigação sanguínea , Células Endoteliais/efeitos dos fármacos , Glioblastoma/irrigação sanguínea , Sunitinibe/farmacologia , Temozolomida/farmacologia , Inibidores da Angiogênese/farmacologia , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Imunológicos/farmacologia , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Células Endoteliais/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/efeitos dos fármacos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteína X Associada a bcl-2/efeitos dos fármacos , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteínas ras/efeitos dos fármacos , Proteínas ras/genética , Proteínas ras/metabolismo
14.
Sci Rep ; 8(1): 8748, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29884885

RESUMO

Gliomas are the most common brain tumors, with diverse biological behaviour. Glioblastoma (GBM), the most aggressive and with the worst prognosis, is characterized by an intense and aberrant angiogenesis, which distinguishes it from low-grade gliomas (LGGs) and benign expansive lesions, as meningiomas (MNGs). With increasing evidence for the importance of vascularization in tumor biology, we focused on the isolation and characterization of endothelial cells (ECs) from primary GBMs, LGGs and MNGs. Gene expression analysis by Real-Time PCR, immunofluorescence and flow cytometry analysis, tube-like structures formation and vascular permeability assays were performed. Our results showed a higher efficiency of ECs to form a complex vascular architecture, as well as a greater impairment of a brain blood barrier model, and an overexpression of pro-angiogenic mediators in GBM than in LGG and MNG. Furthermore, administration of temozolomide, bevacizumab, and sunitinib triggered a different proliferative, apoptotic and angiogenic response, in a dose and time-dependent manner. An increased resistance to temozolomide was observed in T98G cells co-cultured in GBM-EC conditioned media. Therefore, we developed a novel platform to reproduce tumor vascularization as "disease in a dish", which allows us to perform screening of sensitivity/resistance to drugs, in order to optimize targeted approaches to GBM therapy.


Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Glioblastoma/irrigação sanguínea , Glioblastoma/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/patologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Glioblastoma/patologia , Humanos , Pessoa de Meia-Idade , Neovascularização Patológica/patologia , Medicina de Precisão/métodos , Células Tumorais Cultivadas
15.
Oncotarget ; 9(32): 22269-22287, 2018 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-29854277

RESUMO

Probiotics beneficial effects on the host are associated with regulation of the intestinal microbial homeostasis and with modulation of inflammatory immune responses in the gut and in periphery. In this study, we investigated the clinical efficacy of two lactobacillus and two bifidobacterium probiotic strains in experimental autoimmune myasthenia gravis (EAMG) and experimental autoimmune encephalomyelitis (EAE) models, induced in Lewis rats. Treatment with probiotics led to less severe disease manifestation in both models; ex vivo analyses showed preservation of neuromuscular junction in EAMG and myelin content in EAE spinal cord. Immunoregulatory transcripts were found differentially expressed in gut associated lymphoid tissue and in peripheral immunocompetent organs. Feeding EAMG animals with probiotics resulted in increased levels of Transforming Growth Factor-ß (TGFß) in serum, and increased percentages of regulatory T cells (Treg) in peripheral blood leukocyte. Exposure of immature dendritic cells to probiotics induced their maturation toward an immunomodulatory phenotype, and secretion of TGFß. Our data showed that bifidobacteria and lactobacilli treatment effectively modulates disease symptoms in EAMG and EAE models, and support further investigations to evaluate their use in autoimmune diseases.

16.
Oncotarget ; 8(56): 95256-95269, 2017 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-29221125

RESUMO

Tectonic family member 2 (TCTN2) encodes a transmembrane protein that belongs to the tectonic family, which is involved in ciliary functions. Previous studies have demonstrated the role of tectonics in regulating a variety of signaling pathways at the transition zone of cilia. However, the role of tectonics in cancer is still unclear. Here we identify that TCTN2 is overexpressed in colorectal, lung and ovary cancers. We show that different cancer cell lines express the protein that localizes at the plasma membrane, facing the intracellular milieu. TCTN2 over-expression in cancer cells resulted in an increased ability to form colonies in an anchorage independent way. On the other hand, downregulation of TCTN2 using targeted epigenetic editing in cancer cells significantly reduced colony formation, cell invasiveness, increased apoptosis and impaired assembly of primary cilia. Taken together, our results indicate that TCTN2 acts as an oncogene, making it an interesting cancer-associated protein and a potential candidate for therapeutic applications.

17.
Clin Immunol ; 173: 133-146, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27720845

RESUMO

Multiple Sclerosis (MS) is an inflammatory disease with neurodegenerative alterations, ultimately progressing to neurological handicap. Therapies are effective in counteracting inflammation but not neurodegeneration. Biomarkers predicting disease course or treatment response are lacking. We investigated whether altered gene and protein expression profiles were detectable in the peripheral blood of 78 relapsing remitting MS (RR-MS) patients treated by disease-modifying therapies. A discovery/validation study on RR-MS responsive to glatiramer acetate identified 8 differentially expressed genes: ITGA2B, ITGB3, CD177, IGJ, IL5RA, MMP8, P2RY12, and S100ß. A longitudinal study on glatiramer acetate, Interferon-ß, or Fingolimod treated RR-MS patients confirmed that 7 out of 8 genes were downregulated with reference to the different therapies, whereas S100ß was always upregulated. Thus, we identified a peripheral gene signature associated with positive response in RR-MS which may also explain drug immunomodulatory effects. The usefulness of this signature as a biomarker needs confirmation on larger series of patients.


Assuntos
Fatores Imunológicos/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/genética , Transcriptoma/efeitos dos fármacos , Adulto , Feminino , Cloridrato de Fingolimode/uso terapêutico , Perfilação da Expressão Gênica , Acetato de Glatiramer/uso terapêutico , Humanos , Interferon beta/uso terapêutico , Leucócitos Mononucleares/metabolismo , Masculino , Esclerose Múltipla Recidivante-Remitente/sangue , Adulto Jovem
18.
Int J Gen Med ; 9: 53-64, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27019601

RESUMO

Myasthenia gravis (MG) is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction. Antibodies directed against the acetylcholine receptor (AChR) induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in functional reduction in AChR availability. Besides anti-AChR antibodies, other autoantibodies are known to play pathogenic roles in MG. The experimental autoimmune MG (EAMG) models have been of great help over the years in understanding the pathophysiological role of specific autoantibodies and T helper lymphocytes and in suggesting new therapies for prevention and modulation of the ongoing disease. EAMG can be induced in mice and rats of susceptible strains that show clinical symptoms mimicking the human disease. EAMG models are helpful for studying both the muscle and the immune compartments to evaluate new treatment perspectives. In this review, we concentrate on recent findings on EAMG models, focusing on their utility and limitations.

19.
NMR Biomed ; 28(3): 327-37, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25639498

RESUMO

The pool size ratio measured by quantitative magnetization transfer MRI is hypothesized to closely reflect myelin density, but their relationship has so far been confirmed mostly in ex vivo conditions. We investigate the correspondence between this parameter measured in vivo at 7.0 T, with Black Gold II staining for myelin fibres, and with myelin basic protein and beta-tubulin immunofluorescence in a hybrid longitudinal study of C57BL/6 and SJL/J mice treated with cuprizone, a neurotoxicant causing relatively selective myelin loss followed by spontaneous remyelination upon treatment suspension. Our results confirm that pool size ratio measurements correlate with myelin content, with the correlation coefficient depending on strain and staining method, and demonstrate the in vivo applicability of this MRI technique to experimental mouse models of multiple sclerosis.


Assuntos
Fenômenos Magnéticos , Imageamento por Ressonância Magnética , Bainha de Mielina/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cuprizona , Feminino , Masculino , Camundongos Endogâmicos C57BL , Bainha de Mielina/metabolismo
20.
J Am Chem Soc ; 136(24): 8524-7, 2014 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-24884816

RESUMO

(19)F-MRI offers unique opportunities to image diseases and track cells and therapeutic agents in vivo. Herein we report a superfluorinated molecular probe, herein called PERFECTA, possessing excellent cellular compatibility, and whose spectral properties, relaxation times, and sensitivity are promising for in vivo (19)F-MRI applications. The molecule, which bears 36 equivalent (19)F atoms and shows a single intense resonance peak, is easily synthesized via a simple one-step reaction and is formulated in water with high stability using trivial reagents and methods.


Assuntos
Radioisótopos de Flúor/farmacocinética , Hidrocarbonetos Fluorados/farmacocinética , Imageamento por Ressonância Magnética , Animais , Radioisótopos de Flúor/administração & dosagem , Radioisótopos de Flúor/química , Hidrocarbonetos Fluorados/administração & dosagem , Hidrocarbonetos Fluorados/química , Injeções Subcutâneas , Modelos Moleculares , Estrutura Molecular , Ratos , Ratos Endogâmicos Lew , Distribuição Tecidual
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