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1.
Eur Urol Focus ; 2019 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-31813809

RESUMO

BACKGROUND: TCEB1-mutant renal cell carcinoma (RCC) is a rare variant of RCC with clear-cell features. Owing to its unique morphological and molecular features it has recently been proposed as a separate entity. Initial series suggested an indolent, early-stage phenotype. Here we expand our clinical cohort and describe a more detailed genomic analysis looking for potential drivers of aggressiveness. DESIGN, SETTING, AND PARTICIPANTS: We identified five new cases in our institutional sequencing cohort, four of whom were found to have high-stage disease (American Joint Committee on Cancer stage III/IV). Twelve previously reported cases were pooled for comparison purposes (Sato, The Cancer Genome Atlas, TRACERx Renal). OUTCOME MEASURES AND STATISTICAL ANALYSIS: We used our previously validated pipeline to analyze somatic mutations and copy number alterations (CNAs) in seven tumor samples with available data and estimated the number of cancer cells bearing each somatic mutation. The oncogenic potential of mutations was assessed using OncoKB and two other algorithms. Mann-Whitney U tests were used to evaluate differences in genomic markers between stage groups. RESULTS AND LIMITATIONS: All tumors showed biallelic inactivation of the TCEB1 gene according to a combination of somatic mutation and CNA analyses. Mutations were always found in residues involved in hydrophobic interactions with VHL. We found that high-stage tumors had additional oncogenic mutations (median 1, interquartile range [IQR] 1-1 vs 2, IQR 2-2; median difference 1, 95% confidence interval [CI] 1-1; p= 0.002) and showed whole-genome doubling events. They also seemed to have a higher burden of somatic CNAs (median fraction CNA genome 0.10, IQR 0.10-0.15 vs 0.63, IQR 0.58-0.68), however, this finding did not reach statistical significance (median difference 0.49, 95% CI 0.33-0.63; p=0.052). CONCLUSIONS: TCEB1-mutant RCC can show variable behavior ranging from very indolent to aggressive. Specific molecular events leading to high genomic instability seem to be associated with aggressiveness. This study expands the clinical spectrum of TCEB1-mutant RCC. PATIENT SUMMARY: We present four cases of aggressive TCEB1-mutant renal cell carcinoma, a rare type of kidney cancer. In-depth analysis of the genomes of these tumors revealed certain abnormalities that might explain this aggressive behavior.

2.
J Neuroinflammation ; 15(1): 296, 2018 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-30367633

RESUMO

BACKGROUND: Recent studies in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), suggest an involvement of the histone methyltransferase enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) in important processes such as cell adhesion and migration. METHODS: Here, we aimed to expand these initial observations by investigating the role of EZH2 in MS. mRNA expression levels for EZH2 were measured by real-time PCR in peripheral blood mononuclear cells (PBMC) from 121 MS patients (62 untreated and 59 receiving treatment) and 24 healthy controls. RESULTS: EZH2 expression levels were decreased in PBMC from untreated patients compared to that from controls, and treatment significantly upregulated EZH2 expression. Expression of miR-124 was increased in MS patients compared to controls. Blood immunophenotyping revealed EZH2 expression mostly restricted to CD4+ and CD8+ T cells, and circulating EZH2+ CD4+ and CD8+ T cells were decreased in untreated MS patients compared to controls. CD8+ T cells expressing EZH2 exhibited a predominant central memory phenotype, whereas EZH2+ CD4+ T cells were of effector memory nature, and both T cell subsets produced TNF-α. EZH2+ T cells were enriched in the cerebrospinal fluid compartment compared to blood and were found in chronic active lesions from MS patients. EZH2 inhibition and microarray analysis in PBMC was associated with significant downregulation of key T cell adhesion molecules. CONCLUSION: These findings suggest a role of EZH2 in the migration of T cells in MS patients. The observation of TNF-α expression by CD4+ and CD8+ T cells expressing EZH2 warrants additional studies to explore more in depth the pathogenic potential of EZH2+-positive cells in MS.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla Crônica Progressiva/patologia , Esclerose Múltipla Recidivante-Remitente/patologia , Adulto , Animais , Estudos de Coortes , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , Adjuvante de Freund/toxicidade , Humanos , Leucócitos Mononucleares/classificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Esclerose Múltipla Crônica Progressiva/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidade , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Subpopulações de Linfócitos T , Talina/genética , Talina/metabolismo , Adulto Jovem
3.
Front Immunol ; 9: 253, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29491866

RESUMO

Type 1 diabetes (T1D) is a metabolic disease caused by the autoimmune destruction of insulin-producing ß-cells. With its incidence increasing worldwide, to find a safe approach to permanently cease autoimmunity and allow ß-cell recovery has become vital. Relying on the inherent ability of apoptotic cells to induce immunological tolerance, we demonstrated that liposomes mimicking apoptotic ß-cells arrested autoimmunity to ß-cells and prevented experimental T1D through tolerogenic dendritic cell (DC) generation. These liposomes contained phosphatidylserine (PS)-the main signal of the apoptotic cell membrane-and ß-cell autoantigens. To move toward a clinical application, PS-liposomes with optimum size and composition for phagocytosis were loaded with human insulin peptides and tested on DCs from patients with T1D and control age-related subjects. PS accelerated phagocytosis of liposomes with a dynamic typical of apoptotic cell clearance, preserving DCs viability. After PS-liposomes phagocytosis, the expression pattern of molecules involved in efferocytosis, antigen presentation, immunoregulation, and activation in DCs concurred with a tolerogenic functionality, both in patients and control subjects. Furthermore, DCs exposed to PS-liposomes displayed decreased ability to stimulate autologous T cell proliferation. Moreover, transcriptional changes in DCs from patients with T1D after PS-liposomes phagocytosis pointed to an immunoregulatory prolife. Bioinformatics analysis showed 233 differentially expressed genes. Genes involved in antigen presentation were downregulated, whereas genes pertaining to tolerogenic/anti-inflammatory pathways were mostly upregulated. In conclusion, PS-liposomes phagocytosis mimics efferocytosis and leads to phenotypic and functional changes in human DCs, which are accountable for tolerance induction. The herein reported results reinforce the potential of this novel immunotherapy to re-establish immunological tolerance, opening the door to new therapeutic approaches in the field of autoimmunity.


Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Tolerância Imunológica/imunologia , Fosfatidilserinas/imunologia , Adolescente , Adulto , Autoantígenos/imunologia , Células Cultivadas , Feminino , Humanos , Imunoterapia/métodos , Lipossomos , Masculino , Pessoa de Meia-Idade , Mimetismo Molecular/imunologia , Fagocitose , Adulto Jovem
4.
Brain ; 141(4): 1085-1093, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29452342

RESUMO

The prognostic role of cerebrospinal fluid molecular biomarkers determined in early pathogenic stages of multiple sclerosis has yet to be defined. In the present study, we aimed to investigate the prognostic value of chitinase 3 like 1 (CHI3L1), neurofilament light chain, and oligoclonal bands for conversion to clinically isolated syndrome and to multiple sclerosis in 75 patients with radiologically isolated syndrome. Cerebrospinal fluid levels of CHI3L1 and neurofilament light chain were measured by enzyme-linked immunosorbent assay. Uni- and multivariable Cox regression models including as covariates age at diagnosis of radiologically isolated syndrome, number of brain lesions, sex and treatment were used to investigate associations between cerebrospinal fluid CHI3L1 and neurofilament light chain levels and time to conversion to clinically isolated syndrome and multiple sclerosis. Neurofilament light chain levels and oligoclonal bands were independent risk factors for the development of clinically isolated syndrome (hazard ratio = 1.02, P = 0.019, and hazard ratio = 14.7, P = 0.012, respectively) and multiple sclerosis (hazard ratio = 1.03, P = 0.003, and hazard ratio = 8.9, P = 0.046, respectively). The best cut-off to classify cerebrospinal fluid neurofilament light chain levels into high and low was 619 ng/l, and high neurofilament light chain levels were associated with a trend to shorter time to clinically isolated syndrome (P = 0.079) and significant shorter time to multiple sclerosis (P = 0.017). Similarly, patients with radiologically isolated syndrome presenting positive oligoclonal bands converted faster to clinically isolated syndrome and multiple sclerosis (P = 0.005 and P = 0.008, respectively). The effects of high neurofilament light chain levels shortening time to clinically isolated syndrome and multiple sclerosis were more pronounced in radiologically isolated syndrome patients with ≥37 years compared to younger patients. Cerebrospinal fluid CHI3L1 levels did not influence conversion to clinically isolated syndrome and multiple sclerosis in radiologically isolated syndrome patients. Overall, these findings suggest that cerebrospinal neurofilament light chain levels and oligoclonal bands are independent predictors of clinical conversion in patients with radiologically isolated syndrome. The association with a faster development of multiple sclerosis reinforces the importance of cerebrospinal fluid analysis in patients with radiologically isolated syndrome.


Assuntos
Biomarcadores/líquido cefalorraquidiano , Proteína 1 Semelhante à Quitinase-3/líquido cefalorraquidiano , Doenças Desmielinizantes/líquido cefalorraquidiano , Doenças Desmielinizantes/diagnóstico , Proteínas de Neurofilamentos/líquido cefalorraquidiano , Bandas Oligoclonais/líquido cefalorraquidiano , Adulto , Estudos de Coortes , Europa (Continente) , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estatísticas não Paramétricas
5.
J Nutr Biochem ; 48: 36-43, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28692847

RESUMO

The study of biomarkers of dietary patterns including the Mediterranean diet (MedDiet) is scarce and could improve the assessment of these patterns. Moreover, it could provide a better understanding of health benefits of dietary patterns in nutritional epidemiology. We aimed to determine a robust and accurate biomarker associated with a high adherence to a MedDiet pattern that included dietary assessment and its biological effect. In this cross-sectional study, we included 56 and 63 individuals with high (H-MDA) and low (L-MDA) MedDiet adherence categories, respectively, all from the Prevención con Dieta Mediterránea trial. A 1H-NMR-based untargeted metabolomics approach was applied to urine samples. Multivariate statistical analyses were conducted to determine the metabolite differences between groups. A stepwise logistic regression and receiver operating characteristic curves were used to build and evaluate the prediction model for H-MDA. Thirty-four metabolites were identified as discriminant between H-MDA and L-MDA. The fingerprint associated with H-MDA included higher excretion of proline betaine and phenylacetylglutamine, among others, and decreased amounts of metabolites related to glucose metabolism. Three microbial metabolites - phenylacetylglutamine, p-cresol and 4-hydroxyphenylacetate - were included in the prediction model of H-MDA (95% specificity, 95% sensitivity and 97% area under the curve). The model composed of microbial metabolites was the biomarker that defined high adherence to a Mediterranean dietary pattern. The overall metabolite profiling identified reflects the metabolic modulation produced by H-MDA. The proposed biomarker may be a better tool for assessing and aiding nutritional epidemiology in future associations between H-MDA and the prevention or amelioration of chronic diseases.


Assuntos
Biomarcadores/urina , Dieta Mediterrânea , Microbioma Gastrointestinal/fisiologia , Idoso , Idoso de 80 Anos ou mais , Cresóis/urina , Estudos Transversais , Feminino , Glutamina/análogos & derivados , Glutamina/urina , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Fenilacetatos/urina , Curva ROC
6.
Ann Neurol ; 82(2): 186-195, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28681388

RESUMO

OBJECTIVE: To identify biomarkers associated with the development of progressive multifocal leukoencephalopathy (PML) in multiple sclerosis (MS) patients treated with natalizumab (NTZ). METHODS: Relapsing-remitting MS patients who developed PML under NTZ therapy (pre-PML) and non-PML NTZ-treated patients (NTZ-ctr) were included in the study. Cryopreserved peripheral blood mononuclear cells and serum samples collected at baseline, at 1- and 2-year treated time points, and during PML were analyzed for gene expression by RNA sequencing and for serum protein levels by Luminex and enzyme-linked immunosorbent assays, respectively. RESULTS: Among top differentially expressed genes in the RNA sequencing between pre-PML and NTZ-ctr patients, pathway analysis revealed a high representation of genes belonging to the following categories: proangiogenic factors (MMP9, VEGFA), chemokines (CXCL1, CXCL5, IL8, CCL2), cytokines (IL1B, IFNG), and plasminogen- and coagulation-related molecules (SERPINB2, PLAU, PLAUR, TFPI, THBD). Serum protein levels for these candidates were measured in a 2-step manner in a screening cohort and a validation cohort of pre-PML and NTZ-ctr patients. Only matrix metalloproteinase 9 (MMP9) was validated; in pre-PML patients, MMP9 protein levels were significantly reduced at baseline compared with NTZ-ctr patients, and levels remained lower at later time points during NTZ treatment. INTERPRETATION: The results from this study suggest that the proangiogenic factor MMP9 may play a role as a biomarker associated with the development of PML in MS patients treated with NTZ. Ann Neurol 2017;82:186-195.


Assuntos
Leucoencefalopatia Multifocal Progressiva/induzido quimicamente , Metaloproteinase 9 da Matriz/sangue , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Natalizumab/efeitos adversos , Natalizumab/uso terapêutico , Biomarcadores/sangue , Proteínas Sanguíneas/biossíntese , Expressão Gênica/efeitos dos fármacos , Humanos , Fatores Imunológicos/sangue , Leucoencefalopatia Multifocal Progressiva/sangue , Leucoencefalopatia Multifocal Progressiva/complicações , Metaloproteinase 9 da Matriz/biossíntese , Esclerose Múltipla Recidivante-Remitente/complicações , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/sangue
8.
Mol Cell Endocrinol ; 426: 101-12, 2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-26911933

RESUMO

The transmembrane glycoprotein CD26 or dipeptidyl peptidase IV (DPPIV) is a multifunctional protein. In immune system, CD26 plays a role in T-cell function and is also involved in thymic maturation and emigration patterns. In preclinical studies, treatment with DPPIV inhibitors reduces insulitis and delays or even reverses the new -onset of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. However, the specific mechanisms involved in these effects remain unknown. The aim of the present study was to investigate how DPPIV inhibition modifies the expression of genes in the thymus of NOD mice by microarray analysis. Changes in the gene expression of ß-cell autoantigens and Aire in thymic epithelial cells (TECs) were also evaluated by using qRT-PCR. A DPPIV inhibitor, MK626, was orally administered in the diet for 4 and 6 weeks starting at 6-8 weeks of age. Thymic glands from treated and control mice were obtained for each study checkpoint. Thymus transcriptome analysis revealed that 58 genes were significantly over-expressed in MK626-treated mice after 6 weeks of treatment. Changes in gene expression in the thymus were confined mainly to the immune system, including innate immunity, chemotaxis, antigen presentation and immunoregulation. Most of the genes are implicated in central tolerance mechanisms through several pathways. No differences were observed in the expression of Aire and ß-cell autoantigens in TECs. In the current study, we demonstrate that treatment with the DPPIV inhibitor MK626 in NOD mice alters the expression of the immune response-related genes in the thymus, especially those related to immunological central tolerance, and may contribute to the prevention of T1D.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Indinavir/farmacologia , Animais , Apresentação do Antígeno/genética , Feminino , Regulação da Expressão Gênica/imunologia , Redes Reguladoras de Genes , Imunomodulação/genética , Camundongos Endogâmicos NOD , Timo/efeitos dos fármacos , Timo/metabolismo , Transcriptoma
9.
Br J Sports Med ; 49(13): 843-51, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26084524

RESUMO

The health, fitness and other advantages of youth sports participation are well recognised. However, there are considerable challenges for all stakeholders involved-especially youth athletes-in trying to maintain inclusive, sustainable and enjoyable participation and success for all levels of individual athletic achievement. In an effort to advance a more unified, evidence-informed approach to youth athlete development, the IOC critically evaluated the current state of science and practice of youth athlete development and presented recommendations for developing healthy, resilient and capable youth athletes, while providing opportunities for all levels of sport participation and success. The IOC further challenges all youth and other sport governing bodies to embrace and implement these recommended guiding principles.


Assuntos
Esportes Juvenis/fisiologia , Doença Aguda , Adolescente , Desenvolvimento do Adolescente/fisiologia , Aptidão/fisiologia , Traumatismos em Atletas/etiologia , Traumatismos em Atletas/prevenção & controle , Desempenho Atlético/fisiologia , Criança , Doença Crônica , Competência Clínica/normas , Diosgenina , Meio Ambiente , Exercício/fisiologia , Fadiga/fisiopatologia , Feminino , Nível de Saúde , Humanos , Masculino , Força Muscular/fisiologia , Músculo Esquelético/metabolismo , Transtornos Nutricionais/prevenção & controle , Consumo de Oxigênio/fisiologia , Abuso Físico/prevenção & controle , Educação Física e Treinamento/métodos , Aptidão Física/fisiologia , Fitosteróis , Puberdade/fisiologia , Delitos Sexuais/prevenção & controle , Sono/fisiologia , Medicina Esportiva/normas , Estresse Psicológico/etiologia
10.
PLoS One ; 10(6): e0127057, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26039878

RESUMO

INTRODUCTION: The development of new therapies to induce self-tolerance has been an important medical health challenge in type 1 diabetes. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow ß-cell regeneration. Based on the immunomodulatory effects of apoptosis, we hypothesized that apoptotic mimicry can help to restore tolerance lost in autoimmune diabetes. OBJECTIVE: To generate a synthetic antigen-specific immunotherapy based on apoptosis features to specifically reestablish tolerance to ß-cells in type 1 diabetes. METHODS: A central event on the surface of apoptotic cells is the exposure of phosphatidylserine, which provides the main signal for efferocytosis. Therefore, phosphatidylserine-liposomes loaded with insulin peptides were generated to simulate apoptotic cells recognition by antigen presenting cells. The effect of antigen-specific phosphatidylserine-liposomes in the reestablishment of peripheral tolerance was assessed in NOD mice, the spontaneous model of autoimmune diabetes. MHC class II-peptide tetramers were used to analyze the T cell specific response after treatment with phosphatidylserine-liposomes loaded with peptides. RESULTS: We have shown that phosphatidylserine-liposomes loaded with insulin peptides induce tolerogenic dendritic cells and impair autoreactive T cell proliferation. When administered to NOD mice, liposome signal was detected in the pancreas and draining lymph nodes. This immunotherapy arrests the autoimmune aggression, reduces the severity of insulitis and prevents type 1 diabetes by apoptotic mimicry. MHC class II tetramer analysis showed that peptide-loaded phosphatidylserine-liposomes expand antigen-specific CD4+ T cells in vivo. The administration of phosphatidylserine-free liposomes emphasizes the importance of phosphatidylserine in the modulation of antigen-specific CD4+ T cell expansion. CONCLUSIONS: We conclude that this innovative immunotherapy based on the use of liposomes constitutes a promising strategy for autoimmune diseases.


Assuntos
Autoantígenos/imunologia , Autoimunidade , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Fosfatidilserinas/uso terapêutico , Animais , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular , Citocinas/metabolismo , Células Dendríticas/imunologia , Imunoterapia , Injeções Intraperitoneais , Insulina/uso terapêutico , Lipossomos , Camundongos Endogâmicos NOD , Fenótipo
11.
Clin Cancer Res ; 21(16): 3695-704, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-25944804

RESUMO

PURPOSE: The clinical management of colorectal cancer patients has significantly improved because of the identification of novel therapeutic targets such as EGFR and VEGF. Because rapid tumor proliferation is associated with poor patient prognosis, here we characterized the transcriptional signature of rapidly proliferating colorectal cancer cells in an attempt to identify novel candidate therapeutic targets. EXPERIMENTAL DESIGN: The doubling time of 52 colorectal cancer cell lines was determined and genome-wide expression profiling of a subset of these lines was assessed by microarray analysis. We then investigated the potential of genes highly expressed in cancer cells with faster growth as new therapeutic targets. RESULTS: Faster proliferation rates were associated with microsatellite instability and poorly differentiated histology. The expression of 1,290 genes was significantly correlated with the growth rates of colorectal cancer cells. These included genes involved in cell cycle, RNA processing/splicing, and protein transport. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and protoporphyrinogen oxidase (PPOX) were shown to have higher expression in faster growing cell lines and primary tumors. Pharmacologic or siRNA-based inhibition of GAPDH or PPOX reduced the growth of colon cancer cells in vitro. Moreover, using a mouse xenograft model, we show that treatment with the specific PPOX inhibitor acifluorfen significantly reduced the growth of three of the seven (42.8%) colon cancer lines investigated. CONCLUSIONS: We have characterized at the transcriptomic level the differences between colorectal cancer cells that vary in their growth rates, and identified novel candidate chemotherapeutic targets for the treatment of colorectal cancer.


Assuntos
Neoplasias Colorretais/genética , Flavoproteínas/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Proteínas Mitocondriais/biossíntese , Proteínas de Neoplasias/biossíntese , Protoporfirinogênio Oxidase/biossíntese , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Feminino , Flavoproteínas/antagonistas & inibidores , Flavoproteínas/genética , Regulação Neoplásica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Células HCT116 , Humanos , Masculino , Camundongos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/genética , Terapia de Alvo Molecular , Proteínas de Neoplasias/genética , Nitrobenzoatos/administração & dosagem , Transporte Proteico/genética , Protoporfirinogênio Oxidase/antagonistas & inibidores , Protoporfirinogênio Oxidase/genética , Processamento de RNA/genética , RNA Interferente Pequeno , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Alzheimers Dis ; 45(2): 407-21, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25790933

RESUMO

Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AßPP/PS1 mouse model of familial Alzheimer's disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AßPP/PS1 transgenic mice. EPO and CEPO improved memory in AßPP/PS1 animals. However, only EPO decreased amyloid-ß (Aß)plaque burden and soluble Aß(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AßPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AßPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.


Assuntos
Doença de Alzheimer/complicações , Eritropoetina/análogos & derivados , Regulação da Expressão Gênica/efeitos dos fármacos , Transtornos da Memória/tratamento farmacológico , Transtornos da Memória/etiologia , Sinapses/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Modelos Animais de Doenças , Eritropoetina/uso terapêutico , Peptídeo Liberador de Gastrina/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Receptores de Catecolaminas/metabolismo , Sinapses/genética , Fatores de Tempo
13.
Brain ; 138(Pt 4): 918-31, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25688078

RESUMO

Chitinase 3-like 1 (CHI3L1) has been proposed as a biomarker associated with the conversion to clinically definite multiple sclerosis in patients with clinically isolated syndromes, based on the finding of increased cerebrospinal fluid CHI3L1 levels in clinically isolated syndrome patients who later converted to multiple sclerosis compared to those who remained as clinically isolated syndrome. Here, we aimed to validate CHI3L1 as a prognostic biomarker in a large cohort of patients with clinically isolated syndrome. This is a longitudinal cohort study of clinically isolated syndrome patients with clinical, magnetic resonance imaging, and cerebrospinal fluid data prospectively acquired. A total of 813 cerebrospinal fluid samples from patients with clinically isolated syndrome were recruited from 15 European multiple sclerosis centres. Cerebrospinal fluid CHI3L1 levels were measured by enzyme-linked immunosorbent assay. Multivariable Cox regression models were used to investigate the association between cerebrospinal fluid CHI3L1 levels and time to conversion to multiple sclerosis and time to reach Expanded Disability Status Scale 3.0. CHI3L1 levels were higher in patients who converted to clinically definite multiple sclerosis compared to patients who continued as clinically isolated syndrome (P = 8.1 × 10(-11)). In the Cox regression analysis, CHI3L1 levels were a risk factor for conversion to multiple sclerosis (hazard ratio = 1.7; P = 1.1 × 10(-5) using Poser criteria; hazard ratio = 1.6; P = 3.7 × 10(-6) for McDonald criteria) independent of other covariates such as brain magnetic resonance imaging abnormalities and presence of cerebrospinal fluid oligoclonal bands, and were the only significant independent risk factor associated with the development of disability (hazard ratio = 3.8; P = 2.5 × 10(-8)). High CHI3L1 levels were associated with shorter time to multiple sclerosis (P = 3.2 × 10(-9) using Poser criteria; P = 5.6 × 10(-11) for McDonald criteria) and more rapid development of disability (P = 1.8 × 10(-10)). These findings validate cerebrospinal fluid CHI3L1 as a biomarker associated with the conversion to multiple sclerosis and development of disability and reinforce the prognostic role of CHI3L1 in patients with clinically isolated syndrome. We propose that determining cerebrospinal fluid chitinase 3-like 1 levels at the time of a clinically isolated syndrome event will help identify those patients with worse disease prognosis.


Assuntos
Adipocinas/líquido cefalorraquidiano , Doenças Desmielinizantes/líquido cefalorraquidiano , Doenças Desmielinizantes/diagnóstico , Lectinas/líquido cefalorraquidiano , Adipocinas/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/líquido cefalorraquidiano , Encéfalo/metabolismo , Encéfalo/patologia , Proteína 1 Semelhante à Quitinase-3 , Feminino , Seguimentos , Humanos , Lectinas/biossíntese , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
14.
J Proteome Res ; 13(8): 3706-3721, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24897304

RESUMO

Secretome profiling has become a methodology of choice for the identification of tumor biomarkers. We hypothesized that due to the dynamic nature of secretomes cellular perturbations could affect their composition but also change the global amount of protein secreted per cell. We confirmed our hypothesis by measuring the levels of secreted proteins taking into account the amount of proteome produced per cell. Then, we established a correlation between cell proliferation and protein secretion that explained the observed changes in global protein secretion. Next, we implemented a normalization correcting the statistical results of secretome studies by the global protein secretion of cells into a generalized linear model (GLM). The application of the normalization to two biological perturbations on tumor cells resulted in drastic changes in the list of statistically significant proteins. Furthermore, we found that known epithelial-to-mesenchymal transition (EMT) effectors were only statistically significant when the normalization was applied. Therefore, the normalization proposed here increases the sensitivity of statistical tests by increasing the number of true-positives. From an oncology perspective, the correlation between protein secretion and cellular proliferation suggests that slow-growing tumors could have high-protein secretion rates and consequently contribute strongly to tumor paracrine signaling.

15.
Cancer Res ; 74(5): 1416-28, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24390735

RESUMO

Renal cell carcinoma (RCC), the third most prevalent urological cancer, claims more than 100,000 lives/year worldwide. The clear cell variant (ccRCC) is the most common and aggressive subtype of this disease. While commonly asymptomatic, more than 30% of ccRCC are diagnosed when already metastatic, resulting in a 95% mortality rate. Notably, nearly one-third of organ-confined cancers treated by nephrectomy develop metastasis during follow-up care. At present, diagnostic and prognostic biomarkers to screen, diagnose, and monitor renal cancers are clearly needed. The gene encoding the cell surface molecule HAVCR1/KIM-1 is a suggested susceptibility gene for ccRCC and ectodomain shedding of this molecule may be a predictive biomarker of tumor progression. Microarray analysis of 769-P ccRCC-derived cells where HAVCR/KIM-1 levels have been upregulated or silenced revealed relevant HAVCR/KIM-1-related targets, some of which were further analyzed in a cohort of 98 ccRCC patients with 100 month follow-up. We found that HAVCR/KIM-1 activates the IL-6/STAT-3/HIF-1A axis in ccRCC-derived cell lines, which depends on HAVCR/KIM-1 shedding. Moreover, we found that pSTAT-3 S727 levels represented an independent prognostic factor for ccRCC patients. Our results suggest that HAVCR/KIM-1 upregulation in tumors might represent a novel mechanism to activate tumor growth and angiogenesis and that pSTAT-3 S727 is an independent prognostic factor for ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Interleucina-6/genética , Neoplasias Renais/genética , Glicoproteínas de Membrana/genética , Receptores Virais/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-6/metabolismo , Neoplasias Renais/patologia , Glicoproteínas de Membrana/metabolismo , Receptores Virais/metabolismo , Fator de Transcrição STAT3/metabolismo , Regulação para Cima/genética
16.
Bioinformatics ; 30(8): 1104-1111, 2014 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-24389655

RESUMO

Given the inherent dynamics of a viral quasispecies, we are often interested in the comparison of diversity indices of sequential samples of a patient, or in the comparison of diversity indices of virus in groups of patients in a treated versus control design. It is then important to make sure that the diversity measures from each sample may be compared with no bias and within a consistent statistical framework. In the present report, we review some indices often used as measures for viral quasispecies complexity and provide means for statistical inference, applying procedures taken from the ecology field. In particular, we examine the Shannon entropy and the mutation frequency, and we discuss the appropriateness of different normalization methods of the Shannon entropy found in the literature. By taking amplicons ultra-deep pyrosequencing (UDPS) raw data as a surrogate of a real hepatitis C virus viral population, we study through in-silico sampling the statistical properties of these indices under two methods of viral quasispecies sampling, classical cloning followed by Sanger sequencing (CCSS) and next-generation sequencing (NGS) such as UDPS. We propose solutions specific to each of the two sampling methods-CCSS and NGS-to guarantee statistically conforming conclusions as free of bias as possible. CONTACT: josep.gregori@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
Variação Genética , Hepacivirus/genética , Sequenciamento de Nucleotídeos em Larga Escala , Biologia Computacional , RNA Viral/genética , Análise de Sequência de RNA
17.
Liver Int ; 34(6): 896-906, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24134179

RESUMO

BACKGROUND & AIMS: Hepatitis C virus (HCV) transmission from a chronic patient to a susceptible individual is a good opportunity to study viral and host factors that may influence the natural course of hepatitis C infection towards either spontaneous recovery or chronicity. To compare a documented case of a bottleneck event in the sexual transmission of HCV from a chronically infected patient to a recipient host that cleared infection. METHODS: Host genetic components such as Class I and II HLA and IL28B polymorphism (rs12979860 SNPs) were identified by direct sequencing and LightMix analysis, respectively. Deep nucleotide sequence analysis of quasispecies complexity was performed using massive pyrosequencing platform (454 GS-FLX), and the CD4 specific immune response was characterized by ELISPOT. RESULTS AND CONCLUSIONS: Sequencing analysis and CD4 response highlighted several NS3-helicase domains in which an interplay between amino acid variability and CD4 immune response might have contributed either to chronicity in the donor patient or to viral clearance in the receptor (newly infected) patient.


Assuntos
Hepacivirus/patogenicidade , Hepatite C Crônica/transmissão , Interações Hospedeiro-Patógeno , Parceiros Sexuais , Doenças Virais Sexualmente Transmissíveis/transmissão , Abuso de Substâncias por Via Intravenosa/complicações , Adulto , Antivirais/uso terapêutico , Feminino , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C Crônica/diagnóstico , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Humanos , Masculino , Fenótipo , Indução de Remissão , Doenças Virais Sexualmente Transmissíveis/diagnóstico , Doenças Virais Sexualmente Transmissíveis/tratamento farmacológico , Doenças Virais Sexualmente Transmissíveis/imunologia , Doenças Virais Sexualmente Transmissíveis/virologia , Fatores de Tempo , Resultado do Tratamento , Proteínas não Estruturais Virais/genética
18.
J Proteomics ; 95: 55-65, 2013 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-23770383

RESUMO

UNLABELLED: The microarray community has shown that the low reproducibility observed in gene expression-based biomarker discovery studies is partially due to relying solely on p-values to get the lists of differentially expressed genes. Their conclusions recommended complementing the p-value cutoff with the use of effect-size criteria. The aim of this work was to evaluate the influence of such an effect-size filter on spectral counting-based comparative proteomic analysis. The results proved that the filter increased the number of true positives and decreased the number of false positives and the false discovery rate of the dataset. These results were confirmed by simulation experiments where the effect size filter was used to evaluate systematically variable fractions of differentially expressed proteins. Our results suggest that relaxing the p-value cut-off followed by a post-test filter based on effect size and signal level thresholds can increase the reproducibility of statistical results obtained in comparative proteomic analysis. Based on our work, we recommend using a filter consisting of a minimum absolute log2 fold change of 0.8 and a minimum signal of 2-4 SpC on the most abundant condition for the general practice of comparative proteomics. The implementation of feature filtering approaches could improve proteomic biomarker discovery initiatives by increasing the reproducibility of the results obtained among independent laboratories and MS platforms. BIOLOGICAL SIGNIFICANCE: Quality control analysis of microarray-based gene expression studies pointed out that the low reproducibility observed in the lists of differentially expressed genes could be partially attributed to the fact that these lists are generated relying solely on p-values. Our study has established that the implementation of an effect size post-test filter improves the statistical results of spectral count-based quantitative proteomics. The results proved that the filter increased the number of true positives whereas decreased the false positives and the false discovery rate of the datasets. The results presented here prove that a post-test filter applying a reasonable effect size and signal level thresholds helps to increase the reproducibility of statistical results in comparative proteomic analysis. Furthermore, the implementation of feature filtering approaches could improve proteomic biomarker discovery initiatives by increasing the reproducibility of results obtained among independent laboratories and MS platforms. This article is part of a Special Issue entitled: Standardization and Quality Control in Proteomics.


Assuntos
Proteômica/instrumentação , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Proteômica/normas , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
PLoS One ; 8(5): e63296, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23691013

RESUMO

INTRODUCTION: Efferocytosis is a crucial process by which apoptotic cells are cleared by phagocytes, maintaining immune tolerance to self in the absence of inflammation. Peripheral tolerance, lost in autoimmune processes, may be restored by the administration of autologous dendritic cells loaded with islet apoptotic cells in experimental type 1 diabetes. OBJECTIVE: To evaluate tolerogenic properties in dendritic cells induced by the clearance of apoptotic islet cells, thus explaining the re-establishment of tolerance in a context of autoimmunity. METHODS: Bone marrow derived dendritic cells from non-obese diabetic mice, a model of autoimmune diabetes, were generated and pulsed with islet apoptotic cells. The ability of these cells to induce autologous T cell proliferation and to suppress mature dendritic cell function was assessed, together with cytokine production. Microarray experiments were performed using dendritic cells to identify differentially expressed genes after efferocytosis. RESULTS: Molecular and functional changes in dendritic cells after the capture of apoptotic cells were observed. 1) Impaired ability of dendritic cells to stimulate autologous T cell proliferation after the capture of apoptotic cells even after proinflammatory stimuli, with a cytokine profile typical for immature dendritic cells. 2) Suppressive ability of mature dendritic cell function. 3) Microarray-based gene expression profiling of dendritic cells showed differential expression of genes involved in antigen processing and presentation after efferocytosis. 4) Prostaglandin E2 increased production was responsible for immunosuppressive mechanism of dendritic cells after the capture of apoptotic cells. CONCLUSIONS: The tolerogenic behaviour of dendritic cells after islet cells efferocytosis points to a mechanism of silencing potential autoreactive T cells in the microenvironment of autoimmunity. Our results suggest that dendritic cells may be programmed to induce specific immune tolerance using apoptotic cells; this is a viable strategy for a variety of autoimmune diseases.


Assuntos
Autoimunidade , Células Dendríticas/metabolismo , Dinoprostona/biossíntese , Fagocitose , Animais , Proliferação de Células , Células Dendríticas/citologia , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T Reguladores/citologia
20.
Neurology ; 80(22): 2010-6, 2013 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-23624563

RESUMO

OBJECTIVES: To investigate the roles of 2 polymorphisms of the tumor necrosis factor (TNF) receptor superfamily member 1A (TNFRSF1A) gene, rs1800693 (a common variant) and rs4149584 (a coding polymorphism that results in an amino acid substitution-R92Q), as genetic modifiers of multiple sclerosis (MS), and to evaluate their potential functional implications in the disease. METHODS: The effects of rs1800693 and rs4149584 on 2 measures of disease severity, age at disease onset and Multiple Sclerosis Severity Score, were analyzed in 2,032 patients with MS. In a subgroup of patients, serum levels of the soluble form of TNF-R1 (sTNF-R1) were measured by ELISA; mRNA expression levels of the full-length TNF-R1 and Δ6-TNF-R1 isoform were investigated in peripheral blood mononuclear cells (PBMC) by real-time PCR; cell surface expression of the TNF-R1 was determined in T cells by flow cytometry. RESULTS: For rs4149584, R92Q carriers were younger at disease onset and progressed slower compared to noncarriers. However, no association with disease severity was observed for rs1800693. Serum levels of sTNF-R1 and mRNA expression levels of the full-length receptor were significantly increased in patients with MS carrying the R92Q mutation (p = 0.003 and p = 0.011, respectively), but similarly distributed among rs1800693 genotypes; cell surface TNF-R1 expression in T cells did not differ between rs4149584 and rs1800693 genotypes. The truncated soluble Δ6-TNF-R1 isoform was identified in PBMC from patients carrying the risk allele for rs1800693. CONCLUSIONS: These findings suggest that both rs1800693 and rs4149584 TNFRSF1A polymorphisms have functional consequences in the TNF-R1.


Assuntos
Esclerose Múltipla/genética , Polimorfismo Genético/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Adulto , Fatores Etários , Idade de Início , Alelos , Estudos de Coortes , Progressão da Doença , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/sangue , Mutação/genética , Índice de Gravidade de Doença
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