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1.
J Vis Exp ; (168)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33720119

RESUMO

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.


Assuntos
Neoplasias Encefálicas/patologia , Comunicação Celular , Movimento Celular , Técnicas de Cocultura/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Neurônios/patologia , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Rastreamento de Células , Glioma/patologia , Humanos , Processamento de Imagem Assistida por Computador , Laminina/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
2.
Int Rev Cell Mol Biol ; 358: 1-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33707051

RESUMO

Celiac Disease (CeD) is an immune-mediated complex disease that is triggered by the ingestion of gluten and develops in genetically susceptible individuals. It has been known for a long time that the Human Leucocyte Antigen (HLA) molecules DQ2 and DQ8 are necessary, although not sufficient, for the disease development, and therefore other susceptibility genes and (epi)genetic events must participate in CeD pathogenesis. The advances in Genomics during the last 15 years have made CeD one of the immune-related disorders with the best-characterized genetic component. In the present work, we will first review the main Genome-Wide Association Studies (GWAS) carried out in the disorder, and emphasize post-GWAS discoveries, including diverse integrative strategies, SNP prioritization approaches, and insights into the Microbiome through the host Genomics. Second, we will explore CeD-related Epigenetics and Epigenomics, mostly focusing on the emerging knowledge of the celiac methylome, and the vast but yet under-explored non-coding RNA (ncRNA) landscape. We conclude that much has been done in the field although there are still completely unvisited areas in the post-Genomics of CeD. Chromatin conformation and accessibility, and Epitranscriptomics are promising domains that need to be unveiled to complete the big picture of the celiac Genome.

3.
Front Immunol ; 11: 585616, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154756

RESUMO

Glioblastoma (GBM) are the most common tumors of the central nervous system and among the deadliest cancers in adults. GBM overall survival has not improved over the last decade despite optimization of therapeutic standard-of-care. While immune checkpoint inhibitors (ICI) have revolutionized cancer care, they unfortunately have little therapeutic success in GBM. Here, we elaborate on normal brain and GBM-associated immune landscapes. We describe the role of microglia and tumor-associated macrophages (TAMs) in immune suppression and highlight the impact of energy metabolism in immune evasion. We also describe the challenges and opportunities of immunotherapies in GBM and discuss new avenues based on harnessing the anti-tumor activity of myeloid cells, vaccines, chimeric antigen receptors (CAR)-T and -NK cells, oncolytic viruses, nanocarriers, and combination therapies.


Assuntos
Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Glioblastoma/imunologia , Glioblastoma/terapia , Imunoterapia/métodos , Animais , Humanos , Evasão Tumoral/imunologia
4.
Nucleic Acids Res ; 47(19): 10072-10085, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31665742

RESUMO

Mitochondrial dysfunction plays critical roles in cancer development and related therapeutic response; however, exact molecular mechanisms remain unclear. Recently, alongside the discovery of mitochondrial-specific DNA methyltransferases, global and site-specific methylation of the mitochondrial genome has been described. Investigation of any functional consequences however remains unclear and debated due to insufficient evidence of the quantitative degree and frequency of mitochondrial DNA (mtDNA) methylation. This study uses WGBS to provide the first quantitative report of mtDNA methylation at single base pair resolution. The data show that mitochondrial genomes are extensively methylated predominantly at non-CpG sites. Importantly, these methylation patterns display notable differences between normal and cancer cells. Furthermore, knockdown of DNA methyltransferase enzymes resulted in a marked global reduction of mtDNA methylation levels, indicating these enzymes may be associated with the establishment and/or maintenance of mtDNA methylation. DNMT3B knockdown cells displayed a comparatively pronounced global reduction in mtDNA methylation with concomitant increases in gene expression, suggesting a potential functional link between methylation and gene expression. Together these results demonstrate reproducible, non-random methylation patterns of mtDNA and challenge the notion that mtDNA is lowly methylated. This study discusses key differences in methodology that suggest future investigations must allow for techniques that assess both CpG and non-CpG methylation.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , DNA Mitocondrial/genética , Regulação da Expressão Gênica/genética , Animais , Ilhas de CpG/genética , Humanos , Mitocôndrias/genética
5.
Sci Rep ; 9(1): 4220, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862814

RESUMO

N6-methyladenosine (m6A) is the most common and abundant RNA modification. Recent studies have shown its importance in the regulation of several biological processes, including the immune response, and different approaches have been developed in order to map and quantify m6A marks. However, site specific detection of m6A methylation has been technically challenging, and existing protocols are long and tedious and often involve next-generation sequencing. Here, we describe a simple RT-QPCR based approach for the relative quantification of candidate m6A regions that takes advantage of the diminished capacity of BstI enzyme to retrotranscribe m6A residues. Using this technique, we have been able to confirm the recently described m6A methylation in the 3'UTR of SOCS1 and SOCS3 transcripts. Moreover, using the method presented here, we have also observed alterations in the relative levels of m6A in specific motifs of SOCS genes in celiac disease patients and in pancreatic ß-cells exposed to inflammatory stimuli.


Assuntos
Regiões 3' não Traduzidas , Adenosina/análogos & derivados , Desoxirribonuclease BamHI/química , Motivos de Nucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adenosina/genética , Adenosina/metabolismo , Células CACO-2 , Humanos , Metilação , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo
6.
Sci Rep ; 9(1): 1298, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718669

RESUMO

The Human Leucocyte Antigen (HLA) locus and other DNA sequence variants identified in Genome-Wide Association (GWA) studies explain around 50% of the heritability of celiac disease (CD). However, the pathogenesis of CD could be driven by other layers of genomic information independent from sequence variation, such as DNA methylation, and it is possible that allele-specific methylation explains part of the SNP associations. Since the DNA methylation landscape is expected to be different among cell types, we analyzed the methylome of the epithelial and immune cell populations of duodenal biopsies in CD patients and controls separately. We found a cell type-specific methylation signature that includes genes mapping to the HLA region, namely TAP1 and HLA-B. We also performed Immunochip SNP genotyping of the same samples and interrogated the expression of some of the affected genes. Our analysis revealed that the epithelial methylome is characterized by the loss of CpG island (CGI) boundaries, often associated to altered gene expression, and by the increased variability of the methylation across the samples. The overlap between differentially methylated positions (DMPs) and CD-associated SNPs or variants contributing to methylation quantitative trait loci (mQTLs) is minimal. In contrast, there is a notable enrichment of mQTLs among the most significant CD-associated SNPs. Our results support the notion that DNA methylation alterations constitute a genotype-independent event and confirm its role in the HLA region (apart from the well-known, DQ allele-specific effect). Finally, we find that a fraction of the CD-associated variants could exert its phenotypic effect through DNA methylation.


Assuntos
Doença Celíaca/etiologia , Metilação de DNA , Epigenoma , Genótipo , Antígenos HLA/genética , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Biópsia , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Ilhas de CpG , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Antígenos HLA/imunologia , Humanos , Mucosa Intestinal/patologia , Masculino , Regiões Promotoras Genéticas
7.
Front Nutr ; 6: 187, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921880

RESUMO

Celiac disease (CD) patients present a loss of intestinal barrier function due to structural alterations in the tight junction (TJ) network, the most apical unions between epithelial cells. The association of TJ-related gene variants points to an implication of this network in disease susceptibility. This work aims to characterize the functional implication of TJ-related, disease-associated loci in CD pathogenesis. We performed an association study of 8 TJ-related gene variants in a cohort of 270 CD and 91 non-CD controls. The expression level of transcripts located in the associated SNP region was analyzed by RT-PCR in several human tissues and in duodenal biopsies of celiac patients and non-CD controls. (si)RNA-driven silencing combined with gliadin in the Caco2 intestinal cell line was used to analyze the implication of transcripts from the associated region in the regulation of TJ genes. We replicated the association of rs6962966*A variant [p = 0.0029; OR = 1.88 (95%1.24-2.87)], located in an intron of TJ-related MAGI2 coding gene and upstream of RP4-587D13.2 transcript, bioinformatically classified as a long non-coding RNA (lncRNA). The expression of both genes is correlated and constitutively downregulated in CD intestine. Silencing of lncRNA decreases the levels of MAGI2 protein. At the same time, silencing of MAGI2 affects the expression of several TJ-related genes. The associated region is functionally altered in disease, probably affecting CD-related TJ genes.

8.
J Vis Exp ; (137)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30059028

RESUMO

The purpose of this protocol is to fractionate human intestinal tissue obtained by endoscopy into nuclear and cytoplasmic compartments for the localization analysis of specific proteins or protein complexes in different tissue states (i.e., healthy vs. disease). This method is useful for the fractionation of both fresh and frozen intestinal tissue samples; it is easily accessible for all laboratories and not time consuming.


Assuntos
Conteúdo Gastrointestinal/química , Congelamento , Humanos
9.
Genes (Basel) ; 9(5)2018 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-29748492

RESUMO

The aim of this study was to construct celiac co-expression patterns at a whole genome level and to identify transcription factors (TFs) that could drive the gliadin-related changes in coordination of gene expression observed in celiac disease (CD). Differential co-expression modules were identified in the acute and chronic responses to gliadin using expression data from a previous microarray study in duodenal biopsies. Transcription factor binding site (TFBS) and Gene Ontology (GO) annotation enrichment analyses were performed in differentially co-expressed genes (DCGs) and selection of candidate regulators was performed. Expression of candidates was measured in clinical samples and the activation of the TFs was further characterized in C2BBe1 cells upon gliadin challenge. Enrichment analyses of the DCGs identified 10 TFs and five were selected for further investigation. Expression changes related to active CD were detected in four TFs, as well as in several of their in silico predicted targets. The activation of TFs was further characterized in C2BBe1 cells upon gliadin challenge, and an increase in nuclear translocation of CAMP Responsive Element Binding Protein 1 (CREB1) and IFN regulatory factor-1 (IRF1) in response to gliadin was observed. Using transcriptome-wide co-expression analyses we are able to propose novel genes involved in CD pathogenesis that respond upon gliadin stimulation, also in non-celiac models.

10.
J Pediatr Gastroenterol Nutr ; 67(2): 225-231, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29601440

RESUMO

OBJECTIVE: The aim of the study is to identify additional celiac disease associated loci in the major histocompatibility complex (MHC) independent from classical HLA risk alleles (HLA-DR3-DQ2) and to characterize their potential functional impact in celiac disease pathogenesis at the intestinal level. METHODS: We performed a high-resolution single-nucleotide polymorphism (SNP) genotyping of the MHC region, comparing HLA-DR3 homozygous celiac patients and non-celiac controls carrying a single copy of the B8-DR3-DQ2 conserved extended haplotype. Expression level of potential novel risk genes was determined by RT-PCR in intestinal biopsies and in intestinal and immune cells isolated from control and celiac individuals. Small interfering RNA-driven silencing of selected genes was performed in the intestinal cell line T84. RESULTS: MHC genotyping revealed 2 associated SNPs, one located in TRIM27 gene and another in the non-coding gene HCG14. After stratification analysis, only HCG14 showed significant association independent from HLA-DR-DQ loci. Expression of HCG14 was slightly downregulated in epithelial cells isolated from duodenal biopsies of celiac patients, and eQTL analysis revealed that polymorphisms in HCG14 region were associated with decreased NOD1 expression in duodenal intestinal cells. CONCLUSIONS: We have successfully employed a conserved extended haplotype-matching strategy and identified a novel additional celiac disease risk variant in the lncRNA HCG14. This lncRNA seems to regulate the expression of NOD1 in an allele-specific manner. Further functional studies are needed to clarify the role of HCG14 in the regulation of gene expression and to determine the molecular mechanisms by which the risk variant in HCG14 contributes to celiac disease pathogenesis.


Assuntos
Doença Celíaca/genética , Predisposição Genética para Doença , Antígeno HLA-DR3/genética , Proteína Adaptadora de Sinalização NOD1/metabolismo , RNA Longo não Codificante/genética , Estudos de Casos e Controles , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Criança , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único
11.
Eur J Hum Genet ; 24(12): 1831-1834, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27650971

RESUMO

To identify candidate genes in celiac disease (CD), we reanalyzed the whole Immunochip CD cohort using a different approach that clusters individuals based on immunoancestry prior to disease association analysis, rather than by geographical origin. We detected 636 new associated SNPs (P<7.02 × 10-07) and identified 5 novel genomic regions, extended 8 others previously identified and also detected 18 isolated signals defined by one or very few significant SNPs. To test whether we could identify putative candidate genes, we performed expression analyses of several genes from the top novel region (chr2:134533564-136169524), from a previously identified locus that is now extended, and a gene marked by an isolated SNP, in duodenum biopsies of active and treated CD patients, and non-celiac controls. In the largest novel region, CCNT2 and R3HDM1 were constitutively underexpressed in disease, even after gluten removal. Moreover, several genes within this region were coexpressed in patients, but not in controls. Other novel genes like KIF21B, REL and SORD also showed altered expression in active disease. Apart from the identification of novel CD loci, these results suggest that ancestry-based stratified analysis is an efficient strategy for association studies in complex diseases.


Assuntos
Doença Celíaca/genética , Loci Gênicos , Linhagem , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Doença Celíaca/metabolismo , Duodeno/metabolismo , Glutens/metabolismo , Humanos
12.
Eur J Hum Genet ; 23(8): 1100-5, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25388004

RESUMO

Celiac disease is a chronic immune-mediated disorder with an important genetic component. To date, there are 57 independent association signals from 39 non-HLA loci, and a total of 66 candidate genes have been proposed. We aimed to scrutinize the functional implication of 45 of those genes by analyzing their expression in the disease tissue of celiac patients (at diagnosis/treatment) compared with non-celiac controls. Moreover, we investigated the SNP genotype effect in gene expression and performed coexpression analyses. Several genes showed differential expression among disease groups, most of them related to immune response. Multiple trans-eQTLs but only four cis-eQTLs were found, and surprisingly the genotype effect seems to be stimulus dependent as it differs among groups. Coexpression levels vary from higher to lower levels in active patients at diagnosis, treated patients and non-celiac controls respectively. A subset of 18 genes tightly correlated in both groups of patients but not in controls was identified. Interestingly, this subset of genes was influenced by the genotype of three SNPs. One of the SNPs, rs1018326 on chromosome two is on top of a known lincRNA whose function is not yet described, and whose expression seems to be upregulated in active disease when comparing biopsy pairs from the same individuals. Our results strongly suggest that the effects of disease-associated SNPs go far beyond the oversimplistic idea of transcriptional control at a nearby locus. Further investigations are needed to determine how each variant disrupts fine-tuning mechanisms in the genome that eventually lead to disease.


Assuntos
Doença Celíaca/genética , Predisposição Genética para Doença , Mucosa Intestinal/metabolismo , Locos de Características Quantitativas/genética , Doença Celíaca/metabolismo , Doença Celíaca/patologia , Feminino , Regulação da Expressão Gênica , Estudos de Associação Genética , Humanos , Mucosa Intestinal/patologia , Masculino , Polimorfismo de Nucleotídeo Único , RNA Longo não Codificante/genética
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