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1.
EMBO Rep ; 24(6): e56316, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37099396

RESUMO

Spermatozoa have a unique genome organization. Their chromatin is almost completely devoid of histones and is formed instead of protamines, which confer a high level of compaction and preserve paternal genome integrity until fertilization. Histone-to-protamine transition takes place in spermatids and is indispensable for the production of functional sperm. Here, we show that the H3K79-methyltransferase DOT1L controls spermatid chromatin remodeling and subsequent reorganization and compaction of the spermatozoon genome. Using a mouse model in which Dot1l is knocked-out (KO) in postnatal male germ cells, we found that Dot1l-KO sperm chromatin is less compact and has an abnormal content, characterized by the presence of transition proteins, immature protamine 2 forms and a higher level of histones. Proteomic and transcriptomic analyses performed on spermatids reveal that Dot1l-KO modifies the chromatin prior to histone removal and leads to the deregulation of genes involved in flagellum formation and apoptosis during spermatid differentiation. As a consequence of these chromatin and gene expression defects, Dot1l-KO spermatozoa have less compact heads and are less motile, which results in impaired fertility.


Assuntos
Cromatina , Histonas , Animais , Masculino , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Expressão Gênica , Histonas/metabolismo , Proteômica , Sêmen/metabolismo , Espermatogênese/genética , Espermatozoides/metabolismo , Camundongos
2.
Proc Natl Acad Sci U S A ; 115(35): E8246-E8255, 2018 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-30108144

RESUMO

Oligodendrocyte precursor cells (OPCs) constitute the main proliferative cells in the adult brain, and deregulation of OPC proliferation-differentiation balance results in either glioma formation or defective adaptive (re)myelination. OPC differentiation requires significant genetic reprogramming, implicating chromatin remodeling. Mounting evidence indicates that chromatin remodelers play important roles during normal development and their mutations are associated with neurodevelopmental defects, with CHD7 haploinsuficiency being the cause of CHARGE syndrome and CHD8 being one of the strongest autism spectrum disorder (ASD) high-risk-associated genes. Herein, we report on uncharacterized functions of the chromatin remodelers Chd7 and Chd8 in OPCs. Their OPC-chromatin binding profile, combined with transcriptome and chromatin accessibility analyses of Chd7-deleted OPCs, demonstrates that Chd7 protects nonproliferative OPCs from apoptosis by chromatin closing and transcriptional repression of p53 Furthermore, Chd7 controls OPC differentiation through chromatin opening and transcriptional activation of key regulators, including Sox10, Nkx2.2, and Gpr17 However, Chd7 is dispensable for oligodendrocyte stage progression, consistent with Chd8 compensatory function, as suggested by their common chromatin-binding profiles and genetic interaction. Finally, CHD7 and CHD8 bind in OPCs to a majority of ASD risk-associated genes, suggesting an implication of oligodendrocyte lineage cells in ASD neurological defects. Our results thus offer new avenues to understand and modulate the CHD7 and CHD8 functions in normal development and disease.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Oligodendroglia/metabolismo , Células-Tronco/metabolismo , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Transtorno do Espectro Autista/patologia , Síndrome CHARGE/genética , Síndrome CHARGE/metabolismo , Síndrome CHARGE/patologia , Sobrevivência Celular , Proteínas de Ligação a DNA/genética , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio , Camundongos , Camundongos Knockout , Proteínas Nucleares , Oligodendroglia/patologia , Células-Tronco/patologia , Fatores de Transcrição
3.
PLoS One ; 18(4): e0280976, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37093806

RESUMO

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.


Assuntos
Ácidos Nucleicos Livres , Teste Pré-Natal não Invasivo , Gravidez , Feminino , Humanos , Haplótipos , Teste Pré-Natal não Invasivo/métodos , Diagnóstico Pré-Natal/métodos , Genótipo
4.
J Mol Diagn ; 24(11): 1113-1127, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35963522

RESUMO

Several fusion genes such as BCR::ABL1, FIP1L1::PDGFRA, and PML::RARA are now efficiently targeted by specific therapies in patients with leukemia. Although these therapies have significantly improved patient outcomes, leukemia relapse and progression remain clinical concerns. Most myeloid next-generation sequencing (NGS) panels do not detect or quantify these fusions. It therefore remains difficult to decipher the clonal architecture and dynamics of myeloid malignancy patients, although these factors can affect clinical decisions and provide pathophysiologic insights. An asymmetric capture sequencing strategy (aCAP-Seq) and a bioinformatics algorithm (HmnFusion) were developed to detect and quantify MBCR::ABL1, µBCR::ABL1, PML::RARA, and FIP1L1::PDGFRA fusion genes in an NGS panel targeting 41 genes. One-hundred nineteen DNA samples derived from 106 patients were analyzed by conventional methods at diagnosis or on follow-up and were sequenced with this NGS myeloid panel. The specificity and sensitivity of fusion detection by aCAP-Seq were 100% and 98.1%, respectively, with a limit of detection estimated at 0.1%. Fusion quantifications were linear from 0.1% to 50%. Breakpoint locations and sequences identified by NGS were concordant with results obtained by Sanger sequencing. Finally, this new sensitive and cost-efficient NGS method allowed integrated analysis of resistant chronic myeloid leukemia patients and thus will be of interest to elucidate the mutational landscape and clonal architecture of myeloid malignancies driven by these fusion genes at diagnosis, relapse, or progression.


Assuntos
Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva , Humanos , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação/genética , Recidiva
5.
Histopathology ; 54(3): 319-27, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19236508

RESUMO

AIMS: Hepatocellular carcinoma (HCC) results from cirrhosis and, in Western Europe, hepatitis C virus and alcoholism are the predominant causes of this disease. We recently documented a global transcript repression in hepatocarcinoma nodules. The tumour suppressor activated pathway-6 (TSAP6) transcript codes for a transmembrane molecule that is an inducer of a caspase-3-dependent apoptotic pathway. The down-regulation of TSAP6 transcripts in HCC and perinodular cirrhosis, which contrasts with a sustained transcript level in HCC-free cirrhosis, has suggested that this hepatic protein level may provide a prognostic marker for HCC occurrence. METHODS AND RESULTS: This protein was quantified by semiquantitative assessment of immunohistochemistry on samples from 42 cases HCC-free cirrhosis, 49 cases cirrhosis with HCC, 43 HCC associated with healthy liver and 31 controls. TSAP6 expression was linked to the liver state, healthy or cirrhotic without or with an HCC and to tumour grade. CONCLUSIONS: With biopsies periodically performed for surveillance purposes, the decreased expression of TSAP6 in cirrhotic tissue could reflect a decrease in the apoptotic process and could be interpreted as a warning sign. This evaluation of the TSAP6 level in cirrhotic liver conveys predictive information for the development of HCC.


Assuntos
Carcinoma Hepatocelular/patologia , Regulação para Baixo/genética , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Fígado/patologia , Proteínas Oncogênicas/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular , Expressão Gênica , Imuno-Histoquímica , Fígado/metabolismo , Cirrose Hepática/genética , Neoplasias Hepáticas/genética , Proteínas Oncogênicas/metabolismo , Oxirredutases
6.
World J Gastroenterol ; 14(11): 1749-58, 2008 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-18350606

RESUMO

AIM: To look at a comprehensive picture of etiology-dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS: With a liver-oriented microarray, transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism, 12; hepatitis C, 10) and 5 controls. Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set (12 + 10) and a test set (6 + 7). RESULTS: A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism) or -2 (hepatitis C) and ontology indicated a predominant inflammatory response (alcoholism) or changes in cell cycle regulation, transcription factors and interferon responsiveness (hepatitis C). A stringent selection of 23 transcripts whose differences between etiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis. These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION: Etiology-specific abnormalities (chromo-some preference; differences in transcriptomes and related functions) have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C. This may open novel avenues for differential therapies in this disease.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Hepatite C/complicações , Cirrose Hepática Alcoólica/complicações , Cirrose Hepática/complicações , Neoplasias Hepáticas/genética , Adulto , Idoso , Carcinoma Hepatocelular/virologia , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 8 , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica/métodos , Hepatite C/genética , Humanos , Cirrose Hepática/genética , Cirrose Hepática/virologia , Cirrose Hepática Alcoólica/genética , Neoplasias Hepáticas/virologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Reprodutibilidade dos Testes
7.
Oncotarget ; 5(13): 5065-75, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24970810

RESUMO

Little is known about the genomic basis of primary central nervous system lymphoma (PCNSL) tumorigenesis. To investigate the mutational profile of PCNSL, we analyzed nine paired tumor and germline DNA samples from PCNSL patients by high throughput exome sequencing. Eight genes of interest have been further investigated by focused resequencing in 28 additional PCNSL tumors to better estimate their incidence. Our study identified recurrent somatic mutations in 37 genes, some involved in key signaling pathways such as NFKB, B cell differentiation and cell cycle control. Focused resequencing in the larger cohort revealed high mutation rates for genes already described as mutated in PCNSL such as MYD88 (38%), CD79B (30%), PIM1 (22%) and TBL1XR1 (19%) and for genes not previously reported to be involved in PCNSL tumorigenesis such as ETV6 (16%), IRF4 (14%), IRF2BP2 (11%) and EBF1 (11%). Of note, only 3 somatically acquired SNVs were annotated in the COSMIC database. Our results demonstrate a high genetic heterogeneity of PCNSL and mutational pattern similarities with extracerebral diffuse large B cell lymphomas, particularly of the activated B-cell (ABC) subtype, suggesting shared underlying biological mechanisms. The present study provides new insights into the mutational profile of PCNSL and potential targets for therapeutic strategies.


Assuntos
Neoplasias do Sistema Nervoso Central/genética , Exoma/genética , Predisposição Genética para Doença/genética , Linfoma Difuso de Grandes Células B/genética , Mutação , Linfócitos B/metabolismo , Linfócitos B/patologia , Análise Mutacional de DNA/métodos , Humanos , Mutação INDEL , Polimorfismo de Nucleotídeo Único
8.
Oncotarget ; 5(9): 2688-702, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24811913

RESUMO

Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in AlkF1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN/KI AlkR1279Q and MYCN/KI AlkF1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.


Assuntos
Transformação Celular Neoplásica/patologia , Regulação Neoplásica da Expressão Gênica , Mutação/genética , Neuroblastoma/genética , Neurogênese , Proteínas Proto-Oncogênicas c-ret/metabolismo , Receptores Proteína Tirosina Quinases/genética , Sequência de Aminoácidos , Quinase do Linfoma Anaplásico , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Southern Blotting , Western Blotting , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Integrases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-ret/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Células Tumorais Cultivadas
9.
PLoS One ; 8(8): e72182, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23991058

RESUMO

Neuroblastoma is a pediatric cancer of the peripheral nervous system in which structural chromosome aberrations are emblematic of aggressive tumors. In this study, we performed an in-depth analysis of somatic rearrangements in two neuroblastoma cell lines and two primary tumors using paired-end sequencing of mate-pair libraries and RNA-seq. The cell lines presented with typical genetic alterations of neuroblastoma and the two tumors belong to the group of neuroblastoma exhibiting a profile of chromothripsis. Inter and intra-chromosomal rearrangements were identified in the four samples, allowing in particular characterization of unbalanced translocations at high resolution. Using complementary experiments, we further characterized 51 rearrangements at the base pair resolution that revealed 59 DNA junctions. In a subset of cases, complex rearrangements were observed with templated insertion of fragments of nearby sequences. Although we did not identify known particular motifs in the local environment of the breakpoints, we documented frequent microhomologies at the junctions in both chromothripsis and non-chromothripsis associated breakpoints. RNA-seq experiments confirmed expression of several predicted chimeric genes and genes with disrupted exon structure including ALK, NBAS, FHIT, PTPRD and ODZ4. Our study therefore indicates that both non-homologous end joining-mediated repair and replicative processes may account for genomic rearrangements in neuroblastoma. RNA-seq analysis allows the identification of the subset of abnormal transcripts expressed from genomic rearrangements that may be involved in neuroblastoma oncogenesis.


Assuntos
Aberrações Cromossômicas , Pontos de Quebra do Cromossomo , Rearranjo Gênico/genética , Neuroblastoma/genética , Translocação Genética/genética , Hidrolases Anidrido Ácido/genética , Quinase do Linfoma Anaplásico , Sequência de Bases , Linhagem Celular Tumoral , Pré-Escolar , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Neuroblastoma/patologia , Reação em Cadeia da Polimerase , Receptores Proteína Tirosina Quinases/genética , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica
10.
Cancer Res ; 73(1): 195-204, 2013 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23139213

RESUMO

Activating mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma (NB), a cancer of the peripheral nervous system, and are thought to be the primary mechanism of oncogenic activation of this receptor in this pediatric neoplasm. To address the possibility that ALK activation may occur through genomic rearrangements as detected in other cancers, we first took advantage of high-resolution array-comparative genomic hybridization to search for ALK rearrangements in NB samples. Using complementary experiments by capture/paired-end sequencing and FISH experiments, various types of rearrangements were fully characterized, including partial gains or amplifications, in several NB cell lines and primary tumors. In the CLB-Bar cell line, we described a genomic rearrangement associated with an amplification of the ALK locus, leading to the expression of a 170 kDa protein lacking part of the extracellular domain encoded by exons 4 to 11, named ALK(Δ4-11). Analysis of genomic DNA from the tumor at diagnosis and relapse revealed that the ALK gene was amplified at diagnosis but that the rearranged ALK allele was observed at the relapse stage only, suggesting that it may be implicated in tumor aggressiveness. Consistently, oncogenic and tumorigenic properties of the ALK(Δ4-11) variant were shown after stable expression in NIH3T3 cells. Moreover, we documented an increased constitutive kinase activity of this variant, as well as an impaired maturation and retention into intracellular compartments. These results indicate that genomic rearrangements constitute an alternative mechanism to ALK point mutations resulting in receptor activation.


Assuntos
Rearranjo Gênico/genética , Neuroblastoma/genética , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Humanos , Immunoblotting , Imunoprecipitação , Hibridização in Situ Fluorescente
11.
Joint Bone Spine ; 79(6): 574-80, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22459417

RESUMO

OBJECTIVE: Because available biomarkers (rheumatoid factors [RF], anti-cyclic citrullinated autoantibodies [anti-CCP2], erythrocyte sedimentation rate at 1st hour [ESR]/C-reactive peptide [CRP] and bone erosions) are insufficient to predict rheumatoid arthritis (RA) structural damage, to determine whether synovium expression of greater or equal to 1 markers could constitute new prognostic factor(s). METHOD: The study was conducted on 18 prospectively enrolled disease-modifying anti-rheumatic drug (DMARD)- and glucocorticoid-naïve, VErA cohort patients with very-early arthritis (median duration: 4months). Recorded at baseline were: clinical and biological (serum ESR, CRP, RF-isotypes, anti-CCP2, osteoprotegerin, receptor activator of nuclear κB-ligand [RANK-L] and cartilage oligomeric matrix protein [COMP] levels) data; synovium expression (HLA-DR, CD163, CD3, CD20, VEGF, osteoprotegerin, RANK-L, Bcl2 and global inflammation index) for a metacarpophalangeal joint-synovium biopsy. Baseline and 3-year hand-and-foot X-rays were graded with the van der Heijde-modified-Sharp score; the judgment criterion was its progression during follow-up. Pearson's product moment correlation statistics were used to test for association between paired samples. RESULTS: A baseline, a significant relationship was found between erosive damage and markers of B-cell activation, notably the synovium CD20 expression (r=0.68; P=0.0001). Quantified by the modified-Sharp erosion score variation, the 3-year structural damage progression was significantly correlated with: serum levels of RF-IgG (r=0.75; P=0.0003), -IgM (r=0.69; P=0.001), anti-CCP2 (r=0.53; P=0.02) and RANK-L (r=0.61; P=0.007); synovium CD20 expression (r=0.70; P=0.001). CONCLUSION: This analysis of the prognostic value of a large panel of synovium markers in a limited sample of prospectively followed, well-documented patients suggested that both synovial CD20 and serum RANK-L levels might be new predictors of structural damage progression in very-early RA.


Assuntos
Antígenos CD20/metabolismo , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Reabsorção Óssea/metabolismo , Progressão da Doença , Membrana Sinovial/imunologia , Adulto , Idoso , Artrite Reumatoide/diagnóstico , Biomarcadores/metabolismo , Biópsia , Reabsorção Óssea/diagnóstico , Estudos de Coortes , Feminino , Seguimentos , Humanos , Estudos Longitudinais , Masculino , Articulação Metacarpofalângica/patologia , Pessoa de Meia-Idade , Projetos Piloto , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Ligante RANK/sangue , Membrana Sinovial/patologia , Resultado do Tratamento
12.
Arthritis Res Ther ; 11(2): R38, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19284558

RESUMO

INTRODUCTION: The aim of our study was to identify new early rheumatoid arthritis (RA) autoantibodies. METHODS: Sera obtained from 110 early untreated RA patients (<6 months) were analyzed by western blot using HL-60 cell extract, separated on one-dimensional and two-dimensional gel electrophoresis (1-DE, 2-DE). Sera from 50 healthy blood donors and 20 patients with non-RA rheumatisms were used as controls for 1-DE and 2-DE, respectively. The immunoreactive proteins were identified by MALDI-TOF mass spectrometric analysis and the presence of potential sites of citrullination in each of these proteins was evaluated. FT-ICR mass spectrometry was used to verify experimentally the effect of citrullination upon the mass profile observed by MALDI-TOF analysis. RESULTS: The 110 1-DE patterns allowed detection of 10 recurrent immunoreactive bands of 33, 39, 43, 46, 51, 54, 58, 62, 67 and 70 kDa, which were further characterized by 2-DE and proteomic analysis. Six proteins were already described RA antigens: heterogeneous nuclear ribonucleoprotein A2/B1, aldolase, alpha-enolase, calreticulin, 60 kDa heat shock protein (HSP60) and BiP. Phosphoglycerate kinase 1 (PGK1), stress-induced phosphoprotein 1 and the far upstream element-binding proteins (FUSE-BP) 1 and 2 were identified as new antigens. Post-translational protein modifications were analyzed and potentially deiminated peptides were found on aldolase, alpha-enolase, PGK1, calreticulin, HSP60 and the FUSE-BPs. We compared the reactivity of RA sera with citrullinated and noncitrullinated alpha-enolase and FUSE-BP linear peptides, and showed that antigenicity of the FUSE-BP peptide was highly dependent on citrullination. Interestingly, the anti-cyclic citrullinated peptide antibody (anti-CCP2) status in RA serum at inclusion was not correlated to the reactivity directed against FUSE-BP citrullinated peptide. CONCLUSIONS: Two categories of antigens, enzymes of the glycolytic family and molecular chaperones are also targeted by the early untreated RA autoantibody response. For some of them, and notably the FUSE-BPs, citrullination is involved in the immunological tolerance breakdown observed earlier in RA patients. Autoantibodies recognizing a citrullinated peptide from FUSE-BP may enhance the sensibility for RA of the currently available anti-CCP2 test.


Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoanticorpos/análise , Autoanticorpos/sangue , Biomarcadores/sangue , Western Blotting , Ensaio Cometa , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier
13.
J Hepatol ; 42(6): 860-9, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15885357

RESUMO

BACKGROUND/AIMS: Whether the transcriptional reprogramming process induced by hepatocellular carcinoma recapitulates that of the developing liver is at present unclear. METHODS: With a complete coverage of the liver transcriptome by microarray using adult livers as controls, we searched for similarities and differences in mRNA abundances between hepatocellular carcinoma nodules and fetal livers taken before (early) or after (late) the 22-24th week of gestation. Changes in some mRNA levels were studied in further liver samples by quantitative RT-PCR. RESULTS: Altered gene expression in hepatocellular carcinoma mostly results in down-regulated mRNAs which largely overlap with those repressed in the late fetal liver. Different frequencies of transcription factor binding sites in the down-regulated genes vs control genes as well as changes in abundance of mRNAs for relevant transcription factors point to a transcriptional repression. The down-regulated mRNAs code for proteins involved in (i) transcription and translation, (ii) specific functions of the differentiated hepatocyte or (iii) activation of proliferation and apoptosis. CONCLUSIONS: Apoptosis limitation is likely to predominate over active proliferation during liver development and hepatocellular carcinoma. Repression of the apoptosis-associated dudulin-2 mRNA points to a potential marker for the transition from a carcinoma-free to carcinoma-associated cirrhosis.


Assuntos
Carcinoma Hepatocelular/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Fígado/fisiologia , Proteínas Oncogênicas/genética , Idoso , Idoso de 80 Anos ou mais , Sítios de Ligação/genética , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular , Regulação para Baixo/genética , Feminino , Humanos , Fígado/embriologia , Cirrose Hepática/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredutases , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Hepatology ; 42(4): 946-55, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16175611

RESUMO

Given the unknown timing of the onset of an acute systemic inflammation in humans, the fine tuning of cascades and pathways involved in the associated hepatocyte response cannot be appraised in vivo. Therefore, the authors used a genome-wide and kinetic analysis in the human Hep3B hepatoma cell line challenged with a conditioned medium from bacterial lipopolysaccharide-stimulated macrophages. A complete coverage of the liver transcriptome disclosed 648 mRNAs whose change in abundance allowed for their clustering in mRNA subsets with an early, intermediate, or late regulation. The contribution of transcription, stability, or translation was appraised with genome-wide studies of the changes in nuclear primary transcripts, mRNA decay, or polysome-associated mRNAs. A predominance of mRNAs with decreased stability and the fact that translation alone controls a significant number of acute phase-associated proteins are prominent findings. Transcription and stability act independently or, more rarely, cooperate or even counteract in a gene-by-gene manner, which results in a unidirectional change in mRNA abundance. Waves of mRNAs for groups of functionally related proteins are up- or downregulated in an ordered fashion. This includes an early regulation of transcription-associated proteins, an intermediate repression of detoxication and metabolism proteins, and finally an enhanced translation and transport of a number of membranous or secreted proteins along with an enhanced protein degradation. In conclusion, this study provides a comprehensive and simultaneous overview of events in the human hepatocyte during the inflammatory acute phase.


Assuntos
Citocinas/imunologia , Genômica , Hepatócitos/imunologia , Hepatócitos/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação de Fase Aguda/genética , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Genoma Humano , Humanos , Cinética , Neoplasias Hepáticas , Polirribossomos/fisiologia , Biossíntese de Proteínas/genética , Biossíntese de Proteínas/imunologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/imunologia
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