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1.
Blood ; 129(4): 525-532, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-27811020

RESUMO

We evaluated the impact of recipient and cord blood unit (CBU) genetic polymorphisms related to immune response on outcomes after unrelated cord blood transplantations (CBTs). Pretransplant DNA samples from 696 CBUs with malignant diseases were genotyped for NLRP1, NLRP2, NLRP3, TIRAP/Mal, IL10, REL, TNFRSF1B, and CTLA4. HLA compatibility was 6 of 6 in 10%, 5 of 6 in 39%, and ≥4 of 6 in 51% of transplants. Myeloablative conditioning was used in 80%, and in vivo T-cell depletion in 81%, of cases. The median number of total nucleated cells infused was 3.4 × 107/kg. In multivariable analysis, patients receiving CBUs with GG-CTLA4 genotype had poorer neutrophil recovery (hazard ratio [HR], 1.33; P = .02), increased nonrelapse mortality (NRM) (HR, 1.50; P < .01), and inferior disease-free survival (HR, 1.41; P = .02). We performed the same analysis in a more homogeneous subset of cohort 1 (cohort 2, n = 305) of patients who received transplants for acute leukemia, all given a myeloablative conditioning regimen, and with available allele HLA typing (HLA-A, -B, -C, and -DRB1). In this more homogeneous but smaller cohort, we were able to demonstrate that GG-CTLA4-CBU was associated with increased NRM (HR, 1.85; P = .01). Use of GG-CTLA4-CBU was associated with higher mortality after CBT, which may be a useful criterion for CBU selection, when multiple CBUs are available.


Assuntos
Antígeno CTLA-4/imunologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Antígenos HLA/imunologia , Neoplasias Hematológicas/genética , Polimorfismo Genético , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Adolescente , Adulto , Alelos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Antígeno CTLA-4/genética , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Sangue Fetal/citologia , Sangue Fetal/imunologia , Sangue Fetal/transplante , Expressão Gênica , Genótipo , Antígenos HLA/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Teste de Histocompatibilidade , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Agonistas Mieloablativos/uso terapêutico , Proteínas NLR , Modelos de Riscos Proporcionais , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Estudos Retrospectivos , Condicionamento Pré-Transplante , Doadores não Relacionados
2.
Cancer Sci ; 106(10): 1264-77, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26251039

RESUMO

Recent evidence has shown that deregulated expression of members of the microRNA-29 (miR-29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR-29 in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T-ALL) has not been investigated. Here, we show that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T-ALL patients. Furthermore, miR-29a levels are extremely reduced in T-ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR-29a mimics into Jurkat cells revealed the downregulation of several predicted targets (CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR-29a levels in Jurkat and Molt-4 T-ALL cells led to the demethylation of many genes commonly methylated in T-ALL. Overall, our results suggest that reduced miR-29a levels may contribute to the altered epigenetic status of T-ALL, highlighting its relevance in the physiopathology of this disease.


Assuntos
Metilação de DNA/genética , Epigênese Genética/genética , Regulação Leucêmica da Expressão Gênica/genética , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Antígenos de Neoplasias/biossíntese , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Classe Ia de Fosfatidilinositol 3-Quinase , Quinase 6 Dependente de Ciclina/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/biossíntese , Daunorrubicina/farmacologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Células Jurkat , Oxigenases de Função Mista , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Peroxidases , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Receptores de Interleucina-1/biossíntese , DNA Metiltransferase 3B
3.
Immunology ; 143(3): 381-95, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24813052

RESUMO

Pemphigus foliaceus (PF) is a complex autoimmune disease characterized by bullous skin lesions and the presence of antibodies against desmoglein 1. In this study we sought to contribute to a better understanding of the molecular processes in endemic PF, as the identification of factors that participate in the pathogenesis is a prerequisite for understanding its biological basis and may lead to novel therapeutic interventions. CD4+ T lymphocytes are central to the development of the disease. Therefore, we compared genome-wide gene expression profiles of peripheral CD4+ T cells of various PF patient subgroups with each other and with that of healthy individuals. The patient sample was subdivided into three groups: untreated patients with the generalized form of the disease, patients submitted to immunosuppressive treatment, and patients with the localized form of the disease. Comparisons between different subgroups resulted in 135, 54 and 64 genes differentially expressed. These genes are mainly related to lymphocyte adhesion and migration, apoptosis, cellular proliferation, cytotoxicity and antigen presentation. Several of these genes were differentially expressed when comparing lesional and uninvolved skin from the same patient. The chromosomal regions 19q13 and 12p13 concentrate differentially expressed genes and are candidate regions for PF susceptibility genes and disease markers. Our results reveal genes involved in disease severity, potential therapeutic targets and previously unsuspected processes involved in the pathogenesis. Besides, this study adds original information that will contribute to the understanding of PF's pathogenesis and of the still poorly defined in vivo functions of most of these genes.


Assuntos
Perfilação da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Pênfigo/genética , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Análise por Conglomerados , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Pênfigo/imunologia , Pênfigo/metabolismo , Transdução de Sinais , Adulto Jovem
4.
Ann Hematol ; 93(9): 1457-65, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24696091

RESUMO

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.


Assuntos
Adenosina/metabolismo , Anemia Falciforme/metabolismo , Antidrepanocíticos/farmacologia , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/metabolismo , Hidroxiureia/farmacologia , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Adolescente , Adulto , Anemia Falciforme/tratamento farmacológico , Anemia Falciforme/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Antidrepanocíticos/uso terapêutico , Apirase/genética , Apirase/metabolismo , Células Sanguíneas/patologia , Estudos de Casos e Controles , Criança , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Hidroxiureia/uso terapêutico , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Pessoa de Meia-Idade , Adulto Jovem
5.
J Cell Mol Med ; 16(6): 1232-44, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21777379

RESUMO

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Lectinas Tipo C/genética , Análise em Microsséries , NF-kappa B/genética , Nitrilas , Transplante de Células-Tronco de Sangue Periférico/métodos , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Sulfonas , Linfócitos T Reguladores/metabolismo , Fator de Transcrição RelB/genética , Fator de Transcrição RelB/metabolismo
6.
Hepatology ; 53(5): 1600-7, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21520173

RESUMO

UNLABELLED: Some patients with liver disease progress to cirrhosis, but the risk factors for cirrhosis development are unknown. Dyskeratosis congenita, an inherited bone marrow failure syndrome associated with mucocutaneous anomalies, pulmonary fibrosis, and cirrhosis, is caused by germline mutations of genes in the telomerase complex. We examined whether telomerase mutations also occurred in sporadic cirrhosis. In all, 134 patients with cirrhosis of common etiologies treated at the Liver Research Institute, University of Arizona, between May 2008 and July 2009, and 528 healthy subjects were screened for variation in the TERT and TERC genes by direct sequencing; an additional 1,472 controls were examined for the most common genetic variation observed in patients. Telomere length of leukocytes was measured by quantitative polymerase chain reaction. Functional effects of genetic changes were assessed by transfection of mutation-containing vectors into telomerase-deficient cell lines, and telomerase activity was measured in cell lysates. Nine of the 134 patients with cirrhosis (7%) carried a missense variant in TERT, resulting in a cumulative carrier frequency significantly higher than in controls (P = 0.0009). One patient was homozygous and eight were heterozygous. The allele frequency for the most common missense TERT variant was significantly higher in patients with cirrhosis (2.6%) than in 2,000 controls (0.7%; P = 0.0011). One additional patient carried a TERC mutation. The mean telomere length of leukocytes in patients with cirrhosis, including six mutant cases, was shorter than in age-matched controls (P = 0.0004). CONCLUSION: Most TERT gene variants reduced telomerase enzymatic activity in vitro. Loss-of-function telomerase gene variants associated with short telomeres are risk factors for sporadic cirrhosis.


Assuntos
Cirrose Hepática/genética , Mutação de Sentido Incorreto , Telomerase/genética , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Adulto Jovem
7.
Haematologica ; 97(8): 1246-54, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22331265

RESUMO

BACKGROUND: The malignant B cells in chronic lymphocytic leukemia receive signals from the bone marrow and lymph node microenvironments which regulate their survival and proliferation. Characterization of these signals and the pathways that propagate them to the interior of the cell is important for the identification of novel potential targets for therapeutic intervention. DESIGN AND METHODS: We compared the gene expression profiles of chronic lymphocytic leukemia B cells purified from bone marrow and peripheral blood to identify genes that are induced by the bone marrow microenvironment. Two of the differentially expressed genes were further studied in cell culture experiments and in an animal model to determine whether they could represent appropriate therapeutic targets in chronic lymphocytic leukemia. RESULTS: Functional classification analysis revealed that the majority of differentially expressed genes belong to gene ontology categories related to cell cycle and mitosis. Significantly up-regulated genes in bone marrow-derived tumor cells included important cell cycle regulators, such as Aurora A and B, survivin and CDK6. Down-regulation of Aurora A and B by RNA interference inhibited proliferation of chronic lymphocytic leukemia-derived cell lines and induced low levels of apoptosis. A similar effect was observed with the Aurora kinase inhibitor VX-680 in primary chronic lymphocytic leukemia cells that were induced to proliferate by CpG-oligonucleotides and interleukin-2. Moreover, VX-680 significantly blocked leukemia growth in a mouse model of chronic lymphocytic leukemia. CONCLUSIONS: Aurora A and B are up-regulated in proliferating chronic lymphocytic leukemia cells and represent potential therapeutic targets in this disease.


Assuntos
Células da Medula Óssea/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Serina-Treonina Quinases/genética , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/genética , Aurora Quinase A , Aurora Quinases , Células da Medula Óssea/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Análise por Conglomerados , Feminino , Perfilação da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Camundongos , Mitose/genética , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima
8.
Proc Natl Acad Sci U S A ; 106(4): 1187-92, 2009 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-19147845

RESUMO

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P < 0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.


Assuntos
Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Mutação/genética , Telomerase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Estudos de Casos e Controles , Linhagem Celular , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Telomerase/química , Telômero/metabolismo
9.
Am J Hum Genet ; 82(3): 583-92, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18313026

RESUMO

It is well accepted that the Americas were the last continents reached by modern humans, most likely through Beringia. However, the precise time and mode of the colonization of the New World remain hotly disputed issues. Native American populations exhibit almost exclusively five mitochondrial DNA (mtDNA) haplogroups (A-D and X). Haplogroups A-D are also frequent in Asia, suggesting a northeastern Asian origin of these lineages. However, the differential pattern of distribution and frequency of haplogroup X led some to suggest that it may represent an independent migration to the Americas. Here we show, by using 86 complete mitochondrial genomes, that all Native American haplogroups, including haplogroup X, were part of a single founding population, thereby refuting multiple-migration models. A detailed demographic history of the mtDNA sequences estimated with a Bayesian coalescent method indicates a complex model for the peopling of the Americas, in which the initial differentiation from Asian populations ended with a moderate bottleneck in Beringia during the last glacial maximum (LGM), around approximately 23,000 to approximately 19,000 years ago. Toward the end of the LGM, a strong population expansion started approximately 18,000 and finished approximately 15,000 years ago. These results support a pre-Clovis occupation of the New World, suggesting a rapid settlement of the continent along a Pacific coastal route.


Assuntos
Indígena Americano ou Nativo do Alasca/genética , DNA Mitocondrial/genética , Emigração e Imigração , Filogenia , América , Genômica , Haplótipos , Humanos , Análise de Sequência de DNA
10.
Hum Mutat ; 31(2): 184-90, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19953531

RESUMO

Estimating the proportions of different ancestries in admixed populations is very important in population genetics studies, and it is particularly important for detecting population substructure effects in case-control association studies. In this work, a set of 48 ancestry-informative insertion-deletion polymorphisms (INDELs) were selected with the goal of efficiently measuring the proportions of three different ancestries (sub-Saharan African, European, and Native American) in mixed populations. All selected markers can be easily analyzed via multiplex PCR and detected with standard capillary electrophoresis. A total of 593 unrelated individuals representative of European, African, and Native American parental populations were typed, as were 380 individuals from three Brazilian populations with known admixture patterns. As expected, the interethnic admixture estimates show that individuals from southern Brazil present an almost exclusively European ancestry; Afro-descendant communities in the Amazon region, apart from the major African contribution, present some degree of admixture with Europeans and Native Americans; and a sample from Belém, in the northeastern Amazon, shows a significant contribution of the three ethnic groups, although with a greater European proportion. In summary, a panel of ancestry-informative INDELs was optimized and proven to be a valuable tool for estimating individual and global ancestry proportions in admixed populations. The ability to accurately infer interethnic admixtures highlights the usefulness of this marker set for assessing population substructure in association studies, particularly those conducted in Brazilian and other Latin American populations sharing trihybrid ancestry patterns.


Assuntos
Etnicidade/genética , Genealogia e Heráldica , Genética Populacional , Mutação INDEL/genética , Viés , População Negra/genética , Frequência do Gene/genética , Marcadores Genéticos , Humanos , Indígenas Norte-Americanos/genética , População Branca/genética
11.
Blood ; 112(2): 394-7, 2008 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-18434611

RESUMO

To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19(+)). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19(+) libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time reverse transcription-polymerase chain reaction in 78 CLL and 12 nCD19(+) cases (P < .001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6-fold increase of TOSO protein in circulating CLL cells (P = .013) and lymph nodes (P = .006). Our SAGE results have demonstrated that TOSO is a novel over-expressed antiapoptotic gene in CLL.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/etiologia , Proteínas de Membrana/genética , Receptor fas , Proteínas Reguladoras de Apoptose/fisiologia , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas de Membrana/fisiologia
12.
BMC Bioinformatics ; 10: 170, 2009 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-19500384

RESUMO

BACKGROUND: High-throughput molecular approaches for gene expression profiling, such as Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS) or Sequencing-by-Synthesis (SBS) represent powerful techniques that provide global transcription profiles of different cell types through sequencing of short fragments of transcripts, denominated sequence tags. These techniques have improved our understanding about the relationships between these expression profiles and cellular phenotypes. Despite this, more reliable datasets are still necessary. In this work, we present a web-based tool named S3T: Score System for Sequence Tags, to index sequenced tags in accordance with their reliability. This is made through a series of evaluations based on a defined rule set. S3T allows the identification/selection of tags, considered more reliable for further gene expression analysis. RESULTS: This methodology was applied to a public SAGE dataset. In order to compare data before and after filtering, a hierarchical clustering analysis was performed in samples from the same type of tissue, in distinct biological conditions, using these two datasets. Our results provide evidences suggesting that it is possible to find more congruous clusters after using S3T scoring system. CONCLUSION: These results substantiate the proposed application to generate more reliable data. This is a significant contribution for determination of global gene expression profiles. The library analysis with S3T is freely available at http://gdm.fmrp.usp.br/s3t/. S3T source code and datasets can also be downloaded from the aforementioned website.


Assuntos
Análise por Conglomerados , Etiquetas de Sequências Expressas/química , Perfilação da Expressão Gênica/métodos , RNA/química , Animais , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Interpretação Estatística de Dados , Bases de Dados Genéticas , Etiquetas de Sequências Expressas/metabolismo , Expressão Gênica , Humanos , Camundongos , RNA/metabolismo
13.
Exp Hematol ; 36(5): 642-54, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18295964

RESUMO

OBJECTIVE: The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS: We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS: Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION: Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.


Assuntos
Antígeno CD146/genética , Fibroblastos/citologia , Perfilação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Pericitos/citologia , Cordão Umbilical/citologia , Antígeno CD146/fisiologia , Diferenciação Celular/fisiologia , Separação Celular/métodos , Células Cultivadas , Análise por Conglomerados , Fibroblastos/fisiologia , Citometria de Fluxo , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/fisiologia , Pericitos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/genética , Cordão Umbilical/fisiologia
14.
Int J Cancer ; 122(4): 807-15, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17960622

RESUMO

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children.


Assuntos
Astrocitoma/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Proteínas Serina-Treonina Quinases/genética , Adulto , Apoptose , Astrocitoma/patologia , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proliferação de Células , Criança , Metilação de DNA , Dosagem de Genes , Glioblastoma/patologia , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas
15.
BMC Genomics ; 8: 249, 2007 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-17650329

RESUMO

BACKGROUND: The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. RESULTS: Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. CONCLUSION: Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.


Assuntos
Drosophila melanogaster/genética , Éxons , RNA Mensageiro/análise , Serina Endopeptidases/genética , Ferimentos e Lesões/genética , Algoritmos , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Drosophila melanogaster/microbiologia , Regulação Enzimológica da Expressão Gênica , Infecções/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Regulação para Cima , Ferimentos e Lesões/microbiologia
16.
Thromb Res ; 120(2): 221-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17113632

RESUMO

INTRODUCTION: Venous thrombosis (VT) and inflammation are two closely related entities. In the present investigation we assessed whether there is a relation between genetic modifiers of the inflammatory response and the risk of VT. MATERIALS AND METHODS: 420 consecutive and unrelated patients with an objective diagnosis of deep VT and 420 matched controls were investigated. The frequencies of the following gene polymorphisms were determined in all subjects: TNF-alpha-308 G/A, LT-alpha+252 A/G, IL-6-174 G/C, IL1-ra 86 bp VNTR, IL-10-1082 A/G and CD-31 125 C/G. RESULTS: Overall odds ratio (OR) for VT related to TNF-alpha-308 G/A, LT-alpha+252 A/G, IL-6-174 G/C, A1 allele (4 bp repeat) of the IL1-ra 86 bp VNTR, IL-10-1082 A/G and CD-31 125 C/G were respectively: 1.0 (CI95: 0.8-1.5), 1.3 (CI95: 1.0-1.7), 1.1 (CI95: 0.9-1.5), 1.6 (CI95: 1-2.5), 1.2 (CI95: 0.8-1.7) and 0.8 (CI95: 0.6-1.1). A possible interaction between polymorphisms was observed only for the co-inheritance of the mutant alleles of the LT-alpha+252 A/G and IL-10-1082 G/A polymorphisms (OR=2; CI95: 1.1-3.8). The risk of VT conferred by factor V Leiden and FII G20210A was not substantially altered by co-inheritance with any of the cytokine gene polymorphisms. CONCLUSIONS: Cytokine gene polymorphisms here investigated did not significantly influence venous thrombotic risk.


Assuntos
Citocinas/genética , Variação Genética , Trombose Venosa/genética , Trombose Venosa/imunologia , Adolescente , Adulto , Idoso , Alelos , Sequência de Bases , Brasil , Estudos de Casos e Controles , Criança , Pré-Escolar , Primers do DNA/genética , Feminino , Frequência do Gene , Humanos , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-10/genética , Interleucina-6/genética , Linfotoxina-alfa/genética , Masculino , Pessoa de Meia-Idade , Razão de Chances , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Polimorfismo Genético , Fatores de Risco , Fator de Necrose Tumoral alfa/genética , Trombose Venosa/etiologia
17.
Leuk Res ; 30(11): 1333-9, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16620968

RESUMO

The preferentially expressed antigen in melanoma (PRAME) gene is aberrantly expressed in chronic lymphoproliferative disorders (CLD). We produced and characterized an anti-PRAME monoclonal antibody (MoAb), which was then applied in a quantitative flow cytometric (QFC) method to evaluate PRAME expression in leukemic cells from the peripheral blood (PB) of 47 patients with chronic lymphocytic leukemia and seven with mantle cell lymphoma as well as in the PB mononuclear cells (PBMCs) and B lymphocytes from 15 healthy subjects. Approximately 90% of CLD, but none of the normal samples, presented more than 20% of PRAME+ lymphocytes. Moreover, the intensity of PRAME expression was significantly higher in CLD cells compared to normal B lymphocytes and PBMCs. By immunofluorescence microscopy and by permeabilized flow cytometry we demonstrated that PRAME is a membrane antigen and a cytoplasmic protein aberrantly expressed in malignant CLD. Our results suggest that the analysis of PRAME protein may contribute for the distinction between normal and leukemic cells in CLD, and that PRAME may be a potential target for therapy.


Assuntos
Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Célula do Manto/genética , Transtornos Linfoproliferativos/genética , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Antígenos de Neoplasias/imunologia , Linhagem Celular Tumoral , Citometria de Fluxo/métodos , Imunofluorescência/métodos , Humanos , Hibridização in Situ Fluorescente/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
18.
Genet Mol Res ; 5(1): 108-14, 2006 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-16755502

RESUMO

Serial analysis of gene expression (SAGE) technology produces large sets of interesting genes that are difficult to analyze directly. Bioinformatics tools are needed to interpret the functional information in these gene sets. We present an interactive web-based tool, called Gene Class, which allows functional annotation of SAGE data using the Gene Ontology (GO) database. This tool performs searches in the GO database for each SAGE tag, making associations in the selected GO category for a level selected in the hierarchy. This system provides user-friendly data navigation and visualization for mapping SAGE data onto the gene ontology structure. This tool also provides graphical visualization of the percentage of SAGE tags in each GO category, along with confidence intervals and hypothesis testing.


Assuntos
Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , Software , Terminologia como Assunto , Animais , Humanos , Internet , Modelos Estatísticos
19.
Rev Saude Publica ; 39(3): 490-7, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15997328

RESUMO

Individual cancer susceptibility seems to be related to factors such as changes in oncogenes and tumor suppressor genes expression, and differences in the action of metabolic enzymes and DNA repair regulated by specific genes. Epidemiological studies on genetic polymorphisms of human xenobiotics metabolizing enzymes and cancer have revealed low relative risks. Research considering genetic polymorphisms prevalence jointly with environmental exposures could be relevant for a better understanding of cancer etiology and the mechanisms of carcinogenesis and also for new insights on cancer prognosis. This study reviews the approaches of molecular epidemiology in cancer research, stressing case-control and cohort designs involving genetic polymorphisms, and factors that could introduce bias and confounding in these studies. Similarly to classical epidemiological research, genetic polymorphisms requires considering aspects of precision and accuracy in the study design.


Assuntos
Exposição Ambiental , Neoplasias/genética , Polimorfismo Genético/genética , Carcinógenos Ambientais , Métodos Epidemiológicos , Feminino , Predisposição Genética para Doença/genética , Humanos
20.
Brain Pathol ; 14(4): 399-405, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15605987

RESUMO

The distinction of astrocytomas and oligodendrogliomas, mainly pilocytic astrocytomas (PILOs) from infiltrating astrocytomas and oligodendrogliomas (ODs), and high-grade oligodendrogliomas from glioblastomas (GBMs), poses a serious clinical problem. There is no useful immunohistochemical (IHC) marker to differentiate these gliomas, and sometimes the differential diagnosis between them is arbitrary. We identified galectin-3 (Gal-3) as a possible tool to differentiate them based on gene expression profiles of GBMs. We confirmed the differential expression in 45 surgical samples (thirteen GBMs; seven PILOs; 5 grade II ODs; 5 anaplastic oligodendrogliomas [AODs], including 2 Oligo-astrocytomas; 8 diffuse astrocytomas [ASTs], and 7 non-neoplastic samples) by quantification of Gal-3 gene expression by real-time quantitative PCR (rt-PCR). Higher expression of Gal-3 was observed in GBMs and PILOs than in OD, AODs and ASTs. The IHC expression of Gal-3 was evaluated in 90 specimens (fifteen PlLOs, fourteen ASTs, 10 anaplastic astrocytomas, fifteen GBMs, eleven ODs, fifteen AODs, and 10 dysembryoplastic neuroepithelial tumors). The mean labeling score for Gal-3 determined according to the percentage of labeled cells in the tumor bulk was significantly different in GBMs versus AODs and in PILOs versus ASTs. Hence, Gal-3 is differentially expressed in central nervous system tumors, making IHC detection of Gal-3 a useful tool in distinguishing between these gliomas.


Assuntos
Astrocitoma/diagnóstico , Galectina 3 , Glioblastoma/diagnóstico , Oligodendroglioma/diagnóstico , Adolescente , Adulto , Astrocitoma/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Feminino , Galectina 3/genética , Galectina 3/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Glioblastoma/metabolismo , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Oligodendroglioma/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Coloração e Rotulagem/métodos
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