ABSTRACT
Dendritic cells (DCs) are antigen (Ag)-presenting cells that activate and stimulate effective immune responses by T cells, but can also act as negative regulators of these responses and thus play important roles in immune regulation. Pro-angiogenic vascular endothelial growth factor (VEGF) has been shown to cause defective DC differentiation and maturation. Previous studies have demonstrated that the addition of VEGF to DC cultures renders these cells weak stimulators of Ag-specific T cells due to the inhibitory effects mediated by VEGF receptor 1 (VEGFR1) and/or VEGFR2 signalling. As the enzyme indoleamine 2,3-dioxygenase (IDO) is recognised as an important negative regulator of immune responses, this study aimed to investigate whether VEGF affects the expression of IDO by DCs and whether VEGF-matured DCs acquire a suppressor phenotype. Our results are the first to demonstrate that VEGF increases the expression and activity of IDO in DCs, which has a suppressive effect on Ag-specific and mitogen-stimulated lymphocyte proliferation. These mechanisms have broad implications for the study of immunological responses and tolerance under conditions as diverse as cancer, graft rejection and autoimmunity.
Subject(s)
Humans , Cell Proliferation/physiology , Dendritic Cells/drug effects , /metabolism , Lymphocytes/physiology , Vascular Endothelial Growth Factor A/pharmacology , Apoptosis , Antigens, Surface/biosynthesis , Cell Culture Techniques , Cells, Cultured , Cell Differentiation/physiology , Dendritic Cells/metabolism , Dendritic Cells/ultrastructure , Immune Tolerance/physiology , /genetics , Leukocytes, Mononuclear/physiology , Monocytes/cytology , Monocytes/ultrastructure , Necrosis , Real-Time Polymerase Chain Reaction , Signal Transduction/immunologyABSTRACT
An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95 percent concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).
Subject(s)
Humans , DNA, Bacterial , Multilocus Sequence Typing/methods , Stenotrophomonas , Bacterial Typing Techniques , DNA-Directed RNA Polymerases , PhylogenyABSTRACT
A susceptibilidade para desenvolver doenças crônicas na vida adulta, tais como diabetes, doenças cardiovasculares ou distúrbios de comportamento, depende em larga medida da interação entre fatores genéticos e ambientais na vida fetal e nos primeiros anos de vida. A investigação de como alterações nos ambientes pré e pós-natal modificam o funcionamento dos genes, promovendo mudanças permanentes no metabolismo e determinando a susceptibilidade a diferentes doenças, constitui hoje o alvo principal da genômica pediátrica. O conhecimento obtido nessa área da Medicina poderá permitir, futuramente, que intervenções específicas em fases iniciais da vida conduzam à melhoria da saúde humana. Este artigo resumeavanços recentes em genômica pediátrica e as atividades do Departamento de Pediatria da FMUSP nessa área...
The susceptibility for developing chronic diseases in adult life, such as diabetes, cardiovascular diseases or behavioral disorders, depends in a large extension on the interplay between genetic and environmental factors in fetal life and early childhood. The investigation on how changes in pre and post natal environment modify gene function, leading to permanentmetabolic alterations and disease susceptibility is nowadays the main target of pediatric genomics. The knowledge obtained in this area of medical research will render possible in a near future that specific intervention in early life stages may conduct to improvements in human health. This article reviews recent progresses in pediatric genomics and summarizes the activities of the FMUSP Department of Pediatrics in this area...
Subject(s)
Humans , Gene Expression , Genome , Genomics , PediatricsABSTRACT
PURPOSE: Screening for mutations in the entire Cystic Fibrosis gene (CFTR) of Brazilian infertile men with congenital absence of vas deferens, in order to prevent transmission of CFTR mutations to offspring with the use of assisted reproductive technologies. METHOD: Specific polymerase chain reaction (PCR) primers were designed to each of the 27 exons and splicing sites of interest followed by single strand conformational polymorphism and Heteroduplex Analysis (SSCP-HA) in precast 12.5 percent polyacrylamide gels at 7°C and 20°C. Fragments with abnormal SSCP migration pattern were sequenced. RESULTS: Two novel missense mutations (S753R and G149W) were found in three patients (two brothers) together with the IVS8-5T allele in hetrozygosis. CONCLUSION: The available screenings for CF mutations do not include the atypical mutations associated to absence of vas deferens and thus, when these tests fail to find mutations, there is still a genetic risk of affected children with the help of assisted reproduction. We recommend the screening of the whole CFTR gene for these infertile couples, as part of the work-up before assisted reproduction.
OBJETIVO: Pesquisar mutações em toda a extensão do gene que causa a Fibrose Cística (CFTR) de homens brasileiros inférteis por agenesia congênita dos vasos deferentes, com a finalidade de prevenir a transmissão de mutações em CFTR à prole com o uso das tecnologias de reprodução assistida. MÉTODOS: Foram desenhados oligonucleotídeos específicos para realização de reação de polimerização em cadeia (PCR) para cada um dos 27 exons e sítios de processamento de interesse no gene CFTR. O PCR foi seguido pela técnica de SSCP-HA (polimorfismos de conformação no DNA de fita simples e na formação de heteroduplexes) em géis pré-fabricados de poliacrilamida a 12,5 por cento em duas temperaturas, 7°C e 20°C. Os fragmentos com padrão alterado na migração do SSCP foram submetidos a seqüenciamento automatizado. RESULTADOS: Foram identificadas duas mutações novas com alteração de aminoácidos (S753R and G149W) em 3 pacientes (dois irmãos) juntamente com o alelo IVS8-5T em heterozigose. CONCLUSÕES: O rastreamento básico de mutações típicas da Fibrose Cística não inclui as mutações atípicas associadas à ausência dos deferentes. Desta forma, quando esses testes resultam normais, ainda assim existe um risco genético de crianças afetadas serem geradas com auxílio das Assisted Reproduction Technologies. Por este motivo, recomenda-se que a pesquisa de mutações em todo o gene CFTR seja o exame a ser oferecido para todos os casais inférteis em que o homem seja portador de agenesia dos vasos deferentes, antes da realização de reprodução assistida.
Subject(s)
Adult , Humans , Male , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Infertility, Male/genetics , Mutation, Missense/genetics , Vas Deferens/abnormalities , Genetic Counseling , Polymerase Chain Reaction , Polymorphism, Single-Stranded ConformationalABSTRACT
Familial hemiplegic migraine (FHM) is a rare autosomal dominant form of migraine with aura. This disease has been associated with missense mutations in the CACNA1A and ATP1A2 genes. The aim of this study was to identify whether CACNA1A and ATP1A2 are or not related to Brazilian FHM. Here we screened four Brazilian FHM families (total of 26 individuals - 13 affected and 13 asymptomatic or normal) for mutations in both genes. We found an amino acid change in a member of family FHM-D (Arg2206Gly). However since this alteration is not present in all affected individuals and is present in one asymptomatic individual it should be considered a polymorphism. Further studies with additional families will be necessary to reveal the importance of both CACNA1A and ATP1A2 genes on the pathogeneses of FHM in Brazil and to test the third gene (SCN1A) in these FHM families.
A enxaqueca hemiplégica familial (EHF) é uma forma rara de enxaqueca com aura e apresenta herança autossômica dominante. Esta doença está associada com mutações do tipo missense nos genes CACNA1A e ATP1A2. O objetivo deste estudo foi identificar se os genes CACNA1A e ATP1A2 estão ou não relacionados com a enxaqueca hemiplégica familial em famílias brasileiras. Os genes citados acima foram analisados em quatro famílias brasileiras (total de 26 indivíduos - 13 afetados e 13 assintomáticos ou normais) e uma troca de aminoácido em um membro da família FHM-D (Arg2206Gly) foi observada. Porém, esta alteração não foi identificada em todos os indivíduos afetados e está presente em um indivíduo assintomático, devendo, portanto, ser considerada um polimorfismo. Estudos adicionais nas famílias já estudadas e em outras famílias brasileiras afetadas por enxaqueca hemiplégica familial serão necessários para esclarecer a importância dos genes CACNA1A e ATP1A2 na patogênese da EHF no Brasil, bem como para testar o terceiro gene (SCN1A) relacionado à EHF.