RESUMO
Keratolytic winter erythema (KWE) is a rare autosomal-dominant skin disorder characterized by recurrent episodes of palmoplantar erythema and epidermal peeling. KWE was previously mapped to 8p23.1-p22 (KWE critical region) in South African families. Using targeted resequencing of the KWE critical region in five South African families and SNP array and whole-genome sequencing in two Norwegian families, we identified two overlapping tandem duplications of 7.67 kb (South Africans) and 15.93 kb (Norwegians). The duplications segregated with the disease and were located upstream of CTSB, a gene encoding cathepsin B, a cysteine protease involved in keratinocyte homeostasis. Included in the 2.62 kb overlapping region of these duplications is an enhancer element that is active in epidermal keratinocytes. The activity of this enhancer correlated with CTSB expression in normal differentiating keratinocytes and other cell lines, but not with FDFT1 or NEIL2 expression. Gene expression (qPCR) analysis and immunohistochemistry of the palmar epidermis demonstrated significantly increased expression of CTSB, as well as stronger staining of cathepsin B in the stratum granulosum of affected individuals than in that of control individuals. Analysis of higher-order chromatin structure data and RNA polymerase II ChIA-PET data from MCF-7 cells did not suggest remote effects of the enhancer. In conclusion, KWE in South African and Norwegian families is caused by tandem duplications in a non-coding genomic region containing an active enhancer element for CTSB, resulting in upregulation of this gene in affected individuals.
Assuntos
Catepsina B/metabolismo , Elementos Facilitadores Genéticos , Eritema/genética , Duplicação Gênica , Regulação da Expressão Gênica , Ceratose/genética , Dermatopatias Genéticas/genética , Estudos de Casos e Controles , Catepsina B/genética , Mapeamento Cromossômico , Cromossomos Humanos Par 8/genética , Variações do Número de Cópias de DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Epiderme/metabolismo , Epigenômica , Eritema/epidemiologia , Feminino , Marcadores Genéticos , Humanos , Queratinócitos/metabolismo , Ceratose/epidemiologia , Células MCF-7 , Masculino , Noruega/epidemiologia , Linhagem , Dermatopatias Genéticas/epidemiologia , África do Sul/epidemiologiaRESUMO
MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.
Assuntos
MicroRNAs/genética , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Macular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters. METHODS: A multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact. RESULTS: Ophthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity. CONCLUSION: We identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.
Assuntos
Distrofias Hereditárias da Córnea/genética , Mutação , Sulfotransferases/genética , Adulto , Córnea/patologia , Distrofias Hereditárias da Córnea/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , África do Sul , Carboidrato SulfotransferasesRESUMO
BACKGROUND: Due to the unparalleled genetic diversity of its peoples, Africa is attracting growing research attention. Several African populations have been assessed in global initiatives such as the International HapMap and 1000 Genomes Projects. Notably excluded, however, is the southern Africa region, which is inhabited predominantly by southeastern Bantu-speakers, currently suffering under the dual burden of infectious and non-communicable diseases. Limited reference data for these individuals hampers medical research and prevents thorough understanding of the underlying population substructure. Here, we present the most detailed exploration, to date, of genetic diversity in 94 unrelated southeastern Bantu-speaking South Africans, resident in urban Soweto (Johannesburg). RESULTS: Participants were typed for ~4.3 million SNPs using the Illumina Omni5 beadchip. PCA and ADMIXTURE plots were used to compare the observed variation with that seen in selected populations worldwide. Results indicated that Sowetans, and other southeastern Bantu-speakers, are a clearly distinct group from other African populations previously investigated, reflecting a unique genetic history with small, but significant contributions from diverse sources. To assess the suitability of our sample as representative of Sowetans, we compared our results to participants in a larger rheumatoid arthritis case-control study. The control group showed good clustering with our sample, but among the cases were individuals who demonstrated notable admixture. CONCLUSIONS: Sowetan population structure appears unique compared to other black Africans, and may have clinical implications. Our data represent a suitable reference set for southeastern Bantu-speakers, on par with a HapMap type reference population, and constitute a prelude to the Southern African Human Genome Programme.
Assuntos
População Negra/genética , Genética Populacional , Polimorfismo de Nucleotídeo Único , Feminino , Técnicas de Genotipagem , Humanos , Masculino , Análise de Componente Principal , África do SulRESUMO
We describe cell type-specific significance analysis of microarrays (csSAM) for analyzing differential gene expression for each cell type in a biological sample from microarray data and relative cell-type frequencies. First, we validated csSAM with predesigned mixtures and then applied it to whole-blood gene expression datasets from stable post-transplant kidney transplant recipients and those experiencing acute transplant rejection, which revealed hundreds of differentially expressed genes that were otherwise undetectable.
Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Rejeição de Enxerto/sangue , Rejeição de Enxerto/genética , Humanos , Transplante de Rim , Modelos Lineares , RatosRESUMO
CONTEXT: Correct gender assignment in humans at the molecular level is crucial in many scientific disciplines and applied areas. MATERIALS AND METHODS: Candidate gender markers were identified through supervised statistical analysis of genome wide microarray expression data from human blood samples (N = 123, 58 female, 65 male) as a training set. The potential of the markers to predict undisclosed tissue donor gender was tested on microarray data from 13 healthy and 11 cancerous human tissue collections (internal) and external datasets from samples of varying tissue origin. The abundance of some genes in the marker panel was quantified by RT-PCR as alternative analytical technology. RESULTS: We identified and qualified predictive, gender-specific transcript markers based on a set of five genes (RPS4Y1, EIF1AY, DDX3Y, KDM5D and XIST). CONCLUSION: Gene expression marker panels can be used as a robust tissue- and platform-independent predictive approach for gender determination.
Assuntos
Perfilação da Expressão Gênica , RNA Mensageiro/sangue , Análise para Determinação do Sexo/métodos , Biomarcadores/sangue , RNA Helicases DEAD-box/sangue , RNA Helicases DEAD-box/genética , Feminino , Histona Desmetilases/sangue , Histona Desmetilases/genética , Humanos , Masculino , Antígenos de Histocompatibilidade Menor , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , RNA Longo não Codificante/sangue , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Proteínas Ribossômicas/sangue , Proteínas Ribossômicas/genética , TranscriptomaRESUMO
The European InnoMed-PredTox project was a collaborative effort between 15 pharmaceutical companies, 2 small and mid-sized enterprises, and 3 universities with the goal of delivering deeper insights into the molecular mechanisms of kidney and liver toxicity and to identify mechanism-linked diagnostic or prognostic safety biomarker candidates by combining conventional toxicological parameters with "omics" data. Mechanistic toxicity studies with 16 different compounds, 2 dose levels, and 3 time points were performed in male Crl: WI(Han) rats. Three of the 16 investigated compounds, BI-3 (FP007SE), Gentamicin (FP009SF), and IMM125 (FP013NO), induced kidney proximal tubule damage (PTD). In addition to histopathology and clinical chemistry, transcriptomics microarray and proteomics 2D-DIGE analysis were performed. Data from the three PTD studies were combined for a cross-study and cross-omics meta-analysis of the target organ. The mechanistic interpretation of kidney PTD-associated deregulated transcripts revealed, in addition to previously described kidney damage transcript biomarkers such as KIM-1, CLU and TIMP-1, a number of additional deregulated pathways congruent with histopathology observations on a single animal basis, including a specific effect on the complement system. The identification of new, more specific biomarker candidates for PTD was most successful when transcriptomics data were used. Combining transcriptomics data with proteomics data added extra value.
Assuntos
Ciclosporinas/toxicidade , Perfilação da Expressão Gênica/métodos , Marcadores Genéticos/genética , Gentamicinas/toxicidade , Túbulos Renais Proximais/efeitos dos fármacos , Proteômica/métodos , Animais , Biomarcadores/análise , Relação Dose-Resposta a Droga , Túbulos Renais Proximais/patologia , Túbulos Renais Proximais/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
BACKGROUND: Interstitial fibrosis and tubular atrophy (IF/TA) in renal transplants are the major morphological correlates of progressive graft deterioration. Early diagnosis of IF/TA is a pre-requisite for a timely therapeutic intervention in patients at risk. To evaluate events occurring before the overt onset of IF/TA, gene expression profiling of 3-month protocol biopsies from patients with IF/TA was performed in a patient group (n = 8) who developed mild IF/TA [chronic allograft nephropathy (CAN) grade I, by the Banff scoring system] in the subsequent 6-month protocol biopsy ('progressors'), and in 12 patients without IF/TA at 6 months ('non-progressors'). METHODS: RNA was extracted, labelled and hybridized to human specific genome wide DNA microarrays. Normalized data were subjected to gene-centric and pathway-centric statistical methods. RESULTS: Compared to the non-progressors, the 3-month biopsies of the progressor group showed overexpression of several genes that are important in the T- and B-cell activation and immune response. Genes involved in pro-fibrotic processes were identified in the biopsies of the progressors that preceded the observed IF/TA at 6 months. Furthermore, several genes with transporter and metabolic functions were underrepresented in the progressors in the 3-month biopsies. CONCLUSION: Gene expression profiling of early protocol biopsies identified changes in the transcriptome of grafts, which may be important for the development of IF/TA. Such early detection of transcriptome changes can facilitate the identification of patients at risk shifting the intervention time point well before the histological diagnosis of irreversible IF/TA.
Assuntos
Atrofia/genética , Fibrose/genética , Perfilação da Expressão Gênica , Rejeição de Enxerto/genética , Transplante de Rim , Túbulos Renais/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Atrofia/metabolismo , Atrofia/patologia , Biomarcadores/metabolismo , Biópsia , Criança , Feminino , Fibrose/metabolismo , Fibrose/patologia , Genoma Humano , Rejeição de Enxerto/metabolismo , Humanos , Técnicas Imunoenzimáticas , Túbulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Transplante Homólogo , Adulto JovemRESUMO
Hepatocyte transplantation is a promising therapy for acute liver failure in humans. Recently, we succeeded in inducing various acute and chronic liver failures in nude mice. Engraftment of transplanted xenogeneic rat hepatocytes, visualized in the host liver by anti-MHC class I immunohistochemistry, revealed that liver repopulation was limited, and equivalent in nude mice with and without acute liver failure. In the present study, acute liver failure was induced in nude mice by a single injection of sublethal anti-Fas antibody Jo2, followed 24 h later by rat hepatocyte transplantation and than by a weekly repeated injection of Jo2. Rat hepatocyte engraftment into the recipient liver parenchyma 3 weeks following hepatocyte transplantation was about sevenfold increased when nude mice were subsequently subjected to weekly repeated Jo2 injection. Genomic analysis of these mice showed an overall transcriptome profile of upregulation of cellular cycle blocking transcripts, activation of liver injury inducing IFN-gamma/STAT1 pathway, and circadian transcript signature of antiproliferative cell status compared to mice submitted to hepatocyte transplantation only. The findings of the present study suggest that the induction of cell proliferation blockade in recipient livers could promote sufficient engraftment of transplanted hepatocytes to allow transient or definitive treatment of liver failure in humans.
Assuntos
Sobrevivência de Enxerto/imunologia , Hepatócitos/transplante , Falência Hepática Aguda/terapia , Receptor fas/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Proliferação de Células , Feminino , Expressão Gênica , Sobrevivência de Enxerto/genética , Interferon gama/metabolismo , Falência Hepática Aguda/imunologia , Regeneração Hepática/genética , Regeneração Hepática/imunologia , Masculino , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT1/metabolismo , Transplante Heterólogo , Receptor fas/antagonistas & inibidoresRESUMO
In the present study we investigated the effect of a two-year treatment period with a diet containing 3.2g, 0.8 g and 0.15 g Mg/kg, on the rat liver transcriptome. At the end of the study, a treatment-dependent decrease in plasmatic Mg concentration was found (0.86 +/- 0.02 mmol/L, 0.70 +/- 0.02 mmol/L and 0.52 +/- 0.03 mmol/L for groups receiving 3.2g, 0.8 g and 0.15 g Mg/kg diet, respectively). No significant treatment-related effect on body and liver weights was observed, however a dietary Mg intake-dependent increase in mortality rate occurred in animals (11%, 25% and 38% death of animals). Mg content in the diet affected gene expression in rat livers, as assessed by rat specific DNA microarrays. We identified 11 genes up-regulated and 39 genes down-regulated by at least two-fold by a decrease in Mg content and grouped them within five functional pathways: metabolism 20%, cytoarchitecture (connective tissue/cell adhesion/cytoskeleton) 12%, channels/ transporters 20%, turn-over (nucleic acid and protein) 16%, and homeostasis (stress/DNA damage/apoptosis/ageing) 32%. The results of the present study confirm the pleiotropic effects of Mg and provide further evidence that a Mg decrease in the diet may be considered as a promoting factor for pathologies, especially in the liver, during ageing.
Assuntos
Dieta , Perfilação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Magnésio/administração & dosagem , Magnésio/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Magnésio/sangue , Masculino , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Taxa de Sobrevida , Fatores de TempoRESUMO
BACKGROUND AND AIM OF THE WORK: Sarcoidosis is a chronic granulomatous disorder of unknown etiology. In most patients the disease is self-limited, although for reasons unclear, others progress or die from progressive organ fibrosis. Growth factors have been implicated in the pathogenesis of other fibrotic lung conditions. We have, therefore, examined the relationship between growth factor expression and disease phenotype in sarcoidosis. METHODS: Adopting a target gene approach utilizing gene expression arrays, growth factor gene expression profile was analyzed in the peripheral blood of 12 patients and 12 healthy controls. Expression, functional activity and the effect of oligonucleotide antisense treatment on selected proteins differentially expressed in progressive sarcoidosis were then tested in vitro on primary human lung fibroblasts. RESULTS: Genes regulating angiogenesis were preferentially upregulated in the self-limited form of disease, while early growth response-1 and interleukin-6 were predominantly activated in progressive sarcoidosis. Increased expression of early growth response-1 in sarcoid lung was confirmed by immunohistochemistry. Stimulated human fibroblasts also rapidly expressed interleukin-6 and early growth response-1 and these proteins were found to mediate serum-induced fibroblast proliferation as proliferation could be significantly abrogated with interleukin-6 and early growth response-1 antisense oligonucelotides. CONCLUSION: We conclude that progressive pulmonary sarcoidosis is characterized by a fibroproliferative dysregulation potentially triggered by early growth response-1 and interleukin-6. Our disease model underlines the inability of steroids to prevent ongoing fibroproliferation in the lung.
Assuntos
Divisão Celular/fisiologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibroblastos/metabolismo , Interleucina-6/metabolismo , Sarcoidose Pulmonar/patologia , Estudos de Casos e Controles , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Proteína 1 de Resposta de Crescimento Precoce/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucina-6/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologiaRESUMO
Fibroadenomas are considered a benign lesion in rodent carcinogenicity studies. However, the entity adenocarcinoma arising in fibroadenoma does exist and in humans there is evidence of certain forms of fibroadenomas to confer greater risk of subsequent breast cancer. In this study, we aim to elucidate the molecular features of both spontaneous fibroadenomas and adenocarcinomas. The gene expression of the two tumour types is examined and compared to mammary gland in the same developmental state and examined for similarities which might indicate common molecular pathways. In the present study no similarities were discovered. We conclude that in the tumours examined here, no progression to adenocarcinoma is likely. Further studies are needed, examining a greater number of tumours and including cases of adenocarcinoma arising in fibroadenoma.
Assuntos
Adenocarcinoma/genética , Fibroadenoma/genética , Perfilação da Expressão Gênica , Glândulas Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/genética , Animais , Colágeno/biossíntese , Feminino , Imuno-Histoquímica , Fosfopiruvato Hidratase/análise , Fosfopiruvato Hidratase/genética , Ratos , Ratos Sprague-Dawley , Antígenos Thy-1/análise , Antígenos Thy-1/genéticaRESUMO
Breast cancer is the most frequently occurring cancer in women. Treatment options are still an active area of research. Models used for this purpose include induced models in rodents. By the advent of microarrays it has become possible to evaluate models not only for similar morphology or selected markers by polymerase chain reaction (PCR) or immunohistochemistry but also for the expression of thousands of genes at once. This study presents gene expression profiles of the hormone-sensitive 7,12-dimethylbenzanthracene-induced and the metastasising MTLn3-model. The models are discussed for their relevance to breast cancer in humans.
Assuntos
Perfilação da Expressão Gênica , Neoplasias Mamárias Experimentais/genética , 9,10-Dimetil-1,2-benzantraceno , Adenocarcinoma/genética , Animais , Modelos Animais de Doenças , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Metaloproteinases da Matriz/genética , Neoplasias Hormônio-Dependentes/genética , Osteopontina/genética , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-DawleyRESUMO
The differentiation of spontaneous and induced adenocarcinomas is of interest in the setting of carcinogenicity studies. In the experiment reported here, a differentiation of morphologically similar spontaneous and induced mammary adenocarcinomas of the rat is achieved by gene expression profiling. By choosing one marker gene based on the gene expression profile for each tumour type, we were able to distinguish the tumours by real-time quantitative polymerase chain reaction (RT-QPCR) as well.
Assuntos
Adenocarcinoma/genética , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Experimentais/genética , 9,10-Dimetil-1,2-benzantraceno , Animais , Feminino , Perfilação da Expressão Gênica , Neoplasias Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/patologia , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-DawleyRESUMO
Several studies using microarrays have shown that changes in gene expression provide information about the mechanism of toxicity induced by xenobiotic agents. Nevertheless, the issue of whether gene expression profiles are reproducible across different laboratories remains to be determined. To address this question, several members of the Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute evaluated the liver gene expression profiles of rats treated with methapyrilene (MP). Animals were treated at one facility, and RNA was distributed to five different sites for gene expression analysis. A preliminary evaluation of the number of modulated genes uncovered striking differences between the five different sites. However, additional data analysis demonstrated that these differences had an effect on the absolute gene expression results but not on the outcome of the study. For all users, unsupervised algorithms showed that gene expression allows the distinction of the high dose of MP from controls and low dose. In addition, the use of a supervised analysis method (support vector machines) made it possible to correctly classify samples. In conclusion, the results show that, despite some variability, robust gene expression changes were consistent between sites. In addition, key expression changes related to the mechanism of MP-induced hepatotoxicity were identified. These results provide critical information regarding the consistency of microarray results across different laboratories and shed light on the strengths and limitations of expression profiling in drug safety analysis.
Assuntos
Antialérgicos/toxicidade , Regulação da Expressão Gênica , Fígado/efeitos dos fármacos , Fígado/patologia , Metapirileno/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Masculino , Variações Dependentes do Observador , RNA/análise , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos TestesRESUMO
Hexachlorobenzene (HCB) is a persistent environmental pollutant with toxic effects in man and rat. Reported adverse effects are hepatic porphyria, neurotoxicity, and adverse effects on the reproductive and immune system. To obtain more insight into HCB-induced mechanisms of toxicity, we studied gene expression levels using DNA microarrays. For 4 weeks, Brown Norway rats were fed a diet supplemented with 0, 150, or 450 mg HCB/kg. Spleen, mesenteric lymph nodes (MLN), thymus, blood, liver, and kidney were collected and analyzed using the Affymetrix rat RGU-34A GeneChip microarray. Most significant (p < 0.001) changes, compared to the control group, occurred in spleen, followed by liver, kidney, blood, and MLN, but only a few genes were affected in thymus. This was to be expected, as the thymus is not a target organ of HCB. Transcriptome profiles confirmed known effects of HCB such as stimulatory effects on the immune system and induction of enzymes involved in drug metabolism, porphyria, and the reproductive system. In line with previous histopathological findings were increased transcript levels of markers for granulocytes and macrophages. New findings include the upregulation of genes encoding proinflammatory cytokines, antioxidants, acute phase proteins, mast cell markers, complements, chemokines, and cell adhesion molecules. Generally, gene expression data provide evidence that HCB induces a systemic inflammatory response, accompanied by oxidative stress and an acute phase response. In conclusion, this study confirms previously observed (immuno)toxicological effects of HCB but also reveals several new and mechanistically relevant gene products. Thus, transcriptome profiles can be used as markers for several of the processes that occur after HCB exposure.
Assuntos
Fungicidas Industriais/toxicidade , Perfilação da Expressão Gênica , Hexaclorobenzeno/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Reação de Fase Aguda , Administração Oral , Animais , Citocinas/biossíntese , Feminino , Fungicidas Industriais/administração & dosagem , Hexaclorobenzeno/administração & dosagem , Inflamação , Estresse Oxidativo , Ratos , Regulação para CimaRESUMO
BACKGROUND: Sarcoidosis is a systemic disorder of unknown cause, highly variable phenotype and unpredictable outcome. Antigen processing, inflammatory response and immunomodulation appear critical to development and prognosis of the disease. METHODS: We performed a comprehensive genomic analysis, applying high-density human GeneChip probe arrays (HUG95A, Affymetrix Inc.) for gene expression profiling from peripheral blood of patients with acute pulmonary sarcoidosis (n = 12) and matched healthy controls (n = 12), mean age 36 +/- 12 and 33 +/- 10 years respectively. RESULTS: At follow-up (18 [15-24] months), 7 patients had self-limited disease and 5 had persistent disease. Significantly different expression comparing patients and controls was identified for 1,860 (14.9%) and 729 (5.8%) gene products at p = 0.05 and p = 0.01 levels respectively. Genes closely associated with persistent disease included HLA-DRB1*1501 DQB1*0602, TNFA, NFKB, cyclic AMP-responsive element modulator (CREM) and T-cell activation marker CD69. IL1B, IL8, growth related (GRO)-beta/-gamma and CCR 2,5,6 were closely associated with self-limited disease. CONCLUSION: We hypothesize that, in self-limited disease, greater effector cell activation leads to successful antigen elimination/tolerance, whereas HLA-DRB1*1501 DQBI*0602-mediated, probably defective/partial T-lymphocyte activation results in an inefficient primary immune response, antigen intolerance and persistent disease.
Assuntos
Apresentação de Antígeno/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genômica , Sarcoidose Pulmonar/genética , Sarcoidose Pulmonar/imunologia , Adulto , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Sarcoidose Pulmonar/patologiaRESUMO
5-(2-chloroethyl)-2'-deoxyuridine (CEDU) is a pyrimidine nucleoside analogue formerly in development for the treatment of herpes simplex virus infections. The compound proved clearly mutagenic in the mouse spot test and exhibited weak activity in the Salmonella reverse mutation test, which led to the termination of the compound's development. In another study, CEDU, administered orally to beta-galactosidase (lacZ) transgenic mice (Muta Mouse) for five days, induced a clear increase in lacZ mutant frequencies in spleen, lung, and bone marrow. In the present follow-up study, we analyzed 32 of those lacZ mutants isolated from the bone marrow of the Muta Mouse animals of the experiments mentioned above, in order to obtain further information on the type of mutations induced by CEDU. CEDU induced a pronounced increase in A:T to G:C transitions. The distribution of A:T to G:C transitions was clearly non-random, showing a bias towards T to C substitutions in the coding DNA strand and a preference to occur in the sequence motif 5'-(G or C)-T-G-3'. Our data support the hypothesis that CEDU, after being phosphorylated, is incorporated into cellular DNA in place of thymidine, which leads to mispairing with guanosine during subsequent DNA replication. As a result, the compound is thought to exert its mutagenicity by inducing mismatches leading to T to C transitions. Our findings point towards a mode of mutagenic action of CEDU that differs fundamentally from that of other antiviral antinucleosides whose clastogenic and recombinogenic activities prevail.
Assuntos
Antivirais/toxicidade , Reparo do DNA/efeitos dos fármacos , Desoxiuridina/análogos & derivados , Desoxiuridina/toxicidade , Óperon Lac/genética , Mutação/genética , Animais , Antivirais/química , Composição de Bases , Sequência de Bases , Células da Medula Óssea , Análise Mutacional de DNA , Primers do DNA , Desoxiuridina/química , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
5-(2-chloroethyl)-2'-deoxyuridine (CEDU) had been developed for the treatment of herpes simplex infections. In the Salmonella reverse mutation test, the compound was found to be mutagenic in strains TA1535 and TA102 at very high concentrations (> or =2500 micro g/plate), both with and without S9-mix. The mutagenic potential of CEDU was further investigated in vivo and in vitro. It did not induce DNA repair in rat hepatocyte primary cultures, and was negative in the micronucleus test in V79 cells and in the comet assay in human leukocytes. In vivo, CEDU was negative in the bone marrow micronucleus test in CD1 mice. The mouse spot test provided a clearly positive result. Treatment of mice on day 9 of pregnancy with 2000 mg/kg resulted in 5.9% of the F1 animals having genetically relevant spots, whereas the corresponding vehicle control group had a spot rate of 1.9%. Since these data clearly identified CEDU as an inducer of gene mutations in vivo, this potential was further investigated in lacZ transgenic Muta Mouse. Six female animals were treated daily on five consecutive days with 2000 mg/kg/day and sacrificed, after a treatment-free sampling time, 14 days later. The data showed a clear increase in the mutant frequency in the bone marrow, the lung and in the spleen. CEDU is an exception in the group of nucleoside analogues, because it was found to be a strong gene mutagen and, in contrast to the other compounds of this group investigated so far, had no considerable clastogenic effects.
Assuntos
Antivirais/toxicidade , Reparo do DNA/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/toxicidade , Mutação/genética , Animais , Ensaio Cometa , Desoxiuridina/química , Feminino , Masculino , Camundongos , Testes para Micronúcleos , Gravidez , Ratos , Salmonella , Pigmentação da Pele/efeitos dos fármacosRESUMO
We recently found that rat hepatocyte transplantation was efficient (liver repopulation: 2.4%) in a sublethal nude mouse model (less than 33% mortality) of repeated liver injury generated using Jo2, a mouse-specific anti-Fas antibody, at sublethal dose of 250 µg/kg for 3 weeks. Genomic analysis of the livers revealed cell cycle blockade and an antiproliferative status of circadian genes, suggesting a selective advantage. By contrast, in the present study, freshly isolated human hepatocyte transplantation performed in the same mouse model resulted in implantation of less than 6,000 cells per liver (about 0.006% repopulation) in all animals. Genomic analysis of nude mouse livers revealed a lack of P21 upregulation, while a signature of stimulation of liver regeneration was observed, including upregulation of early response genes and upregulation of circadian genes. When we translated this sublethal model to a lethal model (65% mortality) by increasing the Jo2 repeated doses to 375 µg/kg, human hepatocyte engraftment was still very low; however, animal mortality was corrected by transplantation (only 20% mortality). Genomic findings in livers from the mice of the lethal Jo2 transplanted group were similar to those of the sublethal Jo2 transplanted group, that is, no selective advantage genomic signature and signature of mouse liver regeneration. In conclusion, transplanted human hepatocytes acted as if they modified nude mouse liver responses to Jo2 by stimulating liver regeneration, leading to an increased survival rate.