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1.
Nat Commun ; 10(1): 4703, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619666

RESUMO

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

2.
Sci Rep ; 9(1): 13758, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551465

RESUMO

RMRP was the first non-coding nuclear RNA gene implicated in a disease. Its mutations cause cartilage-hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia with growth failure, immunodeficiency, and a high risk for malignancies. This study aimed to gain further insight into the role of RNA Component of Mitochondrial RNA Processing Endoribonuclease (RMRP) in cellular physiology and disease pathogenesis. We combined transcriptome analysis with single-cell analysis using fibroblasts from CHH patients and healthy controls. To directly assess cell cycle progression, we followed CHH fibroblasts by pulse-labeling and time-lapse microscopy. Transcriptome analysis identified 35 significantly upregulated and 130 downregulated genes in CHH fibroblasts. The downregulated genes were significantly connected to the cell cycle. Multiple other pathways, involving regulation of apoptosis, bone and cartilage formation, and lymphocyte function, were also affected, as well as PI3K-Akt signaling. Cell-cycle studies indicated that the CHH cells were delayed specifically in the passage from G2 phase to mitosis. Our findings expand the mechanistic understanding of CHH, indicate possible pathways for therapeutic intervention and add to the limited understanding of the functions of RMRP.

3.
BMC Bioinformatics ; 20(1): 418, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409293

RESUMO

BACKGROUND: Standard RNAseq methods using bulk RNA and recent single-cell RNAseq methods use DNA barcodes to identify samples and cells, and the barcoded cDNAs are pooled into a library pool before high throughput sequencing. In cases of single-cell and low-input RNAseq methods, the library is further amplified by PCR after the pooling. Preparation of hundreds or more samples for a large study often requires multiple library pools. However, sometimes correlation between expression profiles among the libraries is low and batch effect biases make integration of data between library pools difficult. RESULTS: We investigated 166 technical replicates in 14 RNAseq libraries made using the STRT method. The patterns of the library biases differed by genes, and uneven library yields were associated with library biases. The former bias was corrected using the NBGLM-LBC algorithm, which we present in the current study. The latter bias could not be corrected directly, but could be solved by omitting libraries with particularly low yields. A simulation experiment suggested that the library bias correction using NBGLM-LBC requires a consistent sample layout. The NBGLM-LBC correction method was applied to an expression profile for a cohort study of childhood acute respiratory illness, and the library biases were resolved. CONCLUSIONS: The R source code for the library bias correction named NBGLM-LBC is available at https://shka.github.io/NBGLM-LBC and https://shka.bitbucket.io/NBGLM-LBC . This method is applicable to correct the library biases in various studies that use highly multiplexed sequencing-based profiling methods with a consistent sample layout with samples to be compared (e.g., "cases" and "controls") equally distributed in each library.


Assuntos
Biblioteca Gênica , Análise de Sequência de RNA/métodos , Transcriptoma , Linhagem Celular , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Componente Principal , RNA/química , RNA/metabolismo , Interface Usuário-Computador
5.
Sci Rep ; 9(1): 4366, 2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30867451

RESUMO

Systems biology is increasingly being applied in nanosafety research for observing and predicting the biological perturbations inflicted by exposure to nanoparticles (NPs). In the present study, we used a combined transcriptomics and proteomics approach to assess the responses of human monocytic cells to Au-NPs of two different sizes with three different surface functional groups, i.e., alkyl ammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated Au-NPs. Cytotoxicity screening using THP-1 cells revealed a pronounced cytotoxicity for the ammonium-terminated Au-NPs, while no cell death was seen after exposure to the carboxylated or PEG-modified Au-NPs. Moreover, Au-NR3+ NPs, but not the Au-COOH NPs, were found to trigger dose-dependent lethality in vivo in the model organism, Caenorhabditis elegans. RNA sequencing combined with mass spectrometry-based proteomics predicted that the ammonium-modified Au-NPs elicited mitochondrial dysfunction. The latter results were validated by using an array of assays to monitor mitochondrial function. Au-NR3+ NPs were localized in mitochondria of THP-1 cells. Moreover, the cationic Au-NPs triggered autophagy in macrophage-like RFP-GFP-LC3 reporter cells, and cell death was aggravated upon inhibition of autophagy. Taken together, these studies have disclosed mitochondria-dependent effects of cationic Au-NPs resulting in the rapid demise of the cells.

6.
BMC Genomics ; 19(1): 432, 2018 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-29866042

RESUMO

BACKGROUND: CCHCR1 (Coiled-Coil α-Helical Rod protein 1) is a putative psoriasis candidate gene with the risk alleles CCHCR1*WWCC and *Iso3, the latter inhibiting the translation of isoform 1. CCHCR1 was recently shown to be a centrosomal protein, as well as a component of cytoplasmic processing bodies (P-bodies) that regulate mRNA turnover. The function of CCHCR1 has remained unsettled, partly because of the inconsistent findings; it has been shown to play a wide variety of roles in divergent processes, e.g., cell proliferation and steroidogenesis. Here we utilized RNA sequencing (RNAseq) using HEK293 cells overexpressing isoforms 1 or 3 (Iso1, Iso3 cells), in combination with the coding non-risk or risk (*WWCC) haplotype of CCHCR1. Our aim was to study the overall role of CCHCR1 and the effects of its variants. RESULTS: The overexpression of CCHCR1 variants in HEK293 cells resulted in cell line-specific expression profiles though several similarities were observable. Overall the Iso1 and Iso3 cells showed a clear isoform-specific clustering as two separate groups, and the Non-risk and Risk cells often exhibited opposite effects. The RNAseq supported a role for CCHCR1 in the centrosomes and P-bodies; the most highlighted pathways included regulation of cytoskeleton, adherens and tight junctions, mRNA surveillance and RNA transport. Interestingly, both the RNAseq and immunofluorescent localization revealed variant-specific differences for CCHCR1 within the P-bodies. CONCLUSIONS: CCHCR1 influenced a wide variety of signaling pathways, which could reflect its active role in the P-bodies and centrosomes that both are linked to the cytoskeleton; as a centrosomal P-body protein CCHCR1 may regulate diverse cytoskeleton-mediated functions, such as cell adhesion and -division. The present findings may explain the previous inconsistent observations about the functions of CCHCR1.

7.
Sci Rep ; 7: 45771, 2017 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-28361960

RESUMO

Knowledge of the genomic variation among different strains of a pathogenic microbial species can help in selecting optimal candidates for diagnostic assays and vaccine development. Pooled sequencing (Pool-seq) is a cost effective approach for population level genetic studies that require large numbers of samples such as various strains of a microbe. To test the use of Pool-seq in identifying variation, we pooled DNA of 100 Streptococcus pyogenes strains of different emm types in two pools, each containing 50 strains. We used four variant calling tools (Freebayes, UnifiedGenotyper, SNVer, and SAMtools) and one emm1 strain, SF370, as a reference genome. In total 63719 SNPs and 164 INDELs were identified in the two pools concordantly by at least two of the tools. Majority of the variants (93.4%) from six individually sequenced strains used in the pools could be identified from the two pools and 72.3% and 97.4% of the variants in the pools could be mined from the analysis of the 44 complete Str. pyogenes genomes and 3407 sequence runs deposited in the European Nucleotide Archive respectively. We conclude that DNA sequencing of pooled samples of large numbers of bacterial strains is a robust, rapid and cost-efficient way to discover sequence variation.


Assuntos
Streptococcus pyogenes/genética , Variação Genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA
8.
Sci Rep ; 6: 33256, 2016 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-27633116

RESUMO

High-throughput sequencing using pooled DNA samples can facilitate genome-wide studies on rare and low-frequency variants in a large population. Some major questions concerning the pooling sequencing strategy are whether rare and low-frequency variants can be detected reliably, and whether estimated minor allele frequencies (MAFs) can represent the actual values obtained from individually genotyped samples. In this study, we evaluated MAF estimates using three variant detection tools with two sets of pooled whole exome sequencing (WES) and one set of pooled whole genome sequencing (WGS) data. Both GATK and Freebayes displayed high sensitivity, specificity and accuracy when detecting rare or low-frequency variants. For the WGS study, 56% of the low-frequency variants in Illumina array have identical MAFs and 26% have one allele difference between sequencing and individual genotyping data. The MAF estimates from WGS correlated well (r = 0.94) with those from Illumina arrays. The MAFs from the pooled WES data also showed high concordance (r = 0.88) with those from the individual genotyping data. In conclusion, the MAFs estimated from pooled DNA sequencing data reflect the MAFs in individually genotyped samples well. The pooling strategy can thus be a rapid and cost-effective approach for the initial screening in large-scale association studies.


Assuntos
Exoma , Frequência do Gene , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pré-Eclâmpsia/genética , Sequenciamento Completo do Exoma/métodos , Algoritmos , Alelos , Animais , Comportamento Compulsivo/diagnóstico , Comportamento Compulsivo/genética , Comportamento Compulsivo/fisiopatologia , DNA , Doenças do Cão/diagnóstico , Doenças do Cão/genética , Doenças do Cão/fisiopatologia , Cães , Feminino , Finlândia , Estudo de Associação Genômica Ampla , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Pré-Eclâmpsia/diagnóstico , Pré-Eclâmpsia/patologia , Gravidez , Sequenciamento Completo do Exoma/estatística & dados numéricos
9.
Sci Rep ; 6: 22745, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26976200

RESUMO

Psoriatic skin differs distinctly from normal skin by its thickened epidermis. Most gene expression comparisons utilize full-thickness biopsies, with substantial amount of dermis. We assayed the transcriptomes of normal, lesional, and non-lesional psoriatic epidermis, sampled as split-thickness skin grafts, with 5'-end RNA sequencing. We found that psoriatic epidermis contains more mRNA per total RNA than controls, and took this into account in the bioinformatic analysis. The approach highlighted innate immunity-related pathways in psoriasis, including NOD-like receptor (NLR) signaling and inflammasome activation. We demonstrated that the NLR signaling genes NOD2, PYCARD, CARD6, and IFI16 are upregulated in psoriatic epidermis, and strengthened these findings by protein expression. Interestingly, PYCARD, the key component of the inflammasome, showed an altered expression pattern in the lesional epidermis. The profiling of non-lesional skin highlighted PSORS4 and mitochondrially encoded transcripts, suggesting that their gene expression is altered already before the development of lesions. Our data suggest that all components needed for the active inflammasome are present in the keratinocytes of psoriatic skin. The characterization of inflammasome pathways provides further opportunities for therapy. Complementing previous transcriptome studies, our approach gives deeper insight into the gene regulation in psoriatic epidermis.


Assuntos
Epiderme/metabolismo , Perfilação da Expressão Gênica/métodos , Inflamassomos/genética , Proteínas NLR/genética , Psoríase/genética , Transdução de Sinais/genética , Idoso , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Epiderme/patologia , Epiderme/ultraestrutura , Feminino , Humanos , Imuno-Histoquímica , Inflamassomos/metabolismo , Queratinócitos/metabolismo , Masculino , Microscopia Confocal , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Psoríase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/metabolismo , Pele/patologia , Pele/ultraestrutura , Adulto Jovem
10.
Br J Haematol ; 171(4): 478-90, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26255870

RESUMO

Refractory anaemia with ring sideroblasts (RARS) is distinguished by hyperplastic inefficient erythropoiesis, aberrant mitochondrial ferritin accumulation and anaemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of RARS cases. To explore the link between SF3B1 mutations and anaemia, we studied mutated RARS CD34(+) marrow cells with regard to transcriptome sequencing, splice patterns and mutational allele burden during erythroid differentiation. Transcriptome profiling during early erythroid differentiation revealed a marked up-regulation of genes involved in haemoglobin synthesis and in the oxidative phosphorylation process, and down-regulation of mitochondrial ABC transporters compared to normal bone marrow. Moreover, mis-splicing of genes involved in transcription regulation, particularly haemoglobin synthesis, was confirmed, indicating a compromised haemoglobinization during RARS erythropoiesis. In order to define the phase during which erythroid maturation of SF3B1 mutated cells is most affected, we assessed allele burden during erythroid differentiation in vitro and in vivo and found that SF3B1 mutated erythroblasts showed stable expansion until late erythroblast stage but that terminal maturation to reticulocytes was significantly reduced. In conclusion, SF3B1 mutated RARS progenitors display impaired splicing with potential downstream consequences for genes of key importance for haemoglobin synthesis and terminal erythroid differentiation.


Assuntos
Anemia Refratária/genética , Anemia Sideroblástica/genética , Eritropoese/genética , Hemoglobinas/biossíntese , Fosfoproteínas/genética , Processamento de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Idoso , Idoso de 80 Anos ou mais , Anemia Refratária/sangue , Anemia Sideroblástica/sangue , Transporte Biológico/genética , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Heterogeneidade Genética , Humanos , Ferro/metabolismo , Fosfoproteínas/fisiologia , Isoformas de Proteínas/genética , Fatores de Processamento de RNA , RNA Mensageiro/genética , Ribonucleoproteína Nuclear Pequena U2/fisiologia , Análise de Sequência de RNA , Transdução de Sinais/genética
11.
BMC Genomics ; 16: 476, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26108968

RESUMO

BACKGROUND: Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs' lifecycle has been omitted. RESULTS: We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types. CONCLUSIONS: The epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Queratinócitos/metabolismo , RNA/administração & dosagem , Apoptose/genética , Epiderme/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Queratinócitos/citologia , RNA/síntese química , RNA/genética , RNA Mensageiro/genética , Análise de Sequência de RNA , Pele/efeitos dos fármacos , Pele/metabolismo , Transplante de Pele
12.
PLoS One ; 4(12): e8037, 2009 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-19997561

RESUMO

BACKGROUND: Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multiple susceptibility genes. We have previously reported suggestive linkage to the chromosomal region 14q21-q23 in Finnish SLE families. PRINCIPAL FINDINGS: Genetic fine mapping of this region in the same family material, together with a large collection of parent affected trios from UK and two independent case-control cohorts from Finland and Sweden, indicated that a novel uncharacterized gene, MAMDC1 (MAM domain containing glycosylphosphatidylinositol anchor 2, also known as MDGA2, MIM 611128), represents a putative susceptibility gene for SLE. In a combined analysis of the whole dataset, significant evidence of association was detected for the MAMDC1 intronic single nucleotide polymorphisms (SNP) rs961616 (P -value = 0.001, Odds Ratio (OR) = 1.292, 95% CI 1.103-1.513) and rs2297926 (P -value = 0.003, OR = 1.349, 95% CI 1.109-1.640). By Northern blot, real-time PCR (qRT-PCR) and immunohistochemical (IHC) analyses, we show that MAMDC1 is expressed in several tissues and cell types, and that the corresponding mRNA is up-regulated by the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) in THP-1 monocytes. Based on its homology to known proteins with similar structure, MAMDC1 appears to be a novel member of the adhesion molecules of the immunoglobulin superfamily (IgCAM), which is involved in cell adhesion, migration, and recruitment to inflammatory sites. Remarkably, some IgCAMs have been shown to interact with ITGAM, the product of another SLE susceptibility gene recently discovered in two independent genome wide association (GWA) scans. SIGNIFICANCE: Further studies focused on MAMDC1 and other molecules involved in these pathways might thus provide new insight into the pathogenesis of SLE.


Assuntos
Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Moléculas de Adesão de Célula Nervosa/genética , Linhagem Celular , Cromossomos Humanos Par 14/genética , Citocinas/genética , Citocinas/metabolismo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Ligação Genética , Loci Gênicos/genética , Humanos , Lúpus Eritematoso Sistêmico/patologia , Monócitos/metabolismo , Moléculas de Adesão de Célula Nervosa/química , Razão de Chances , Filogenia , Polimorfismo de Nucleotídeo Único/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos
13.
Virchows Arch ; 455(6): 495-503, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19921252

RESUMO

Merkel cell carcinoma (MCC) is an aggressive cutaneous tumor with poor outcome and increasing incidence. We examined by immunohistochemistry the expression of three novel matrix metalloproteinases (MMPs)-MMP-21, MMP-26, and MMP-28-in 44 primary MCC tumors and six lymph node metastases while MMP-10 served as a positive control. Their mRNA expression was also studied in the UISO MCC cell line basally and after various stimulations using quantitative real-time PCR. MMP-28 was observed in tumor cells of 15/44 samples especially in tumors <2 cm in diameter (p = 0.015) while 21/44 specimens showed MMP-28 in the tumor stroma. Expression of MMP-21 was demonstrated in tumor cells of 13/43 samples. MMP-26, instead, was positive in stromal cells (17/44) and its expression associated with tumors >or=2 cm in diameter (p = 0.006). Stromal expression of MMP-10 was the most frequent finding of the studied samples (31/44), but MMP-10 was detected also in tumor cells (17/44). Most of the metastatic lymph nodes expressed MMP-10 and MMP-26. MMP-10, MMP-21, and MMP-28 mRNAs were basally expressed by the UISO cells, and the corresponding proteins were detectable by immunostaining of cultured cells. IFN-alpha and TNF-alpha downregulated MMP-21 and MMP-28 expression. Our results suggest that novel MMPs may have a role in MCC pathogenesis: especially that MMP-26 expression in stroma is associated with larger tumors with poor prognosis. Expression of MMP-21 and MMP-28 seems to associate with the tumors of lesser malignant potential. We also confirm the previous finding on the role of MMP-10 in MCC pathogenesis.


Assuntos
Carcinoma de Célula de Merkel/genética , Metaloproteinase 10 da Matriz/genética , Metaloproteinases da Matriz Secretadas/genética , Metaloproteinases da Matriz/genética , Neoplasias Cutâneas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Célula de Merkel/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Neoplasias Cutâneas/patologia
14.
Exp Dermatol ; 18(12): 1044-52, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19601983

RESUMO

The squamous cell cancers (SCC) of renal transplant recipients are more aggressive and metastasize earlier than those of the non-immunocompromised population. Matrix metalloproteinases (MMPs) have a central role in tumor initiation, invasion and metastasis. Our aim was to compare the expression of MMPs-10, -12 and -21 in SCCs from immunosuppressed (IS) and control patients and the contribution of MMPs-10 and -21 to SCC development in the FVB/N-Tg(KRT5-Nfkbia)3Rto mouse line. Immunohistochemical analysis of 25 matched pairs of SCCs, nine of Bowen's disease and timed back skin biopsies of mice with selective inhibition of Rel/NF-kappaB signalling were performed. Semiquantitatively assessed stromal MMP-10 expression was higher (P = 0.009) in the control group when compared with IS patients. Tumor cell-derived MMP-10, -12 and -21 expression did not differ between the groups but stromal fibroblasts of the control SCCs tended to express MMP-21 more abundantly. MMP-10 expression was observed already in Bowen's disease while MMP-21 was absent. MMP-10 and -21 were present in inflammatory or stromal cells in ageing mice while dysplastic keratinocytes and invasive cancer were negative. Our results suggest that MMP-10 may be important in the initial stages of SCC progression and induced in the stroma relating to the general host-response reaction to skin cancer. MMP-21 does not associate with invasion of SCC but may be involved in keratinocyte differentiation.


Assuntos
Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinases da Matriz Secretadas/metabolismo , Neoplasias de Células Escamosas/metabolismo , Neoplasias Cutâneas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Doença de Bowen/etiologia , Doença de Bowen/metabolismo , Doença de Bowen/patologia , Ciclosporina/efeitos adversos , Ciclosporina/uso terapêutico , Modelos Animais de Doenças , Endotélio/metabolismo , Receptores ErbB/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Imunossupressão/efeitos adversos , Queratinócitos/metabolismo , Queratinócitos/patologia , Transplante de Rim/efeitos adversos , Linfócitos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Metaloproteinase 12 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Neoplasias de Células Escamosas/etiologia , Neoplasias de Células Escamosas/patologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Pele/metabolismo , Pele/patologia , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Células Estromais/metabolismo , Células Estromais/patologia
15.
PLoS One ; 4(6): e6030, 2009 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-19551138

RESUMO

Despite chronic inflammation, psoriatic lesions hardly ever progress to skin cancer. Aberrant function of the CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1, HCR) within the PSORS1 locus may contribute to the onset of psoriasis. As CCHCR1 is expressed in certain cancers and regulates keratinocyte (KC) proliferation in a transgenic mouse model, we studied its relation to proliferation in cutaneous squamous cell cancer (SCC) cell lines by expression arrays and quantitative RT-PCR and in skin tumors by immunohistochemistry. CCHCR1 protein was detected in the pushing border of SCC and lining basal cell carcinoma islands. Different from psoriasis, Ki67 had a similar expression pattern as CCHCR1. The most intense CCHCR1 staining occurred in areas positive for epidermal growth factor receptor (EGFR). Expression of CCHCR1 mRNA was upregulated 30-80% in SCC lines when compared to normal KCs and correlated positively with Ki67 expression. The most aggressive and invasive tumor cell lines (RT3, FaDu) expressed CCHCR1 mRNA less than non-tumorigenic HaCaT cells. Moreover, the tumor promoters okadaic acid and menadione downregulated CCHCR1 mRNA. We conclude that both in psoriasis and the early stages of KC transformation, CCHCR1 may function as a negative regulator of proliferation, but beyond a certain point in oncogenesis cannot control this phenomenon any longer.


Assuntos
Receptores ErbB/biossíntese , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Psoríase/metabolismo , Neoplasias Cutâneas/metabolismo , Regulação para Cima , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Imuno-Histoquímica/métodos , Inflamação , Antígeno Ki-67/biossíntese , Linfócitos/metabolismo , Modelos Biológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
16.
J Invest Dermatol ; 129(1): 119-30, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18633436

RESUMO

In the skin, expression of several matrix metalloproteinases (MMPs) occurs in response to tissue injury, tumorigenesis, angiogenesis, apoptosis, and inflammation. The recently cloned MMP-21 has been implicated in skin development and various epithelial cancers. In this study, we found that it is also expressed by differentiated keratinocytes (KCs) in various benign skin disorders, in which it was not associated with KC apoptosis or proliferation, and in organotypic cultures. Furthermore, MMP-21 was induced in keratinocytes in association with increased calcium and presence of the differentiation marker filaggrin. In stably transfected A431 and HEK293 cell lines, MMP-21 increased invasion of cells but did not associate with increased apoptosis, proliferation, or epithelial-to-mesenchymal transition. Of various agents tested in HaCaT cell cultures, only retinoic acid (10(-6) M) and staurosporine (2.5 x 10(-8) M) upregulated MMP-21 mRNA and protein expression, whereas tumor promoters, hormones, or dexamethasone were without effect. Our results suggest that MMP-21 may be an important protease in the terminal differentiation of keratinocytes.


Assuntos
Regulação Enzimológica da Expressão Gênica , Queratinócitos/metabolismo , Metaloproteinases da Matriz Secretadas/biossíntese , Tretinoína/metabolismo , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular , Colágeno/metabolismo , Dexametasona/farmacologia , Humanos , Ratos , Estaurosporina/farmacologia , Tretinoína/farmacologia
17.
Mod Pathol ; 20(11): 1128-40, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17873896

RESUMO

Pancreatic adenocarcinoma is known for early aggressive local invasion, high metastatic potential, and a low 5-year survival rate. Matrix metalloproteinases (MMPs) play important roles in tumor growth and invasion. Earlier studies on pancreatic cancer have found increased expression of certain MMPs to correlate with poorer prognosis, short survival time or presence of metastases. We studied the expression of MMP-21, -26, and tissue inhibitor of matrix metalloproteinases (TIMP)-4 in 50 tissue samples, including 25 adenocarcinomas, seven other malignant pancreatic tumors, and 18 control samples of non-neoplastic pancreatic tissue with immunohistochemistry. The regulation of MMP-21, -26, and TIMP-4 mRNAs by cytokines was studied with RT-PCR in pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1. MMP-21, -26, and TIMP-4 were detected in cancer cells in 64, 40, and 60% of tumors, respectively. MMP-21 expressed in well-differentiated cancer cells and occasional fibroblasts, like TIMP-4, tended to diminish in intensity from grade I to grade III tumors. Patients with metastatic lymph nodes had increased expression of MMP-26 in actual tumor samples. All cultured cancer cell lines expressed MMP-21 basally at low levels, and presence of the protein was confirmed immunohistochemically in cultured cells. MMP-21 expression was induced by epidermal growth factor (EGF) in PANC-1 cells. MMP-26 was neither expressed basally nor induced by tumor necrosis factor alpha, transforming growth factor beta-1 (TGFbeta1), EGF, or interferon gamma. Basal TIMP-4 expression was lowest in the poorly differentiated cancer cell line PANC-1 compared to better-differentiated BxPC-3 and AsPC-1 cells. TIMP-4 expression was induced by TGFbeta1 in PANC-1 cells and by EGF in BxPC-3 cells. Our findings suggest that MMP-21 is not a marker of invasiveness, but rather of differentiation, in pancreatic cancer and it may be upregulated by EGF. The putative role of MMP-26 as a marker of metastases warrants further studies. Unlike other TIMPs, TIMP-4 was not upregulated in relation to aggressiveness of pancreatic cancer.


Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/análise , Metaloproteinases da Matriz Secretadas/biossíntese , Metaloproteinases da Matriz/biossíntese , Neoplasias Pancreáticas/metabolismo , Inibidores Teciduais de Metaloproteinases/biossíntese , Adenocarcinoma/patologia , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica/fisiopatologia , Neoplasias Pancreáticas/patologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Acta Derm Venereol ; 87(3): 223-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17533487

RESUMO

Allograft inflammatory factor-1 (AIF-1) is an evolutionarily conserved, inflammatory protein produced by activated macrophages during chronic transplant rejection and in inflammatory brain lesions. Since T-cell-mediated inflammation is common to various dermatoses and nothing is known about AIF-1 in skin, we studied its protein expression at the tissue level and regulation in monocytic cell lines by various agents. Using immunohistochemistry, we found that AIF-1 is expressed at low levels in normal skin, but is highly upregulated in various inflammatory skin disorders, such as psoriasis, lichen planus, graft-versus-host disease and mycosis fungoides. The main cell types expressing AIF-1 in affected skin are macrophages and Langerhans' cells. We also show by real-time PCR that AIF-1 mRNA levels in monocytic THP-1 and U937 cell lines are significantly upregulated by retinoic acid as well as a number of cytokines. We conclude that AIF-1 may mediate survival and pro-inflammatory properties of macrophages in skin diseases.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Dermatite/metabolismo , Anti-Inflamatórios/farmacologia , Linhagem Celular , Citocinas/metabolismo , Células Dendríticas/metabolismo , Dexametasona/farmacologia , Reação Enxerto-Hospedeiro , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ceratolíticos/farmacologia , Células de Langerhans/metabolismo , Macrófagos/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Pele/lesões , Pele/metabolismo , Tretinoína/farmacologia , Regulação para Cima
19.
J Med Genet ; 44(5): 314-21, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17220214

RESUMO

BACKGROUND: Several members of the GIMAP gene family have been suggested as being involved in different aspects of the immune system in different species. Recently, a mutation in the GIMAP5 gene was shown to cause lymphopenia in a rat model of autoimmune insulin-dependent diabetes. Thus it was hypothesised that genetic variation in GIMAP5 may be involved in susceptibility to other autoimmune disorders where lymphopenia is a key feature, such as systemic lupus erythematosus (SLE). MATERIAL AND METHODS: To investigate this, seven single nucleotide polymorphisms in GIMAP5 were analysed in five independent sets of family-based SLE collections, containing more than 2000 samples. RESULT: A significant increase in SLE risk associated with the most common GIMAP5 haplotype was found (OR 1.26, 95% CI 1.02 to 1.54, p = 0.0033). In families with probands diagnosed with trombocytopenia, the risk was increased (OR 2.11, 95% CI 1.09 to 4.09, p = 0.0153). The risk haplotype bears a polymorphic polyadenylation signal which alters the 3' part of GIMAP5 mRNA by producing an inefficient polyadenylation signal. This results in higher proportion of non-terminated mRNA for homozygous individuals (p<0.005), a mechanism shown to be causal in thalassaemias. To further assess the functional effect of the polymorphic polyadenylation signal in the risk haplotype, monocytes were treated with several cytokines affecting apoptosis. All the apoptotic cytokines induced GIMAP5 expression in two monocyte cell lines (1.5-6 times, p<0.0001 for all tests). CONCLUSION: Taken together, the data suggest the role of GIMAP5 in the pathogenesis of SLE.


Assuntos
Proteínas de Ligação ao GTP/genética , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Poliadenilação/genética , Polimorfismo Genético , Citocinas/farmacologia , Éxons/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Frequência do Gene/efeitos dos fármacos , Marcadores Genéticos/efeitos dos fármacos , Haplótipos/efeitos dos fármacos , Humanos , Metanálise como Assunto , Monócitos/efeitos dos fármacos , Razão de Chances , Poliadenilação/efeitos dos fármacos , Polimorfismo Genético/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Risco , Células U937
20.
Biochem Biophys Res Commun ; 351(3): 777-83, 2006 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-17084384

RESUMO

The -863 C/A polymorphism in the tumor necrosis factor-alpha (TNF-alpha) promoter has been suggested to influence TNF-alpha expression. Here we elucidated the molecular mechanisms underlying the allele-specific regulation of TNF-alpha gene expression under basal and LPS-stimulated conditions in THP-1 cells and in human primary macrophages. We show that the binding of two NF-kappaB complexes, the p50/p50 homodimer and the p50/p65 heterodimer, was upregulated upon LPS stimulation. Both complexes bound to the C-allele whereas the A-allele only bound the p50/p65 complex. Two DNase I hypersensitive sites appeared in the TNF-alpha promoter after LPS stimulation of THP-1 cells. DNase I hypersensitivity of the TNF-alpha promoter was also analyzed in human monocytes prepared from individuals of different -863C/A genotype. Hypersensitivity was increased in the promoter harboring the mutant A-allele, particularly after LPS stimulation. In summary, binding of transcription factor NF-kappaB to the TNF-alpha promoter is associated with allele-specific remodeling of chromatin structure.


Assuntos
Montagem e Desmontagem da Cromatina/genética , Monócitos/fisiologia , Regiões Promotoras Genéticas/genética , Fator de Necrose Tumoral alfa/genética , Alelos , Células Cultivadas , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Humanos , Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/genética
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