Irritant contact dermatitis (ICD) pathogenesis is not completely understood and the genes participating in the epidermal response towards chemical irritants are only partly known. It is commonly accepted that different irritants have different mechanisms of action in the development of ICD. To define the differential molecular events induced in the epidermis by different irritants, we collected sequential biopsies (, 4 and 24 hours after a single exposure and at day 11 after repeated exposure) from human volunteers exposed to sodium lauryl sulphate (SLS) or nonanoic acid (NON). Gene expression analysis using high-density oligonucleotide microarrays revealed essentially different pathway responses h after exposure: NON transiently induced the IL-6 pathway as well as a number of mitogen activated signalling cascades including ERK and growth factor receptor signalling, whereas SLS transiently downregulated cellular energy metabolism pathways. Differential expression of the cyclooxygenase-2 and matrix metalloproteinase 3 transcripts was confirmed immunohistochemically. After cumulative exposure, 883 genes were differentially expressed while 26 suggested common biomarkers were identified . In conclusion, we bring new insights into two hitherto less well elucidated phases of skin irritancy: the very initial as well as the late phase after single and cumulative exposure, respectively.
Genome-wide expression analysis of human in vivo irritated epidermis: differential profiles induced by sodium lauryl sulfate and nonanoic acid.
Specimen part
View SamplesTo obtain a separation of the epidermal and dermal compartments in order to examine compartment specific biological mechanisms in the skin we incubated 4 mm human skin punch biopsies in ammonium thiocyanate (NH4SCN). We wanted to test 1) the histological quality of the dermo-epidermal separation obtained by different incubation times 2) the amount and quality of extractable epidermal RNA, and 3) its impact on sample RNA expression profiles assessed by large-scale gene expression microarray analysis in both normal and inflamed skin. At 30 minutes incubation, the split between dermis and epidermis was not always histologically well-defined (i.e. occurred partly intra-epidermally) but varied between subjects. Consequently, curettage along the dermal surface of the biopsy was added to the procedure. This modified method resulted in an almost perfect separation of the epidermal and dermal compartments and satisfactory amounts of high-quality RNA were obtained. Hybridization to Affymetrix HG_U133A 2.0 GeneChips showed that ammonium thiocyanate incubation had a minute effect on gene expression resulting in only one significantly downregulated gene (cystatin E/M). We conclude that epidermis can be reproducibly and almost completely separated from the dermis of 4 mm skin biopsies by 30 min incubation in 3.8% ammonium thiocyanate combined with curettage of the dermal surface, producing high-quality RNA suitable for transcriptional analysis. Our refined method of dermo-epidermal separation will undoubtedly prove valuable in the many different settings, where the epidermal and dermal compartments need to be evaluated separately.
Extraction of high-quality epidermal RNA after ammonium thiocyanate-induced dermo-epidermal separation of 4 mm human skin biopsies.
Specimen part, Subject
View SamplesMicroarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independent of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin-responsiveness in primary human muscle cells from patients with type 2 diabetes.
Transcriptional profiling of myotubes from patients with type 2 diabetes: no evidence for a primary defect in oxidative phosphorylation genes.
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View SamplesGene expression profiling is a promising diagnostic and prognostic tool. Expression profiles are snap-shots of mRNA levels at time of extraction and they have been shown to be affected by tissue handling during sample collection. The effect of cold (room temperature) ischemia in the time interval between surgical removal of the specimen and freezing has been described in a number of studies. However, not much is known about the effect of warm (body temperature) ischemia during surgery.
Differential effect of surgical manipulation on gene expression in normal breast tissue and breast tumor tissue.
Sex, Specimen part, Disease, Subject
View SamplesGlioblastoma (GBM) is the most frequent and malignant brain tumor with an overall survival of only 14.6 months. Although these tumors are treated with surgery, radiation and chemotherapy recurrence is inevitable. A critical population of tumor cells in terms of therapy, the so-called cancer stem cells (CSCs), has been identified in gliomas and many other cancers. These tumor cells have a stem cell-like phenotype and are suggested to be responsible for tumor growth, chemo- and radio-resistance as well as recurrence. However, functional evidence for migrating glioma cells having a stem cell-like phenotype is currently lacking.
No associated publication
Specimen part
View SamplesKnowing the effect of autoimmune CNS-targeting T cells on the expression of neurogenesis-associated genes is essential to understand their putative effect on hippocampal functioning. Here, we used RNA microarray analysis to study the effect of myelin-specific T cells on the gene profile in the dentate gyrus 2 days after a perforant pathway transection, when neural precursor cells are highly proliferating. By the use of Gene Ontology analysis, the MANGO database and the Allan Mouse Brain Atlas, we identified dihydropyrimidinase-like 3 (Dpysl3), due to its expression in the subgranular zone of the dentate gyrus. In situ hybridizations and quantification of Dpysl3 mRNA+ grains in the subgranular zone and granular cell layer showed a significant increase in Dpysl3 mRNA in axonal lesioned compared to control mice, and no effect of the myelin-specific T cells. Dpysl3 mRNA was not expressed in Ki-67 mRNA+ proliferating cells and NeuN mRNA+ mature neurons, but it was expressed in Nestin mRNA+ neural precursor cells and even more so in Dcx mRNA+ migrating neuroblasts. These results suggest that the regulation of Dpysl3 mRNA expression in immature granular neurons is refractory to inflammatory mediators secreted by the myelin-specific T cells, while being highly responsive to neuronal stimuli. Comparable mechanisms may support normal hippocampal functioning in diseases characterized by infiltration of autoimmune CNS-targeting T cells, such as multiple sclerosis.
No associated publication
Sex, Specimen part, Treatment, Time
View SamplesThe transcriptional repressor Zbtb20 is essential for specification of hippocampal CA1 pyramidal neurons. Moreover, ectopic expression of Zbtb20 is sufficient to transform subicular and retrosplenial areas of D6/Zbtb20S mice to CA1. We used microarrays to identify genes that are repressed by Zbtb20 in developing CA1 pyramidal neurons in the CA1-transformed cortex of D6/Zbtb20S mice.
Zbtb20 defines a hippocampal neuronal identity through direct repression of genes that control projection neuron development in the isocortex.
Specimen part
View SamplesDLK1/FA-1 (delta-like 1/fetal antigen-1) is a transmembrane protein belonging to Notch/Delta family that acts as a membrane-associated or a soluble protein to regulate regeneration of a number of adult tissues. Here, we examined the role of DLK1/FA-1 in bone biology using osteoblast-specific-Dlk1 over-expressing mice (Col1-Dlk1). Col1-Dlk1 mice displayed growth retardation and significantly reduced total body weight and bone mineral density (BMD). CT-scanning revealed a reduced trabecular and cortical bone volume fraction. Tissue-level histomorphometric analysis demonstrated decreased bone formation rate and enhanced bone resorption in Col1-Dlk1 as compared to WT. At a cellular level, DLK1 markedly reduced the total number of bone marrow (BM)-derived CFU-F, as well as their osteogenic capacity. In a number of in vitro culture systems, DLK1 stimulated osteoclastogenesis indirectly through osteoblast-dependent increased production of pro-inflammatory bone resorbing cytokines (e.g, Il7, Tnfa and Ccl3). We found that ovariectomy (ovx)-induced bone loss was associated with increased production of DLK1 in bone marrow by activated T-cells. However, Dlk1-/- mice were protected from ovx-induced bone loss. Thus, we identified DLK1 as a novel regulator of bone mass that function to inhibit bone formation and to stimulate bone resorption. Increasing DLK1 production by T-cells under estrogen deficiency suggests its possible use as a therapeutic target for preventing postmenopausal bone loss.
DLK1 is a novel regulator of bone mass that mediates estrogen deficiency-induced bone loss in mice.
Specimen part
View SamplesThe level of reactivity in pre-sensitized individuals following repeated epicutaneous challenges with diphenylcyclopropenone (DPCP) remains unknown. We studied the kinetics and time-course of the developing reactivity to this potent contact sensitizer using genome-wide gene expression data
No associated publication
Specimen part
View SamplesDirecting differentiation of human embryonic stem cells (hESC) into specific cell types using an easy and reproducible protocol is a perquisite for the clinical use of hESC in regenerative medicine protocols. Here, we report the generation of mesodermal cells with differentiation potential to myocytes, osteoblasts, chondrocytes and adipocytes. We demonstrate that during hESC differentiation as embryoid bodies (EB), inhibition of TGF-b/Activin/Nodal signaling using SB-431542 (SB) markedly up-regulated paraxial mesodermal markers (TBX6, TBX5), early myogenic transcriptional factors (Myf5, Pax7) as well as myocyte committed markers (NCAM, CD34, Desmin, MHC (fast), alpha-smooth muscle actin, Nkx2.5, cTNT). Establishing EB outgrowth cultures (SB-OG) in the presence of SB (1 uM) led to further enrichment of cells expressing markers for myocyte progenitor cell: CD34+ (33%), NCAM+ (CD56) (73%), PAX7 (25%) and mature myocyte proteins (MYOD1, tropomyocin, fast MHC an
Enhanced differentiation of human embryonic stem cells to mesenchymal progenitors by inhibition of TGF-beta/activin/nodal signaling using SB-431542.
Cell line
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