Project description:Gene expression profiling was performed on biopsies of affected and unaffected psoriatic skin and normal skin from seven Japanese patients to obtain insights into the pathways that control this disease. U95A Affymetrix DNA chips that contain oligonucleotide arrays of approximately 12,000 well-characterized human genes were used in the study. The statistical analysis of the Affymetrix data, based on the ranking of the Student-test statistic, revealed a complex regulation of molecular stress and immune gene responses. The majority of the 266 induced-genes in affected and unaffected psoriatic skin were involved with interferon mediation, immunity, cell-adhesion, cytoskeleton restructuring, protein trafficking and degradation, RNA regulation and degradation, signaling transduction, apoptosis and atypical epidermal cellular proliferation and differentiation. The disturbances in the normal protein degradation equilibrium of skin were reflected by the significant increase in the gene expression of various protease inhibitors and proteinases including the induced components of the ATP/ubiquitin-dependent non-lysosomal proteolytic pathway that is involved with peptide processing and presentation to T-cells. Some of the upregulated genes, such as TGM1, IVL, CSTA, FABP5 and SPRR, are well known psoriatic markers involved in atypical epidermal cellular organization and differentiation. In the comparison between the affected and unaffected psoriatic skin, the transcription factor JUNB was found at the top of the statistical rankings for the 51 significantly upregulated genes in affected skin, suggesting that it has an important but as yet undefined role in psoriasis. Our gene expression data and analysis suggest that psoriasis is a chronic IFN and T-cell-mediated immune disease of the skin where the imbalance in epidermal cellular structure, growth and differentiation arises from the molecular antiviral stress signals initiating inappropriate immune responses. Experiment Overall Design: Analysis of the expression profile of skin samples for each of three conditions (states) of psoriasis activity Experiment Overall Design: 3 samples were from psoriasis negative individuals (biological replicates) Experiment Overall Design: 4 samples were from psorasis free skin areas of psoriasis active individuals (biological replicates) Experiment Overall Design: 4 samples were from psorasis active skin areas of psoriasis active individuals (biological replicates)

Project description:Cultured skin substitutes, prepared using keratinocytes, fibroblasts and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. However, because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between cultured skin substitutes and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Goals: Our analysis focused on identifying gene signatures that were highly characteristic of each cell and tissue type, and those that are regulated by the formation of cultured skin substitute from the individual components. Normalization: We used a normalization and referencing strategy that consisted of BioConductor/RMA Express RMA processing of the entire series of cel files followed by a per gene normalization in which the median value of expression for each gene was derived from the cultured samples only, and this was used as a reference for all samples including the cultured skin substitute. This approach allowed for the identification of genes that were higher and lower-expressed in the cultured skin relative to the individual cell types that were also expressed strongly or weakly in normal skin relative to the median value established by the three cell types. Results Summary:We identified six major clusters of coordinately regulated genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up-regulated or down-regulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in cultured skin substitutes compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that cultured skin substitutes in vitro, which display a well-differentiated epidermal layer, exhibit skin-like differentiation relative to gene expression patterns in the individual cells. This consists of both the activation of normal skin signature genes and the suppression of keratinocyte and fibroblast signatures. There is also a signature consistent with a hyperproliferative phenotype similar to wounded native skin. Keywords: Cell interaction and co-culture response expression profile

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Single-Cell RNA-seq immune profile of normal and inflammed mouse skin

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