Project description:Gene expression profiles of reconstructed human epidermis following exposure to interleukins 4, 13, and 25 for 48 hours We used microarrays to study the changes in gene expression of reconstructed human epidermis after 48 hours of exposure to interleukins 4, 13, and 25
Project description:Gene expression profiles of reconstructed human epidermis following exposure to lactic acid or methylparaben for 1, 6, or 24 hours We used microarrays to study the changes in gene expression of reconstructed human epidermis after exposure to lactic acid or methylparaben during 1, 6, and 24 hours
Project description:The aim of this study was to analyze the in vitro biological epidermal processes occurring in reconstructed skins using cells from breast skin of African and Caucasian skin type color. A exploration of mRNA expression levels in the epidermis of reconstructed skin was undertaken to elucidate the differential in vitro functions of keratinocytes. The reconstruction of skin models was made with keratinocytes and fibroblasts from four different donors per skin type and experiments were conducted in triplicate for each donor. At the end of culture, the epidermis from reconstructed skin was manually separated from the dermal equivalent part in order to analyse gene expression in keratinocytes only. RNA samples were labelled with biotin and hybridation was performed on Affymetrix Human Genome U133 + PM Array Plates.
Project description:This study aimed to understand the in vitro behaviour of epidermal cells of African and Caucasian skin types in the context of 3D reconstructed skin. Reconstructed skins epidermis made with cells isolated from skin of African or Caucasian skin type exhibited high differences in stratification and differentiation. The objective of this study is a first global approach to identify at the protein level the differences between reconstructed skins.
Project description:The cytoskeletal mechanisms regulating inflammatory responses in the epidermis are not well described. Here we addressed this question by inducing a psoriatic phenotype in human keratinocytes and reconstructed human epidermis using a cytokine stimulation model.We report the results from RNA-seq in N/TERT keratinocytes treated with M5 or both M5 and ROCK2 inhibitor KD025.
Project description:Effect of EGFR inhibitors on the epidermis Epidermal Growth Factor inhibitors (EGFRi) used in oncology therapy modify the keratinocyte differentiation processes, impairing proper skin barrier formation and leading to Cutaneous Adverse Drug Reactions (CADR). To uncover the molecular signatures associated with CADRs, we applied phospho-proteomic and transcriptomic assays on Reconstructed Human Epidermis (RHE) tissues exposed to a therapeutically relevant concentration of afatinib, a second EGFRi generation. Following drug exposure, we observed an increased expression of gene families involved in keratinocyte differentiation, senescence, oxidative stress and alterations in the epidermal immune-related markers. Furthermore, our results show that afatinib may interfere with Vitamin D3 (VD3) metabolism, acting via CYP27A1 and CYP24A1 to regulate calcium concentration through the PI3K/AKT pathway. Consequently, basal layer keratinocytes switch from a pro-proliferating to a pro-differentiative program, characterized by upregulation of biomarkers associated with increased keratinization, cornification, Th2 response and decreased innate immunity. Such effects may increase the skin susceptibility to cutaneous penetration of irritants and pathogens.
Project description:Chemical skin and respiratory allergies affect many people around the world and are becoming a major public health problem. To date our knowledge on the process of protein haptenation is still limited and mainly derived from studies performed in solution using either amino acids, model peptides, isolated proteins or cell lysates. In order to better understand chemical interactions occurring between respiratory allergens and the skin, we have investigated the reactivity of phthalic anhydride 1 (PA), a chemical respiratory allergen, towards a reconstructed human epidermis model. This study was performed using a new approach combining reconstructed human epidermis (RHE) as a skin model, HRMAS NMR technique to investigate the in situ chemical reactivity and LC-MS/MS to identify modified epidermal proteins. The reaction of PA appeared to be quite fast in RHE compared to model experiments because after 30 min of incubation, no residual signal corresponding to (13C)-1 could be detected. The major product formed could be assigned to phthalic acid, the hydrolysis product of PA. Two amide type adducts on lysine residues were observed and after 8h of incubation, we also observed the formation of an imide type cyclized adducts with lysine. In parallel, RHE samples topically exposed to phthalic anhydride (13C)-1 were analyzed using the shotgun proteomics method. Thus, 948 different proteins were extracted and identified, 135 of which being modified by PA i.e. 14.2% of the extracted proteome. A total of 886 amino acids were modified by PA, of which 211 modifications were validated by fragmentation spectra. We thus identified 475 modified lysine residues (154 validated modifications associated with 122 proteins i.e. 12.9% of the proteome), 183 modified His residues (22 validated modifications associated with 42 proteins i.e. 4.4% of the proteome), 119 modified Tyr residues (30 validated modifications associated with 58 proteins i.e. 6.1% of the proteome) and 109 modified Arg residues (5 validated modifications associated with 25 proteins i.e. 2.6% of the proteome). The rate of modified residues, as a proportion of the total number of modifiable nucleophilic residues in RHE, was rather low (1%) and PA modifications were mainly observed on lysine residues with the formation of amide type adducts with a mass increase of +149 Da and a small number of imide type adducts with an increase mass of +131 Da. At the protein level, modified proteins were mainly type I and Type II keratins and other proteins which are abundant in the epidermis such as protein S100A, Caspase 14, annexin A2, serpin B3, Fatty-acid binding protein 5, histone H2, H3, H4 etc. However, the most modified protein, mainly on histidine residues, was filaggrin, a protein that is sparse (0.0266 mol%) and rich in histidine.
Project description:TNFα-stimulated gene 6 (TSG-6) is an anti-inflammatory protein. In human epidermis, TSG-6 is secreted in extracellular matrix where it interacts with hyaluronan (HA). However, their functions are not well understood. In this study, immortalized N/TERT keratinocytes were edited by CRISPR/Cas9, used to reconstruct in vitro epidermis (RHE) and stimulated or not with interleukins 4 and 13 for 48h to mimic atopic dermatitis (AD). Two TSG-6-/- clones (respectively TSG-6-/- (a) and TSG-6-/- (b)) harboring major deletions in both alleles were selected and compared to TSG-6+/+ cells. Epidermis reconstructed from TSG-6-/- (a) and TSG-6-/- (b) exhibit normal differentiation process and morphology but show an increased HA leakage in the underlying culture medium of both TSG-6-/- RHE compared to TSG-6+/+ tissues, especially in conditions that mimic AD. While IL-4/13 treatment of RHE induces regulation of genes associated with AD, the absence of TSG-6 don’t seem to show any major transcriptomic regulation.