Oligomeric α-Synuclein induces skin degeneration in reconstructed human epidermis.

Aged and photoaged skin exhibit fine wrinkles that are signs of epidermal inflammation and degeneration. It has been shown that healthy elderly skin expresses amyloidogenic proteins, including α-Synuclein, which are known to oligomerize and trigger inflammation and neurodegeneration. However, little is known about their putative role in skin physiology and sensitivity. To unravel this possible role, we investigated the impact of oligomeric α-Synuclein (Oα-Syn) in 2D and 3D keratinocyte human models. Exogenous Oα-Syn caused degeneration of reconstructed human epidermis (RHE) by diminishing proliferation and thickness of the stratum basale. Oα-Syn also increased NF-kB nuclear translocation in keratinocytes and triggered inflammation in the RHE, by increasing expression of interleukin-1ß and tumor necrosis factor-alpha, and the release of tumor necrosis factor-alpha in a time-dependent manner. Dexamethasone and an IL-1ß inhibitor partially diminished RHE degeneration caused by Oα-Syn. These findings suggest that Oα-Syn induces epidermal inflammation and decreases keratinocyte proliferation, and therefore might contribute to epidermal degeneration observed in human skin aging.

Characterization of a New Reconstructed Full Thickness Skin Model, T-Skin™, and its Application for Investigations of Anti-Aging Compounds.

BACKGROUND: We have characterized a new reconstructed full-thickness skin model, T-Skin™, compared to normal human skin (NHS) and evaluated its use in testing anti-aging compounds. METHODS: The structure and layer-specific markers were compared with NHS using histological and immunohistological staining. In anti-aging experiments, T-SkinTM was exposed to retinol (10 µM) or vitamin C (200 µM) for 5 days, followed by immunohistological staining evaluation. RESULTS: T-Skin™ exhibits a well stratified, differentiated and self-renewing epidermis with a dermal compartment of functional fibroblasts. Epidermal (cytokeratin 10, transglutaminase 1), dermo-epidermal junction (DEJ) (laminin 5, collagen-IV, collagen VII) and dermally-located (fibrillin 1, procollagen I) biomarkers were similar to those in NHS. Treatment of T-Skin™ with retinol decreased the expression of differentiation markers, cytokeratin 10 and transglutaminase 1 and increased the proliferation marker, Ki67, in epidermis basal-layer cells. Vitamin C increased the expression of DEJ components, collagen IV and VII and dermal procollagen 1. CONCLUSIONS: T-Skin™ exhibits structural and biomarker location characteristics similar to NHS. Responses of T-Skin™ to retinol and vitamin C treatment were consistent with those of their known anti-aging effects. T-Skin™ is a promising model to investigate responses of epidermal, DEJ and dermal regions to new skin anti-ageing compounds.

In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic HCE model: application to 435 substances from consumer products industry.

The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation.

The EpiSkin phototoxicity assay (EPA): development of an in vitro tiered strategy using 17 reference chemicals to predict phototoxic potency.

The aim of the study was to investigate the ability of human reconstructed epidermis EpiSkin(LM) to identify the phototoxic potency of topically or systemically applied chemicals (EPA: EpiSkin phototoxicity assay). Three classes, according to their available human phototoxic potential, were evaluated: systemic phototoxic compounds, topical phototoxic chemicals and non-phototoxic compounds. Non-cytotoxic concentrations of chemicals were applied topically or directly added to the underlying culture medium in order to mimic a systemic-like administration. Following treatment, tissues were exposed to non-cytotoxic dose of UVA (50 J cm(-2)). Cell viability and pro-inflammatory mediators (IL-1alpha) were investigated 22 h after UVA exposure. Our results show that the phototoxic potential of chemicals can be determined using cell viability combined with inflammatory mediator measurements (cytokine IL-1alpha) in a 3-D epidermis model. Moreover, the EPA was able to discriminate efficiently between phototoxic and non-phototoxic products. Furthermore, the EPA is sensitive to the administration route in the prediction of the phototoxic potency of the tested chemical. Differences observed between the two routes of administration (topical or systemic-like) may be linked in part to chemicals bioavailability which depends on specific penetration potential, epidermis barrier function and also on keratinocytes absorption/metabolization processes. Results were very promising and showed a very good sensitivity (92.3%) and an excellent specificity (100%) with an overall accuracy of 94.1%. The performances of the EPA showed that the EpiSkin(LM) model is an interesting tool able to integrate decision-making processes to address the question of phototoxicity linked to the application site.