OBJECTIVE:Existing methods to evaluate skin care products suffer limitations. This is the case for ex vivo skin explants, a first-choice 3D model. While essential to analyse mid- to long-term biological effects, this classical model hinders assessing microrelief variations. To circumvent these limitations, we developed an ex vivo PERFused EXplant setup (Perfex) that maintains the outer skin surface in the open air, closely mirroring physiological conditions. METHODS:A custom-designed reservoir enables perfusing the dermal side of explants with buffered, temperature-controlled medium, while the epidermis is subjected to "normal" conditions. Skin tension and characteristics of the stratum corneum, microrelief, histology and immunohistology (collagen types I and III, elastin and fibrillin-1) were analysed and compared to those of explants maintained under conventional conditions or in vivo skin. The effects of skin care formulas intended to induce short- and/or mid- to long-term effects were also assessed. RESULTS:Skin explants maintained with the Perfex setup exhibit characteristics (firmness, elasticity, hydration and barrier function) closer to those of in vivo skin than with conventional conditions. Moreover, Perfex-maintained explants present no alteration in histology after 7 days and slight variation in the expression of key protein markers. Microrelief characteristics also remain mostly stable over 7 days. Formula applications corroborate that skin tensor-containing products primarily induce short-term changes in the microrelief, while those with biologically active ingredients mainly lead to mid- to long-term effects on the histology and expression of molecular markers. Furthermore, maintaining skin explants with a physiologically relevant skin surface enabled analysing the relationship between microrelief and key markers, showing that fibrillin-1 is the protein most correlated with microrelief characteristics. CONCLUSIONS:The Perfex setup allows for similar preservation of skin explant histology and key protein expression as the conventional system, yet it maintains a skin surface close to that of in vivo skin. Therefore, it is valuable to analyse both the short- and mid- to long-term impacts of skin care formulas and better comprehend their effects. The Perfex system also offers a new tool for investigating fundamental questions, such as the link that can exist between dermal proteins and skin surface properties.

The skin is a formidable physical and biological barrier which communicates continuously with the outside of the body. And the stratum corneum, the outermost layer of human epidermis, plays a central role in the interaction between the cutaneous tissue and the external environment. The horny layer, and more generally the whole skin layers, avoid the penetration of harmful exogenous agents, produce molecules named anti-microbial peptides which impact the composition of the cutaneous microbiota, regulate the internal corporal temperature, avoid the water loss from the inside of the body and constitute an incredible efficient anti-oxidant network. Nevertheless, nowadays, the skin is more and more solicited by the different elements of the cutaneous exposome, including atmospheric pollution and solar radiations, which can cause a dramatic acceleration of the skin ageing process. As a consequence, due to the multifunctional protective role of the skin, during the recent decade the cosmetic industry invested massively in the development of new raw materials and end-products (dermo-cosmetics) able to preserve an optimal state of the skin regarding the external environment. Based on their physical-chemical properties thermal spring waters, which are extremely rich in inorganics ions, are interesting and powerful candidates to be part, as integral component, of new efficient dermo-cosmetic formulations dedicated to protect the skin from the external stimuli. The aim of the present work was to investigate and characterize the activity of Jonzac thermal spring water on the skin. Using different models, we proved for the first time that Jonzac thermal spring water reinforces the barrier function of the skin by modulating the expression of key markers including filaggrin and human beta defensin 2 on ex vivo human skin. The ex vivo and in vivo hydration activity, by Raman spectroscopy and corneometry respectively, has been also demonstrated. We have also shown that Jonzac thermal spring water ameliorates significantly the cutaneous microrelief in vivo. To conclude, we characterize the soothing effect of Jonzac thermal spring water by the analysis of histamine release in Substance P treated skin explants and by measuring the redness of the skin following UV exposure of the skin in vivo. We observed that both parameters decreased following a preventive treatment of the skin with Jonzac thermal spring water. Taken together our results indicate that Jonzac thermal spring water is a promising and powerful dermo-cosmetic which can be used to preserve an optimal state of the cutaneous tissue.

Background This is a study about the skin ageing exposome, focusing on the effect of cigarette smoke. Human living skin explants (HSE) were exposed to cigarette smoke (CS) of two cigarettes for 2 hours using a custom-made exposure chamber, the Pollubox(R). Effects on the surface physico-chemistry and molecular properties of the skin were analyzed and reported for the first time. Basic Procedures To this end, transcriptomic study followed by immunohistochemistry, MDA (Malondialdehyde Dosage), and surface physio-chemistry data: surface free energy determination, TEWL (Trans Epidermal Water Loss), skin pH and FT-IR (Fourier Transform-Infrared) spectroscopy of the explant were collected from untreated and treated HSE. Main Findings Results showed a decrease of the total surface free energy of the treated HSE. This decrease reflected higher interactions with polar compounds from the environment and consequently a decrease of the surface hydrophobicity. Additionally, an increase of TEWL and skin pH was observed after treatment. The transcriptomic analysis showed downregulation of mitochondrial genes (PON2-NDUFA4L2-ATP1A1-ALDH2-PRODH) combined with an increase of MDA in CS-treated HSE. Conclusions CS-induced oxidation of lipids at HSE surface alters the skin barrier: interactions with polar products are enhanced and the lipid chain packing at the surface is modified. Consequently, skin permeability could increase which correlated with repression of CA9 and AQP1 genes. Beside activation of AHR-NRF2 pathway in CS-exposed HSE, our results suggested that mitochondrial functions were strongly impacted and oxidized lipids failed to be eliminated promoting skin barrier alteration. A mitophagy activity was suggested through the confirmation of PINK1 accumulation in the epidermis by immunostaining.

OBJECTIVE: The skin interacts strictly with the surrounding environment. Despite an efficient system of protection, its integrity is continuously assaulted by a massive group of external stresses. UV irradiations represent one of the most harmful factors for the cutaneous tissue. Both UV-A and UV-B can induce deep modifications of the different layers of the skin, including a weakening of its barrier function properties, DNA damages and degradation of the extracellular matrix. The aim of this project was to assess the UV protection activity of two natural compounds, the birch sap from Betula alba and organic extract from Inonotus obliquus (chaga mushroom) used separately or in a complex. METHODS: The anti-oxidant (ROS and MDA quantification, catalase and SOD activity measurement), anti-inflammatory (IL-1β, IL-6, IL-8, IL-10, TNF-α and INF-γ dosages) and the DNA protection/repair activities (DNA lesion site analysis) of birch sap and chaga mushroom extracts tested separately or in a complex containing organic birch sap 5% and Inonotus obliquus extracts 2% were evaluated in vitro after exposure of cultured keratinocytes and fibroblasts or reconstructed epidermis to UV-A/UV-B irradiations. RESULTS: We observed that birch sap from Betula alba and extracts from Inonotus obliquus prevent the formation of ROS and decrease the oxidative stress induced under UV irradiations, suggesting a strong anti-oxidant activity. In addition, the tested products showed an immunomodulatory effect by reducing the quantity of pro-inflammatory cytokines upon UV irradiations. UV-induced DNA damages of keratinocytes were also reduced by birch sap and chaga mushroom extracts. CONCLUSION: Here, for the first time, we have shown the photo-protection activity of extracts obtained from Betula alba and Inonotus obliquus mushroom on skin cells exposed to UV-A and UV-B irradiations. Due to their anti-oxidant, anti-inflammatory and DNA protection/repair activities, the tested products represent promising candidates in the development of cosmetic products with anti-photo-aging activity.

Background: The skin is the outer shell of the mammalian body and it is continuously exposed to a large spectrum of external stimuli including sun irradiation and atmospheric pollutants. These factors present deleterious effects on the cutaneous compartment by altering the skin barrier functions and accelerating the aging of the skin. Objectives: The goal of this study was to investigate the activity of Seaberry and Water Lily extracts, here called Dermina complex, against different external stresses. Methods: Human skin explants were exposed to different stimuli including delipidation by organic solvents, blue light, atmospheric pollutants and UV-A. The activity of the Seaberry and Water Lily extracts was assessed by immunohistochemistry and by biochemical assays. Results: We showed that Dermina complex prevents the delipidation-induced filaggrin decrease, suggesting that these plant extracts exhibited barrier function protecting properties. Also, we observed that Dermina complex showed an antioxidant and DNA protection activity by decreasing the activated form of Nrf2, the oxidized proteins and the formation of γ-H2AX induced upon stress conditions. The Dermina complex also decreased the pro-inflammatory cytokine IL-1 alpha released in the culture medium following atmospheric pollutants and UV-A exposure confirming its anti-inflammatory activity. Moreover, Dermina complex reduced the blue light-induced overexpression of opsin 3, indicating that its skin protection activities may be due, in part, to filter property against environmental stresses. Conclusions: Dermina complex shows a protective activity of the skin against different environmental stresses and these extracts may be able to slow down the aging process of the cutaneous compartment.

Staphylococcus aureus and Staphylococcus epidermidis are two major skin associated bacteria, and Substance P (SP) is a major skin neuropeptide. Since bacteria are known to sense and response to many human hormones, we investigated the effects of SP on Staphylococci virulence in reconstructed human epidermis model and HaCaT keratinocytes. We show that SP is stimulating the virulence of S. aureus and S. epidermidis in a reconstructed human epidermis model. qRT-PCR array analysis of 64 genes expressed by keratinocytes in the response to bacterial infection revealed a potential link between the action of SP on Staphylococci and skin physiopathology. qRT-PCR and direct assay of cathelicidin and human β-defensin 2 secretion also provided that demonstration that the action of SP on bacteria is independent of antimicrobial peptide expression by keratinocytes. Considering an effect of SP on S. aureus and S. epidermidis, we observed that SP increases the adhesion potential of both bacteria on keratinocytes. However, SP modulates the virulence of S. aureus and S. epidermidis through different mechanisms. The response of S. aureus is associated with an increase in Staphylococcal Enterotoxin C2 (SEC2) production and a reduction of exolipase processing whereas in S. epidermidis the effect of SP appears mediated by a rise in biofilm formation activity. The Thermo unstable ribosomal Elongation factor Ef-Tu was identified as the SP-interacting protein in S. aureus and S. epidermidis. SP appears as an inter-kingdom communication factor involved in the regulation of bacterial virulence and essential for skin microflora homeostasis.

The skin is a natural barrier between the body and the environment and is colonised by a large number of microorganisms. Here, we report a complete analysis of the response of human skin explants to microbial stimuli. Using this ex vivo model, we analysed at both the gene and protein level the response of epidermal cells to Staphylococcus epidermidis (S. epidermidis) and Pseudomonas fluorescens (P. fluorescens), which are present in the cutaneous microbiota. We showed that both bacterial species affect the structure of skin explants without penetrating the living epidermis. We showed by real-time quantitative polymerase chain reaction (qPCR) that S. epidermidis and P. fluorescens increased the levels of transcripts that encode antimicrobial peptides (AMPs), including human β defensin (hBD)2 and hBD3, and the pro-inflammatory cytokines interleukin (IL)-1α and (IL)-1-β, as well as IL-6. In addition, we analysed the effects of bacterial stimuli on the expression profiles of genes related to innate immunity and the inflammatory response across the epidermal layers, using laser capture microdissection (LCM) coupled to qPCR. We showed that AMP transcripts were principally upregulated in suprabasal keratinocytes. Conversely, the expression of pro-inflammatory cytokines was upregulated in the lower epidermis. These findings were confirmed by protein localisation using specific antibodies coupled to optical or electron microscopy. This work underscores the potential value of further studies that use LCM on human skin explants model to study the roles and effects of the epidermal microbiota on human skin physiology.

We describe, for the first time, an efficient protocol based on laser capture microdissection (LCM) for the isolation of human epidermal layers for gene expression profiling using quantitative real-time PCR. Two areas enriched either in basal or granular layers were isolated by LCM. Skin biopsies were fixed in dry icecooled isopentane, cryosectioned and stained before the laser procedure. High-quality total RNA was extracted from each microdissected sample, which allowed the analysis of the spatial distribution of mRNA transcripts from 10 innate immunity-related genes within the epidermal layers. Using integrin alpha-6/integrin beta-4 and corneodesmosin/filaggrin-2 sets as gene markers for the basal and granular layers, respectively, we showed that Toll-like receptor 2, RNase 7, human beta-defensin-2 and -3, psoriasin and nucleotide-binding oligomerization domain 1 are upregulated in the suprabasal layer of normal human epidermis. Our protocol, which is based on the rapid isolation of epidermal layers, can be used to follow transcriptional processes in specific areas of the epidermis and is a very promising tool to use in the study of numerous aspects of dermatology.

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Roots of many plants are known to produce large numbers of 'border' cells that play a central role in root protection and the interaction of the root with the rhizosphere. Unlike border cells, border-like cells were described only recently in the model plant Arabidopsis thaliana and other Brassicaceae species and very little is known about the functional properties of border-like cells as compared with 'classical' border cells. To stimulate discussion and future research on this topic, the function of border cells and the way border-like cells are organized, maintained, and possibly involved in plant protection is discussed here.