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Introduction: The stratum corneum (SC) matrix is composed of free fatty acids, cholesterol and ceramides (CERs), which play a key role in the skin barrier function. Changes in the composition and content of skin lipids will affect the function of the skin barrier. The effect of a glycerol/petrolatum-based emollient (G/P-emollient) cream on the lipid profiles of isolated ex vivo human SC and the SC of a reconstructed human epithelial (RHE) model were measured. Methods: The spatial organization of the cream and the isolated SC intercellular matrix was studied using X-ray diffraction. The inter-bilayer distance in the multi-lamellar lipid structures and lattice type were analyzed using small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS), respectively. Lipidomic analysis using Shotgun Lipidomics was performed on reconstructed human epidermis (RHE) to quantify CER classes and chain lengths. This technology enables the analysis of thousands of lipids in a single biological sample. Results: The crystallized components of the cream are lipids, which were mainly packed in orthorhombic lattices, as well as hexagonal lattices and were similar to the SC structure. The cream penetrated the SC but did not alter the WAXS profile. It increased the amount of higher carbon number ceramides (> 42 carbons) and decreased lower carbon number ceramides (

Deleterious effects of pollutants and ultraviolet radiation on the skin can be attenuated using formulations containing antioxidants. However, these have disadvantages, including chemical instability, photodegradation, poor bioavailability or biological activity. Here, two commercial formulations were evaluated: one optimized to stabilize and deliver ascorbic acid (AA) at 15% and the other containing a glucoside form of AA, namely ascorbic acid 2-glucoside (AA2G), at 1.8% and at a physiological pH. We compared the skin delivery, antioxidative effects and chemical stability of AA2G with AA in their respective formulations.Skin delivery was measured using fresh viable human skin explants, and oxidative stress was measured using a human reconstructed epidermal (RHE) model according to levels of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase.Ascorbic acid 2-glucoside was completely metabolized to AA by the skin before entering the receptor compartment. The skin contained parent and AA, indicating a reserve of AA2G was present for further metabolism. For AA2G and AA, maximum flux of AA-equivalents was at 12 h, with continued absorption over 24 h. The absolute amount in µg was higher in the skin after application of AA than after application of AA2G. This may suggest a greater antioxidative effect; however, according to all three measurements of oxidative stress, the protective effect of AA and AA2G was similar. Unlike AA, AA2G was chemically stable under storage conditions.A lower concentration of AA2G is as effective as the active metabolite, AA, in terms of antioxidant effects. AA2G was chemically stable and can be applied at a lower concentration than AA, thus avoiding the need for an acidic formulation with a pH below 3.5.Les effets délétères des polluants et des rayonnements ultraviolets au niveau cutané peau peuvent être atténués avec des formulations contenant des antioxydants. Cependant, ceux-ci peuvent présenter des inconvénients comme une instabilité chimique, une photodégradation, une faible biodisponibilité ou une faible activité biologique. Nous avons évalué deux formulations commerciales: l'une optimisée pour stabiliser et libérer de l'acide ascorbique (AA) à 15 % et l'autre contenant une forme conjugué de l'AA, à savoir l'acide ascorbique 2-glucoside (AA2G), à 1.8% et formulée à un pH physiologique. Nous avons comparé le passage percutané, les effets antioxydants et la stabilité chimique de l'AA2G avec l'AA dans leurs formulations respectives. MÉTHODES: Le passage percutané a été évalué avec des explants de peau humaine maintenus en survie et le stress oxydatif a été évalué à l'aide d'un modèle d'épiderme reconstruit humain (RHE) en mesurant les niveaux de malondialdéhyde (MDA), de superoxyde dismutase (SOD) et de catalase. RÉSULTATS: L'AA2G a été complètement métabolisé en AA par la peau avant d'atteintre le compartiment récepteur. Le composé parent et l’AA ont été retrouvé dans la peau, indiquant qu'une réserve d'AA2G était présente pour une libération prolongée. Pour l’AA2G et l’AA, le flux maximal d'équivalents AA était à 12 h, avec une absorption continue sur 24 h. La quantité absolue en µg était plus élevée dans la peau après application de la formulation contenant 15% d'AA qu'après application de la formule contenant 1.8% d'AA2G. Cela peut suggérer un effet antioxydant plus important ; cependant, selon les trois paramètres évalués pour le stress oxydatif, l'effet protecteur de l'AA et de l'AA2G était similaire. Contrairement à l'AA, l'AA2G est chimiquement plus stable dans des conditions de stockage.Une concentration plus faible d'AA2G est aussi efficace que le métabolite actif, l'AA, en termes d'effets antioxydants. L'AA2G est chimiquement plus stable et peut être appliqué à une concentration inférieure à l'AA, évitant ainsi le besoin d'une formulation acide avec un pH inférieur à 3.5.

Sunlight induces actinic keratosis, skin cancers and photoaging. Photoprotection is thus a major issue in public health to prevent the harmful effects of solar ultraviolet (UV) radiations. Recent data have shown that the visible (VIS) and infrared (IR) radiations can lead to skin damage by oxidative stress, suggesting that a balanced protection across the entire spectrum of sunlight is necessary to prevent cutaneous alterations. In this context, we developed a new generation of sunfilter called Phenylene Bis-Diphenyltriazine or TriAsorB (CAS N°55514-22-2). The aim of the present study was to assess the photoprotective efficacy of TriAsorB from UV to IR light. Spectrophotometric assays were performed to measure absorption and reflectance of TriAsorB in the different spectral ranges of sunlight: UV, VIS including blue light or high energy visible (HEV) and IR. DNA damage was evaluated using reconstructed human epidermis (RHE): 8-hydroxy-2'-deoxyguanosine (8OHdG) in response to HEV exposure, pyrimidine dimers (CPDs) and (6-4) photoproducts following solar-simulated radiation (SSR). TriAsorB is a broad spectrum UVB + UVA filter including long UVA. Interestingly, it also absorbs VIS radiations, especially in the HEV region. These radiations are also reflected. Protection in the IR spectral range is weak. Furthermore, the sunfilter specifically protects the skin against the oxidative lesions 8OHdG induced by HEV and prevents SSR-induced DNA damage. Thus, TriAsorB is an innovative sunfilter that might be used in sun care products for skin photoprotection from UV to VIS radiations. Finally, it prevents sunlight genotoxicity and protected the skin against solar radiations, especially blue light.

Background Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions and defects of the skin barrier. Bacterial infection with Staphylococcus aureus contributes to increased severity of AD by compromising the barrier further. A microorganism component of Avene Thermal Spring Water, Aquaphilus dolomiae, is thought to contribute to some of its beneficial effects to skin, eg AD alleviation. Aims Here, we have investigated the effects of an extract of A. dolomiae, A. dolomiae extract-G1 (ADE-G1), on the structural barrier function of keratinocytes, tight junction (TJ) protein expression and the expression of several genes altered in AD patients. Methods An epidermal cell culture model mimicking the AD environment and phenotype was used, in which S. aureus-infected cell cultures of normal human epidermal keratinocytes were exposed to a proinflammatory environment. Endpoints measured included the transepithelial electrical resistance (TER) and immunohistological staining of the epidermal TJ proteins, claudin and occludin. Additional analysis was made of several genes known to be differentially regulated in skin from AD patients (C-C motif chemokine ligand 20 (CCL20), interleukin-8 (IL-8), S100 calcium binding protein A7 (S100A7), defensin beta 4 (DEFB4) and filaggrin). Results Aquaphilus dolomiae extract-G1 strongly increased TER in non-infected cells and provided protection against infection by overcoming the decrease in TER induced by the infection with S. aureus. In infected cells exposed to a pro-inflammatory environment - depicting AD-like conditions - TER protection by ADE-G1 was still observed. Gene expression analysis of infected and pro-inflammatory stimulated cells indicated that ADE-G1 modulated the inflammatory response (induced IL-8 and attenuated CCL20 expression), increased antimicrobial activities (induced DEFB4 and A100A7) and strengthened barrier function (restored filaggrin expression). Conclusions ADE-G1 reinforces barrier function and strongly protects TJ barrier disruption induced by bacterial infection and inflammation.

Background Inflammatory skin disorders, including atopic dermatitis (AD), associated pruritus and sensitive skin, have a complex multifactorial pathogenesis including neurogenic inflammation involving the release in blood and skin of neurotransmitters such as substance P (SP). Aims and methods In vitro models evaluated the effect of the original biological extract of Aquaphilus dolomiae extract-G3 (ADE-G3) on cutaneous neurogenic inflammation. Results ADE-G3 significantly inhibited SP-stimulated release of IL-1β and TNF-α from normal human epidermal keratinocytes; significantly and dose-dependently inhibited SP-stimulated activation of human mast cells; significantly inhibited veratridine-stimulated release of SP from human sensory neurons; modulated expression of genes involved in lipid synthesis, innate immunity, corneocyte scaffolding and epidermal differentiation in a histamine-sensitized reconstructed human epidermis model; and, when applied topically to ex vivo human explants, inhibited IL-8 and histamine release. Conclusions Topically applied ADE-G3, once formulated, may improve neuro-inflammation in patients with inflammatory skin disorders.

Background Thermal Spring Water (TSW) has been recognized to have beneficial effects on skin; however, the mechanisms underlying these are not completely elucidated. Aims We compared the effects of Avene TSW with mineral-rich (MR) TSW on the biomechanical properties of the skin using mechanistic ex vivo assays and clinical studies. Methods Ex vivo studies included the effect of both TSWs on the structure of the surface of human skin explants using scanning electron microscopy (SEM); mineral elemental content on the skin surface using SEM coupled to energy dispersing X-ray spectroscopy; and the stress properties of the stratum corneum (SC) when exposed to dehydration. Human clinical studies were conducted to compare the soothing effect of TSWs after a dermatological chemical peeling of face skin and to evaluate the overall sensitive scale of consumers using Avene TSW for 7 days. Results Both TSWs preserved surface skin ultrastructure; however, crystals formed from MR-TSW were needle-like and formed small grains, present in clusters heterogeneously spread over the surface. Needle crystals were mainly composed of calcium, while small clusters were mainly composed of sulphur. By contrast, Avene TSW-formed crystals composed of sodium and chlorine only were regular in shape and homogeneously distributed across the skin surface. Peak stress of SC layers was increased by MR-TSW, whereas Avene TSW showed a comparatively reduced effect on dehydration and stress. The difference in the two TSW types was reflected in clinical findings comparing postpeeling redness after TSW application. Avene TSW significantly decreased postpeeling redness, while MR-TSW increased it. The overall sensitive scale of consumers was decreased by 47% using Avene TSW for 7 days. Conclusions Avene TSW decreases postpeeling redness and soothes sensitive skin in human volunteers. Mechanistic studies suggested that differences in biomechanical effects could be linked to differences in calcium content of the TSW.

Aims: Among in vitro skin models, the scaffold-free skin equivalent (SFSE), without exogenous material, is interesting for pharmacotoxicological studies. Our aim was to adapt in vivo biophysical methods to study the structure, thickness, and extracellular matrix of our in vitro model without any chemical fixation needed as for histology. Methods: We evaluated 3 batches of SFSE and characterized them by histology, transmission electron microscopy (TEM), and immunofluorescence. In parallel, we investigated 3 biophysical methods classically used for in vivo evaluation, optical coherence tomography (OCT), and laser scanning microscopy (LSM) imaging devices as well as the cutometer suction to study the biomechanical properties. Results: OCT allowed the evaluation of SFSE total thickness and its different compartments. LSM has a greater resolution enabling an evaluation at the cell scale and the orientation of collagen fibers. The viscoelasticity measurement by cutometry was possible on our thin skin model and might be linked with mature collagen bundles visible in TEM and LSM and with elastic fibers seen in immunofluorescence. Conclusion: Our data demonstrated the simplicity and sensitivity of these different in vivo biophysical devices on our thin skin model. These noninvasive tools allow to study the morphology and the biomechanics of in vitro models.

The stratum corneum (SC) is key in the maintenance of the biomechanical barrier and hydration of skin. Our previous investigations showed beneficial effects of a combination of emollients on water capture and retention and protein and lipid organization, all of which are linked to dryness and dry skin damage. Here, we show how a formulation containing an emollient combination ("Trio") and its basal formulation (placebo) impacted the descriptors of SC hydration in SC layers. Only the Trio formulation-not its placebo formulation-modified SC biomechanical drying stress behaviour and imparted a high capacity to protect it from dehydration. This was in accordance with findings at the molecular level using Raman analyses and at the structural level using cryo-scanning electron microscopy (SEM). After topical application, only the Trio formulation profoundly increased lateral packing of lipids and their compactness. Cryo-SEM showed that, unlike the placebo formulation, the Trio formulation prevented the water loss when applied before the dehydration process. In conclusion, these studies demonstrate that stresses in the SC due to dehydration can be alleviated using a formulation containing emollients that interact with the SC lipid components.

International audience; As a result of the cosmetics testing ban, safety evaluations of cosmetics ingredients must now be conducted using animal-free methods. A common approach is read across, which is mainly based on structural similarities but can also be conducted using biological endpoints. Here, metabolomics was used to assess biological effects to enable a read across between a candidate cosmetic ingredient, DIV665, only studied using in vitro assays, and a structurally similar reference compound, PA102, previously investigated using traditional in vivo toxicity methods. The (1) cutaneous distribution after topical application, (2) skin metabolism, (3) liver metabolism and (4) effect on the intracellular metabolomic profiles of in vitro skin and hepatic models, SkinEthic®RHE model and HepaRG® cells were investigated. The compounds exhibited similar skin penetration and skin and liver metabolism, with small differences attributed to their physicochemical properties. The effects of both compounds on the metabolome of RHE and HepaRG® cells were similarly small, both in terms of the metabolites modulated and the magnitude of changes. The patterns of metabolome changes did not fit with any known signature relating to a mode of action known to be linked to liver toxicity e.g. modification of the Krebs cycle, urea synthesis and lipid metabolism, were more reflective of transient adaptive responses. Overall, these studies indicate that PA102 is biologically similar to DIV665, allowing read across of safety endpoints, such as in vivo sub-chronic (but not reproduction toxicity) studies, for the former to be applied to DIV665. Based on this, in the absence of animal data (which is prohibited for new chemicals), it could be concluded that DIV665 applied according to the consumer topical use scenario, is similar to PA102, and is predicted to exhibit low local skin and systemic toxicity

Daniel Bacqueville,1,* Anne Maret,2,* Maïté Noizet,1 Laure Duprat,1 Christine Coutanceau,3 Victor Georgescu,3 Sandrine Bessou-Touya,1 Hélène Duplan1 1Department of Pharmacology, Pierre Fabre Dermo-Cosmétique, Toulouse, France; 2Clinical Skin Research Center, Pierre Fabre Dermo-Cosmétique, Toulouse, France; 3Laboratoires Dermatologiques Avène, Lavaur, France*These authors contributed equally to this workCorrespondence: Daniel BacquevilleDepartment of Pharmacology, Pierre Fabre Dermo-Cosmétique, Avenue Hubert Curien, BP 13562, 31035 Toulouse Cedex 1, FranceTel +33 5 34 50 64 27Fax +33 5 34 50 34 24Email daniel.bacqueville@pierre-fabre.comBackground: Skin aging is characterized by slacking and loss of density, especially under ultraviolet (UV) radiation exposure.Objective: To investigate the beneficial effects of a combination containing bakuchiol (BK) and vanilla tahitensis extract (VTE) to prevent skin photoaging in vitro and to improve clinical outcomes for naturally aged skin.Materials and Methods: Human dermal fibroblasts were treated with active compounds, exposed to an acute dose of UVA and analyzed by confocal microscopy: actin network for morphology, interleukin-8 (IL-8) for inflammation and p16 for senescence. Human skin was used to evaluate chronic UVA-induced glycosaminoglycan (GAG) loss and to assess the benefit of topical application of a BK+VTE serum (Alcian blue staining). An open-label clinical trial was conducted in women applying the serum twice daily for 56 days (n=43). Skin remodeling was assessed by FaceScan®. Firmness was evaluated through Dynaskin® and clinical scoring. Skin radiance was also rated on standardized full-face photographs.Results: UVA induced a significant increase in IL-8 and p16 expression and marked morphological changes in fibroblasts. Treatment with BK or VTE alone prevented both actin network alteration and IL-8 upregulation. Interestingly, BK+VTE demonstrated synergistic protection against IL-8 and p16 overexpression. Serum application prevented GAG loss at the dermo-epidermal junction and increased dermal GAG in UVA-exposed skin explants. In the clinical trial, face ptosis was reduced by 11% on average for 26 responsive subjects and up to 23%. Depth of skin deformation was also reduced by 24% on average for 30 responsive subjects and up to 30%. This firming effect was confirmed by clinical scoring. Radiance was significantly improved by 29% on average for 33 responsive subjects. The serum demonstrated good tolerance/safety.Conclusion: BK+VTE combination demonstrated anti-aging efficacy and might provide a substantial benefit in the daily care of naturally aged skin in women, through their synergistic effect on inflammaging and senescence.Keywords: anti-aging therapy, clinical trial, inflammaging, senescence, skin model

Background A biological concentrate was produced from cultures of an Avene aquatic microflora isolate, namely Aquaphilus dolomiae. Some of the beneficial effects on diseased and damaged skin are thought to be due to the presence of this microorganism. Aims An extract of A. dolomiae (A. dolomiae extract-G2, ADE-G2) was evaluated for its wound-healing effects using in vitro and ex vivo models of injured skin. Methods The effect of ADE-G2 on the proliferation of fibroblasts, migration of keratinocytes and re-epithelialization of ex vivo wounded skin explants was measured. Antimicrobial protection by ADE-G2 was measured by analysing the gene expression of a panel of antimicrobial proteins (AMPs) in keratinocytes (RNASE7, S100A7, DEFB4A/B and DEFb103B), as well as the protein encoded by DEFB4A-B (hBD2) in the medium. Results ADE-G2 increased fibroblast proliferation and keratinocyte migration, as well as re-epithelialization of wounded ex vivo skin. ADE-G2 induced the expression of all AMP genes analysed in keratinocytes, as well as stimulated the release in to the medium of hBD2 peptide, encoded by DEFB4A/B. Conclusions We have shown the broad spectrum of the repairing properties of the A. dolomiae extract, ADE-G2. These results support the use of ADE-G2 as a promising component for use in formulations aimed at repairing skin, limiting wound superinfection and preventing complicated wounds.

Hélène Hernandez-Pigeon, Lucile Garidou, Marie‑Florence Galliano, Hélène Delga, Marie‑Françoise Aries, Hélène Duplan, Sandrine Bessou-Touya, Nathalie Castex-Rizzi Department ofPharmacology, Pierre FABRE Dermo-Cosmétique R&D Center, Toulouse, France Background: Rosacea is a chronic facial skin disorder characterized by inflammation and vascular abnormalities. The pathophysiology of rosacea involves increased activation of the capsaicin receptor, TRPV1, the vascular endothelial growth factor (VEGF) pathway, and cathelicidin LL-37, MMP-9, and KLKs. We evaluated the activity of four compounds (dextran sulfate, 4-t-butylcyclohexanol [BCH; TRP-regulin®], pongamia oil, and hesperidin methyl chalcone [HMC]) on inflammatory and vascular responses implicated in rosacea.Materials and methods: The anti-inflammatory activity of dextran sulfate was evaluated on PGE2 production after PMA stimulation of NCTC-2544 keratinocytes, and on normal human epidermal keratinocytes (NHEKs) after proinflammatory stimulation to mimic a rosacea environment. The anti-angiogenic activity of dextran sulfate was measured by analyzing pseudotube formation in co-cultured human microvascular endothelial cells/normal human dermal fibroblasts. HMC modulation of vascular responses and IL-8 cytokine production after SP stimulation was evaluated in human skin explants. We also assessed the effect of BCH on TRPV1 activation, and the effect of combined BCH and pongamia oil on the inflammatory response of NHEKs.Results: Dextran sulfate strongly and significantly inhibited PMA-induced PGE2 production, inhibited KLK5 and MMP-9 mRNA expression, and IL-8, IL-1α and VEGF production, and displayed a highly significant inhibitory effect on VEGF-induced pseudotube formation. In SP-stimulated human skin explants, HMC significantly decreased the proportion of dilated vessels, total vessel area, and IL-8 production. BCH significantly and dose-dependently inhibited TRPV1 activation, and BCH and pongamia oil inhibited CXCL1 and CXCL6 mRNA expression and IL-8 production in NHEKs. Combined BCH/pongamia oil inhibited IL-8 production synergistically.Conclusion: These in vitro results showed that dextran sulfate, BCH, pongamia oil and HMC, possess complementary soothing and anti-redness properties, supporting their combination in Avène redness-relief cosmetic products for sensitive skin prone to redness, and for topical adjunctive rosacea treatment. Keywords: rosacea, keratinocyte, inflammation, redness, VEGF

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs.

Atopic dermatitis (AD) is a common skin inflammatory disease characterized by the production of thymic stromal lymphopoietin (TSLP) and marked TH 2 polarization. Recent studies suggest that IL-1β contributes to the development of AD skin inflammation. Here, we have investigated the impact of IL-1β signalling on the epidermal homeostasis of both healthy subjects and AD patients [with functional filaggrin (FLG) alleles], with particular attention to TSLP production and keratinocyte differentiation. In healthy reconstructed human epidermis (RHE), IL-1β promoted (i) robust secretion of TSLP in an NF-κB-dependent manner and (ii) a significant decrease in the expression of filaggrin and other proteins of the epidermal differentiation complex. These effects were prevented by treatment of RHE with the anti-IL-1β mAb canakinumab and by the IL-1 receptor antagonist anakinra. Interestingly, RHE generated from AD donors behaved like that of healthy individuals and showed comparable responses to IL-1β signals. Collectively, our results suggest that IL-1β may be an early key mediator for the acquisition of an AD phenotype through induction of TSLP and alteration of the epidermal homeostasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background: Information is lacking on the dermal penetration of topically applied formulations on in vitro skin models, under conditions where the stratum corneum (SC) is damaged. Therefore, we have developed a standardized in vitro barrier-disrupted skin model using tape stripping. Methods: Different tape stripping conditions were evaluated using histology, transepidermal water loss, infrared densitometry, and caffeine absorption. Results: The effects of tape stripping were comparable using pig and human skin. Optimized conditions were used to test the effect of SC damage and UV irradiation on the absorption of an UV filter combination present in a sunscreen. The bioavailability of the filters was extremely low regardless of the extent of skin damage, suggesting bioavailability would not be increased if the consumer applied the sunscreen to sun-damaged skin. Conclusion: This standardized in vitro methodology using pig or human skin for damaged skin will add valuable information for the safety assessment of topically applied products.

Background Atopic dermatitis (AD) is a common skin disease characterized by recurrent pruritic inflammatory skin lesions resulting from structural and immune defects of the skin barrier. Previous studies have shown the clinical efficacy of Avene thermal spring water in AD, and a new microorganism, Aquaphilus dolomiae was suspected to contribute to these unique properties. The present study evaluated the anti-inflammatory, antipruritic, and immunomodulatory properties of ES0, an original biological extract of A. dolomiae, in immune and inflammatory cell models in order to assess its potential use in the treatment of AD. Materials and methods An ES0 extract containing periplasmic and membrane proteins, peptides, lipopolysaccharides, and exopolysaccharides was obtained from A. dolomiae. The effects of the extract on pruritus and inflammatory mediators and immune mechanisms were evaluated by using various AD cell models and assays. Results In a keratinocyte model, ES0 inhibited the expression of the inflammatory mediators, thymic stromal lymphopoietin, interleukin (IL)-18, IL-4R, IL-8, monocyte chemoattractant protein-3, macrophage inflammatory protein-3α, and macrophage-derived chemokine and induced the expression of involucrin, which is involved in skin barrier keratinocyte terminal differentiation. In addition, ES0 inhibited protease-activated receptor-2 activation in HaCaT human keratinocytes stimulated by stratum corneum tryptic enzyme and T helper type (Th) 1, Th2, and Th17 cytokine production in Staphylococcal enterotoxin B-stimulated CD4+ lymphocytes. Lastly, ES0 markedly activated innate immunity through toll-like receptor (TLR) 2, TLR4, and TLR5 activation (in recombinant human embryonic kidney 293 cells) and through antimicrobial peptide induction (psoriasin, human beta-defensin-2, and cathelicidin), mainly through TLR5 activation (in normal human keratinocytes). Conclusion Overall, these in vitro results confirm the marked regulatory activity of this A. dolomiae extract on inflammatory and immune responses, which may be of value by virtue of its potential as an adjunctive treatment of AD inflammatory and pruritic lesions.

International audience; The stratum corneum, the outermost layer of the epidermis, is the most important skin barrier against exogenous physical and chemical effects, in addition to protecting against dehydration. Ceramides are integral parts of the intercellular lipid lamellae of the stratum corneum and play an important role in the barrier function of mammalian skin. Ceramides are sphingolipids consisting of sphingoid bases linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine, sphingosine, phytosphingosine, and 6-hydroxysphingosine, and the fatty acid acyl chains are composed of non-hydroxy fatty acid, alpha-hydroxy fatty acid, omega-hydroxy fatty acid, and esterified omega-hydroxy fatty acid. Analytical methods, such as gas chromatography/mass spectrometry, high performance thin layer chromatography with UV detection, and liquid chromatography/mass spectrometry, have been developed for the identification and quantification of ceramides in the stratum corneum. However, only a few publications relate to the mass fragmentation patterns specific to ceramide types to determine the structure of skin ceramides. Moreover, these studies provide very limited structural information and only for some ceramides. Therefore, the aim of our study was to develop a quick and easy method of quantification of ceramides, cholesterol, and free fatty acids by high performance thin layer chromatography with ultraviolet detection. High performance thin layer chromatography with ultraviolet detection was also coupled with mass spectrometry using negative ionization by electrospray and tandem mass spectrometry (MS/MS) for identification of ceramides' structure.

Patrick Bogdanowicz,Marie-José Haure,Isabelle Ceruti,Sandrine Bessou-Touya,Nathalie Castex-Rizzi Department of Pharmacology,Pierre Fabre Dermo-Cosmétique, Toulouse,France Background: Glycation is an aging reaction of naturally occurring sugars with dermal proteins. Type I collagen and elastin are most affected by glycation during intrinsic chronological aging. Aim: To study the in vitro and ex vivo assays in human skin cells and explants and the antiaging effects of glycylglycine oleamide (GGO). Materials and methods: The antiglycation effect of GGO was assessed in a noncellular in vitro study on collagen and, ex vivo, by immunohistochemical staining on human skin explants (elastin network glycation). The ability of GGO to contract fibroblasts was assessed in a functional assay, and its anti-elastase (MMP-12) activity was compared to that of oleic acid alone, glycylglycine (GG) alone, and oleic acid associated with GG. Results: In vitro, GGO reduced the glycation of type I collagen. Ex vivo, GGO restored the expression of fibrillin-1 inhibited by glycation. Furthermore, GGO induced a tissue retraction of almost 30%. Moreover, the MMP-12 activity was inhibited by up to 60%. Conclusion: Under the present in vitro and ex vivo conditions, GGO prevents glycation of the major structural proteins of the dermis, helping to reduce the risk of rigidification. By maintaining the elastic function of the skin, GGO may be a promising sparring partner for other topical antiaging agents. Keywords: extracellular matrix, glycylglycine oleamide, glycation, fibrillin-1, matrix metalloproteinase-12, skin aging