Topical niacinamide enhances hydrophobicity and resilience of corneocyte envelopes on different facial locations.

Age-related differences in maturation parameters of corneocyte envelopes (size, hydrophobicity and rigidity) were examined at several facial test sites in young and old female Caucasians. In addition, the effect of topically applied niacinamide on these parameters was evaluated in a 4-week placebo-controlled study.

An image-based mapping of significance and relevance of facial skin colour changes of females living in Thailand.

OBJECTIVE: There are methods to evaluate skin colour on defined areas over the face but no approach automatically and accurately evaluates skin colour variations on large facial areas, comparing subjects, treatments and/or time points. We propose such an image-based approach to visualize quickly the outcome of clinical studies on colour variations. METHODS: Among 54 Asian women, one group applied a vehicle twice daily, during 28 days, and the other group an anti-ageing emulsion, taking facial images at baseline and after treatment. Changes in L*a*b* values were studied on four pre-selected facial regions. We also reconstructed average facial images from which the L*a*b* parameters were extracted for every pixel, computing relevance (ΔE) and significance data. Using colour gradients, we mapped these results onto the average facial images. RESULTS: After treatment, L*a*b* parameters show no statistically relevant colour changes in the vehicle group. In the 'active' group, skin was lighter at the upper cheek and, overall, redness decreased. Relevance and significance maps confirmed no visible colour changes in the vehicle group. In the 'active' group, the mapping approach revealed colour changes and their location. Skin became lighter below the eye, cheek and forehead. It was less red below the eyes, on the cheek, jawline and forehead, and generally more yellow. CONCLUSION: Our image-based mapping approach proves to be powerful. It enables us to identify precise facial regions of relevant and statistically significant colour changes after a topical treatment, regions that would have otherwise been undetected.

OBJECTIF: Il existe des méthodes pour évaluer la couleur de la peau sur des zones pré-définies du visage mais aucune approche n'évalue de manière automatique et précise les variations de couleur de peaux sur de large régions du visage, en comparant les sujets, les traitements et/ou les temps d'analyse. Nous proposons une telle méthode basée sur l'analyse d'images pour visualiser de manière rapide les résultats des études cliniques portant sur des variations colorimétriques. MÉTHODES: Parmi 54 femmes d'origine asiatique, un premier groupe a appliqué un véhicule deux fois par jour, pendant 28 jours. Un deuxième groupe a, lui, appliqué une émulsion anti-âge. Des images de visage ont été réalisées avant et après traitement. Les variations des valeurs L*a*b* ont été étudiées sur quatre régions du visage pré-sélectionnées. Nous avons également reconstruit des images de visages moyens pour lesquelles les paramètres L*a*b* ont été extraits pour chaque pixel. Pour ces mêmes pixels, les valeurs de pertinence (delta E) et significativité ont été calculées. A l'aide d'un gradient de couleur, nous avons représenté ces résultats sur les images de visages moyens. RÉSULTATS: Après traitement, les paramètres L*a*b* n'ont montré aucun résultat significativement pertinent pour le groupe ayant appliqué le véhicule. Pour le groupe "actif", la peau est devenue plus claire sur la partie supérieure de la joue et globalement moins rouge. Les cartographies de pertinence et de significativité ont confirmée l'absence de variation colorimétrique sur le groupe véhicule. Sur le groupe "actif", l'approche par cartographie a révélé les changements de couleurs et leur localisation. La peau est devenue plus claire sous les yeux, sur les joue et le front. Le dessous des yeux, les joues, la mâchoire et le front sont devenues moins rouges et généralement plus jaunes. CONCLUSION: Notre approche de cartographie basé sur l'analyse d'images s'est montrée pertinente. Elle nous permet d'identifier de manière précise les régions du visage sur lesquelles des changements pertinents et significatifs de couleur ont eu lieu après l'application d'un traitement topique. Ces régions n'auraient pas été détectées sans cette technique.

Expression and ultrastructural localization of plasmin(ogen) in the terminally differentiated layers of normal human epidermis.

OBJECTIVE: Plasmin, a relatively unspecific trypsin-like serine protease, is involved in many physiological and pathological conditions, particularly in dermatoses with barrier impairment. It is secreted as the inactive zymogen plasminogen and is activated to plasmin by plasminogen activators, such as urokinase. There still exists a paucity of data on the precise localization of epidermal plasmin(ogen) within the epidermis and the stratum corneum. The aim of the present study was to get information about its origin and ultrastructural localization within normal human epidermis. METHOD: We performed immunoelectron transmission electron microscopy immunogold labelling in normal abdominal human skin. RESULT: Plasmin was only observed in the terminally differentiated cell layers of the epidermis and was largely associated with the corneocyte envelopes and to some extent with the intercellular lipid matrix in the stratum corneum. CONCLUSION: Our results indicate that in normal human skin, plasmin(ogen) is synthesized by differentiated epidermal keratinocytes of the stratum granulosum and is not serum-born.

OBJECTIF: Plasmine, une relativement peu spécifique ' trypsin-like' protéase sérine, participe aux plusieurs processus physiologiques et pathologiques et, plus particulièrement, à la physiopathologie des dermatoses caractérisées par l'altération de la barrière de perméabilité. Elle est sécrétée sous forme d'un zymogene inactif, plasminogène, et devient activée par les activateurs du plasminogène, telle urokinase. A l'heure actuelle, on manque de précision quant à la localisation de plasmine (ou son précurseur) dans l'épiderme et le stratum corneum. Le but du présent travail a été de d'apporter l'information sur la provenance et la localisation ultrastructurale de plasmine/plasminogène présents dans l'épiderme humain. MÉTHODE: L'étude ultrastructurale de l'épiderme humain normal (plastie abdominale) a fait appel à l'immunomarquage à l'or colloïdal sur coupes ultrafines des tissus inclus à froid dans des résines acryliques. RÉSULTAT: L'anticorps monoclonal anti -plasmine/plasminogène a détecté l'antigène situé exclusivement dans la partie la plus différenciée de l'épiderme et persistant dans la couche cornée. Il n'y a pas eu de réactivité dans les couches épineuse et basale. Le marquage a été prédominant sur les enveloppes cornifiées des kératinocytes granuleux et cornéocytes. Des foyers du marquage ont été également présents dans le cytoplasme et les espaces intercellulaires de la couche granuleuse, ainsi que dans la matrice lipidique de la couche cornée profonde. CONCLUSION: Nos résultats indiquent la production de novo de plasmine/plasminogène dans les kératinocytes le plus différenciés et ne suggèrent pas l'origine sérique de cette enzyme dans l'épiderme.

The importance of 12R-lipoxygenase and transglutaminase activities in the hydration-dependent ex vivo maturation of corneocyte envelopes.

BACKGROUND: Terminally differentiated keratinocytes acquire corneocyte protein envelopes (CPE) complexed with corneocyte lipid envelopes (CLE). These two structural components of the corneocyte envelopes (CEs) undergo maturation by gaining in hydrophobicity, rigidity and surface area. Linoleoyl acylceramides are processed by 12R-lipoxygenase (12R-LOX) and other enzymes before transglutaminase (TG) attaches ω-hydroxyceramides to involucrin in the CPE. Concurrently, structural proteins are cross-linked by TG that has been activated by cathepsin D (CathD). OBJECTIVES: The primary aim of this work was to demonstrate the impact of relative humidity (RH) during ex vivo CE maturation. Low, optimal and high RH were selected to investigate the effect of protease inhibitors (PIs) on CE maturation and TG activity; in addition, 12R-LOX and CathD activity were measured at optimal RH. Finally, the effect of glycerol on ex vivo CE maturation was tested at low, optimal and high RH. METHODS: The first and ninth tape strip of photo-exposed (PE) cheek and photo-protected (PP) post-auricular sites of healthy volunteers were selected. Ex vivo CE maturation was assessed via the relative CE maturity (RCEM) approach based on CE rigidity and hydrophobicity. The second and eighth tapes were exposed to RH in the presence of inhibitors. RESULTS: Irrespective of tape stripping depth, CEs from PE samples attained CE rigidity to the same extent as mature CEs from the PP site, but such improvement was lacking for CE hydrophobicity. 70% RH was optimal for ex vivo CE maturation. The inhibition of 12R-LOX activity resulted in enhanced CE rigidity which was reduced by the TG inhibitor. CE hydrophobicity remained unchanged during ex vivo maturation in the presence of TG or 12R-LOX inhibition. CE hydrophobicity was enhanced in the presence of glycerol at 44% RH and 100% RH but not at 70% RH. Furthermore, TG activity was significantly diminished at 100% RH compared to the commercial inhibitor LDN-27219. However, a protease inhibitor mix reversed the negative effect of overhydration. CONCLUSION: The study adds to the understanding of the roles of 12R-LOX and TG activity in CE maturation and gives further insight into the effect of glycerol on the SC.

CONTEXTE: Les kératinocytes à différenciation terminale acquièrent les enveloppes protéiniques des cornéocytes (ECP) complexées aux enveloppes lipidiques des cornéocytes (ELC). Ces deux composants structurels des enveloppes cornéocytaires (EC) subissent un processus de maturation en gagnant en hydrophobicité, en rigidité et en surface. Les linoléoyl-acyle-céramides sont traités par 12R-lipoxygénase (12R-LOX) et d'autres enzymes avant que la transglutaminase (TG) ne fixe les x-hydroxy-céramides à l'involucrine dans les ECP. Les protéines structurelles sont simultanément réticulées par la TG qui a été activée par la cathepsine D (CathD). OBJECTIFS: L'objectif principal de ces travaux visait à démontrer l'impact de l'humidité relative (HR) pendant la maturation de l'EC ex vivo. Des humidités relatives faible, optimale et élevée ont été retenues pour étudier l'effet des inhibiteurs de la protéase (IP) sur la maturation de l'EC et l'activité de la TG ; l'activité de CathD et 12R-LOX a également été mesurée à une HR optimale. Finalement, l'effet du glycérol sur la maturation de l'EC ex vivo a été testé à des humidités relatives faible, optimale et élevée. MÉTHODES: La première et neuvième bandes adhésives sur un site à l'arrière de l'oreille protégé de la lumière (photo-protégé, PP) et sur une joue exposée à la lumière (photo-exposée, PE) de volontaires sains ont été sélectionnées. La maturation de l'EC ex vivo a été évaluée par l'approche de la maturité relative d'EC (RCEM) reposant sur l'hydrophobicité et la rigidité de l'EC. Les deuxième et huitième bandes ont été exposées à l'humidité relative en présence d'inhibiteurs. RÉSULTATS: Indépendamment de la profondeur de bande adhésive, les EC des échantillons EP ont atteint la rigidité d'EC de la même manière que les EC matures du site PP, mais ces améliorations faisaient défaut en ce qui concerne l'hydrophobicité des EC. Une HR à 70 % était optimale pour la maturation de l'EC ex vivo. L'inhibition de l'activité du 12R-LOX a entraîné une rigidité accrue de l'EC, laquelle était réduite par l'inhibiteur de la TG. L'hydrophobicité des EC est restée inchangée pendant la maturation ex vivo en présence de l'inhibition de la TG ou du 12R-LOX. L'hydrophobicité des EC a été améliorée en présence de glycérol à une HR de 44 % et à une HR de 100 %, mais non à une HR de 70 %. L'activité de la TG a par ailleurs significativement diminué à une HR de 100 % par rapport à l'inhibiteur commercial LDN-27219. Cependant, un mélange inhibiteur de la protéase a inversé l'effet négatif de la surhydratation. CONCLUSION: L'étude renforce la compréhension des rôles de l'activité de la TG et du 12R-LOX dans la maturation de l'EC et donne de plus amples détails sur l'effet du glycérol sur la couche cornée (stratum corneum, SC).

Facial skin mapping: from single point bio-instrumental evaluation to continuous visualization of skin hydration, barrier function, skin surface pH, and sebum in different ethnic skin types.

Dry skin is one of the most important concerns of consumers worldwide. Despite huge efforts over several decades, the personal care industry still does not offer a perfect solution to satisfy the unmet needs of consumers for moisturising treatments in different ethnic groups. The paucity of data for the underlying cellular and biochemical problems in, and the effects of moisturisers on photodamaged facial skin may partly explain this. Mainly, single point measurements are used to understand the effects of products on skin physiology even on surrogate skin sites such as the non-photodamaged volar forearm. Some groups have developed discontinuous facial maps of skin biophysical properties, however, in 2014 a continuous facial analysis of bio-instrumental evaluations was developed using a heat map approach. These maps enabled a continuous visualization of features that not only revealed an unexpected complexity of facial skin but also indicated that use of surrogate skin sites for facial skin is inappropriate. We have demonstrated that remarkable gradients of skin hydration, TEWL, skin surface pH and sebum exist within short distances across the face and the gradients are distinctive among different ethnic groups. In addition, these studies have demonstrated that darkly-pigmented individuals do not necessarily have a better skin barrier function than their less-pigmented counterparts and that Caucasians have a lower facial skin surface pH compared with more pigmented subjects. Overall, there are no correlations between capacitance, TEWL and skin surface pH including individual topology angle values. Novel 3D camera approaches have also been used to facilitate a more precise assignment of measurement sites and visualisation. The 3D facial colour mappings illustrated precisely the local moisturising effects of a moisturising cream. There were subtle ethnic differences in efficacy that may be related to underlying skin biochemistry and/or ethnic differences in product application. A placebo-controlled study using conductance measurements in Chinese subjects is also reported. Finally, a new whole face statistical approach has been taken to prove differences in skin parameters but also of moisturiser treatment that adds further to our understanding of the ethnic differences in skin physiology and product application. This paper reviews the background of the development and application of this methodology.

L'assèchement de la peau est l'un des problèmes les plus importants chez les consommateurs à travers le monde. En dépit des efforts fournis dans les dernières décennies, l'industrie du soin ne propose pas encore une solution parfaite qui répond aux attentes des consommateurs de différentes ethnies pour des traitements hydratants. Le manque de données concernant les problèmes de mécanisme cellulaire et biochimique, ainsi que les effets des soins hydratants sur la peau du visage photo-endommagée peuvent en partie expliquer cela. En général, une mesure ponctuelle est réalisée pour comprendre les effets des produits sur la physiologie de la peau sur des sites de substitution tels que l'avant-bras non photo-endommagé. Certains groupes ont développé des cartographies du visages discontinues des propriétés biophysiques de la peau, mais ce n'est qu'en 2014 qu'une analyse continue du visage de l'évaluation bio-instrumentale a été proposée en utilisant une approche par cartographie de chaleur. Ces cartographies permettent une visualisation continue des caractéristiques qui ne révèlent pas seulement une complexité inattendue de la peau du visage mais indique également que l'utilisation de sites de substitution est inappropriée. Nous avons démontré que certains gradients liés à l'hydratation de la peau, à la PIE, au pH à la surface de la peau et au sébum sont présents sur de faibles distances à travers le visage et que ces gradients sont différents selon les groupes ethniques. De plus, ces études ont démontré que les individus ayant une pigmentation de peau importante n'ont pas nécessairement une meilleure fonction de barrière cutanée que leurs homologues ayant une peau moins pigmentée et que les Caucasiens ont une plus faible surface de pH sur le bas du visage en comparaison avec des sujets ayant plus de pigmentation. Globalement, en incluant les aspects typologiques individuels, il n'y a pas de corrélation entre la capacitance, la PIE et le pH à la surface de la peau. Une nouvelle approche par caméra 3D à également été utilisée pour faciliter l'attribution et la visualisation plus précise de la mesure par site. Les cartographies du visage 3D en couleur illustrent précisément les effets hydratants localisés d'une crème hydratante. Il y avait des différences ethniques subtiles dans l'efficacité qui peuvent être liées au mécanisme de la biochimie cutanée et/ou dans l'application des produits des différentes ethnies. Une étude contrôlée par placebo utilisant une mesure de conductance chez les sujets d'origine chinoise est également communiquée. Enfin, une nouvelle approche statistique sur le visage complet a été adoptée afin de prouver les différences dans les paramètres de la peau mais aussi dans le traitement hydratant, ce qui nous permet de mieux comprendre les différences ethniques dans la physiologie de la peau et l'application des produits. Cette publication retrace les éléments de développement ainsi que l'application des méthodologies.

Clinical and in vitro evaluation of new anti-redness cosmetic products in subjects with winter xerosis and sensitive skin.

OBJECTIVE: To demonstrate the in vitro activities of panthenol, palmitoylethanolamide (PEA), and niacinamide (NAM) and determine the biophysical properties, clinical safety, tolerability together with efficacy of two developmental anti-redness (AR) formulations containing these ingredients, in alleviating facial redness associated with winter xerosis in healthy volunteers with sensitive skin. METHODS: The anti-inflammatory and skin protective properties of panthenol, PEA and NAM were evaluated in vitro. The physical properties of the AR formulations were analysed using measurement of water vapour transport rate (WVTR) and infrared spectroscopy. Clinical studies were performed between the months of December and April (2014-2015) with efficacy assessed during the winter. Facial redness, irritation, sensitization potential, photo-irritation, and photo-sensitization were evaluated. Self-assessed adverse reactions were reported in diaries of use. RESULTS: Panthenol and PEA reduced prostaglandin E2 , interleukin-6, and thymic stromal lymphopoietin levels in vitro, while NAM induced nicotinamide adenine dinucleotide (NAD) levels and the keratinocyte differentiation markers: filaggrin (2-fold increase, P < 0.001), loricrin (2-fold increase, P < 0.05), involucrin (2 fold increase, P < 0.001) & peroxisomal proliferator activated receptor-alpha (1.5 fold increase, P < 0.05). The two AR products exhibited low WVTR vs. no treatment (P < 0.001) and displayed an ordered lipid structure. The day cream formulation protected against ultraviolet B radiation in vitro. A total of 382 participants were included in clinical studies which showed the AR formulations significantly improved facial redness associated with winter xerosis (Day 29 mean change from baseline: AR day cream 0.77 (P < 0.001); AR serum 0.67 (P < 0.001)). No irritation, sensitization, photo-irritation, photo-sensitization or product-related adverse reactions were observed or reported in the clinical studies. CONCLUSION: The new products significantly improved skin redness associated with winter xerosis in participants with self-perceived sensitive skin. Both products were well tolerated with a suitable safety profile for topical use in subjects with sensitive skin.

OBJECTIF: Démontrer l'activité in vitro du panthénol, du palmitoyléthanolamide (PEA), et du nicotinamide (NAM) et déterminer les propriétés biophysiques, la sécurité clinique, la tolérance ainsi que l'efficacité de deux formulations anti-rougeurs (AR) en développement contenant ces ingrédients pour atténuer les rougeurs faciales associées à la xérose hivernale chez des volontaires sains présentant une peau sensible. MÉTHODES: Les propriétés anti-inflammatoires et protectrices du panthénol, du PEA et du NAM ont été évaluées in vitro. Les propriétés physiques des formulations AR ont été analysées en mesurant le taux de transport de vapeur d'eau (WVTR) et par spectroscopie infrarouge. Des études cliniques ont été réalisées entre décembre et avril (2014-2015) et l'efficacité a été évaluée pendant l'hiver. Les rougeurs, l'irritation, le potentiel de sensibilisation, la photo-irritation et la photosensibilisation au niveau du visage ont été évalués. Des effets indésirables auto-évalués ont été signalés dans des journaux d'utilisation. RÉSULTATS: Le panthénol et le PEA ont réduit les niveaux de prostaglandine E2 , d'interleukine-6 et de lymphopoiétine stromale thymique in vitro, tandis que le NAM a généré une augmentation des taux de nicotinamide adénine dinucléotide (NAD) et des marqueurs de différenciation kératinocytaire : filaggrine (multiplication des taux par 2, P < 0,001), loricrine (multiplication des taux par 2, P < 0,05), involucrine (multiplication des taux par 2, P < 0,001) et du récepteur alpha activé de la prolifération peroxysomale (multiplication des taux par 1,5, P < 0,05). Les deux produits antirétroviraux présentaient un faible taux de WVTR par rapport à l'absence de traitement (P < 0,001) et présentaient une structure lipidique ordonnée. La formulation de la crème de jour protège contre le rayonnement ultraviolet B in vitro. Un total de 382 participants ont été inclus dans les études cliniques qui ont montré que les formulations AR amélioraient significativement les rougeurs faciales associées à la xérose hivernale (changement moyen du jour 29 par rapport à la référence : crème de jour AR 0,77 (P < 0,001) ; sérum AR 0,67 (P < 0,001)). Aucune irritation, sensibilisation, photo-irritation, photosensibilisation ni effet indésirable lié au produit n'a été observé ou signalé dans les études cliniques. CONCLUSION: Les nouveaux produits ont considérablement amélioré la rougeur de la peau associée à la xérose hivernale chez les participants présentant une peau sensible auto-perçue. Les deux produits ont été bien tolérés avec un profil de sécurité approprié pour un usage topique chez les sujets présentant une peau sensible.

Bio-derived hydroxystearic acid ameliorates skin age spots and conspicuous pores.

INTRODUCTION: We report on the preparation and efficacy of 10-hydroxystearic acid (HSA) that improves facial age spots and conspicuous pores. METHODS: The hydration of oleic acid into HSA was catalyzed by the oleate hydratase from Escherichia coli. Following treatment with HSA, collagen type I and type III was assessed in primary human dermal fibroblasts together with collagen type III, p53 protein levels and sunburn cells (SBC) after UVB irradiation (1 J cm-2 ) by immunohistochemistry on human ex vivo skin. UVB-induced expression of matrix metalloprotease-1 (MMP-1) was determined from full thickness skin by RT-qPCR. Modification of the fibroblast secretome by HSA was studied by mass-spectrometry-based proteomics. In a full-face, double blind, vehicle-controlled trial HSA was assessed for its effects on conspicuous facial pore size and degree of pigmentation of age spots in Caucasian women over an 8-week period. RESULTS: HSA was obtained in enantiomeric pure, high yield (≥80%). Collagen type I and type III levels were dose-dependently increased (96% and 244%; P < 0.01) in vitro and collagen type III in ex vivo skin by +57% (P < 0.01) by HSA. HSA also inhibited UVB-induced MMP-1 gene expression (83%; P < 0.01) and mitigated SBC induction (-34% vs. vehicle control) and reduced significantly UV-induced p53 up-regulation (-46% vs. vehicle control; P < 0.01) in irradiated skin. HSA modified the fibroblast secretome with significant increases in proteins associated with the WNT pathway that could reduce melanogenesis and proteins that could modify dermal fibroblast activity and keratinocyte differentiation to account for the alleviation of conspicuous pores. Docking studies in silico and EC50 determination in reporter gene assays (EC50 5.5 × 10-6  M) identified HSA as a peroxisomal proliferator activated receptor-α (PPARα) agonist. Clinically, HSA showed a statistically significant decrease of surface and volume of skin pores (P < 0.05) after 8 weeks of application and age spots became significantly less pigmented than the surrounding skin (contrast, P < 0.05) after 4 weeks. CONCLUSION: HSA acts as a PPARα agonist to reduce the signs of age spots and conspicuous pores by significantly modulating the expression of p53, SBC, MMP-1 and collagen together with major changes in secreted proteins that modify keratinocyte, melanocyte and fibroblast cell behavior.

INTRODUCTION: voici notre rapport sur la préparation et l'efficacité de l'acide 10-hydroxystéarique (AHS) qui atténue les taches de vieillesse faciale et améliore l'apparence des pores. MÉTHODES: l'hydratation de l'acide oléique en AHS a été catalysée par l'hydratase d'oléate à partir de l'Escherichia coli. Après un traitement par AHS, les collagènes de type I et de type III ont été analysés dans des fibroblastes dermiques humains primaires, ainsi que le taux de collagène de type III et de protéine p53, et les cellules provenant de coups de soleil (sunburn cells, SBC) après irradiation par UVB (1 J cm−2 ) par immunohistochimie sur de la peau humaine ex vivo. L'expression de la matrice métalloprotéase-1 (MMP-1) induite par les UVB a été déterminée à partir d'un échantillon de pleine épaisseur de peau par RT-qPCR. La modification du sécrétome des fibroblastes par l'AHS a été étudiée par analyse protéomique basée sur une spectrométrie de masse. Dans une étude du visage entier, en double aveugle, contrôlée par excipient, l'AHS a été évaluée pour ses effets sur la taille des pores apparents du visage et sur le degré de pigmentation de taches de vieillesse chez des femmes de race blanche sur une période de 8 semaines. RÉSULTATS: l'AHS a été obtenu à un haut rendement, énantiomérique pur (≥80 %). Les taux de collagènes de type I et de type III ont augmenté in vitro en fonction de la dose (96 % et 244 %; P < 0.01) et le collagène de type III dans de la peau ex vivo de +57 % (P < 0.01) lors d'un traitement par AHS. L'AHS a également inhibé l'expression génique MMP-1 induite par les UVB (83%; P < 0.01) et a atténuée l'induction des SBC (−34 % par rapport à l'excipient), et a réduit significativement la régulation à la hausse du p53 induite par les UV (−46% par rapport à l'excipient; P < 0.01) sur de la peau irradiée. L'AHS a modifié le sécrétome des fibroblastes avec des augmentations significatives des protéines associées à la voie WNT qui pouvaient réduire la mélanogenèse et des protéines qui pouvaient modifier l'activité des fibroblastes dermiques et la différenciation des kératinocytes pour une atténuation des pores apparents. Des études de docking in silico et la détermination de l'EC50 dans les dosages des gènes rapporteurs (EC50 5.5 × 9 10−6 M) ont identifié l'AHS comme un agoniste du récepteur-α activé par les proliférateurs de peroxysomes (peroxisomal proliferator activated receptor-α, PPARα). Cliniquement, l'AHS a permis une diminution statistiquement significative de la surface et du volume des pores de la peau (P < 0.05) après 8 semaines d'application, et les taches de vieillesse sont devenues significativement moins pigmentées par rapport à la peau environnante (contraste, P < 0,05) après 4 semaines. CONCLUSION: l'AHS agit comme un agoniste du PPARα pour réduire les signes de taches de vieillesse et l'apparence des pores par une modulation significative de l'expression de la protéine p53, des SBC, de la MMP-1 et du collagène avec des changements majeurs dans les protéines sécrétées qui modifient le comportement cellulaire des kératinocytes, des mélanocytes et des fibroblastes.

12R-lipoxygenase activity is reduced in photodamaged facial stratum corneum. A novel activity assay indicates a key function in corneocyte maturation.

BACKGROUND: During the late stage of keratinocyte differentiation, corneocytes gain a strong protein-lipid structure: the corneocyte envelopes (CE), composed of the inner corneocyte protein envelope (CPE) and the outer corneocyte lipid envelope (CLE). The hydrophobicity of CEs depends on the covalent attachment of linoleoyl-acyl-ceramides by transglutaminases (TG). These ceramides are processed by a range of other enzymes, including 12R-lipoxygenase (12R-LOX), before the covalent attachment of the free ω-hydroxyceramides to the CPE surface to form the CLE. The mechanical strength of CE is obtained with the formation of isodipeptide bonds by TG. The increase in hydrophobicity and rigidity leads to CE maturation which supports the integrity and mechanical resistance of the stratum corneum (SC). OBJECTIVES: The aim of this work was to develop and validate a novel enzyme activity assay for 12R-LOX in tape strippings of photo-exposed (PE) cheek and photo-protected (PP) post-auricular SC of healthy Chinese volunteers (n = 12; age 25 ± 3 years). RESULTS: A fluorescence-based assay was developed with ethyl linoleic acid as the substrate and a polyclonal antibody against 12R-LOX as an inhibitor. The specificity was shown by the lack of effect by a LOX inhibitor (ML351) and an epidermal-type lipoxygenase 3 (eLOX3) antibody on the acquired 12R-LOX activity. Reduced 12R-LOX activity was observed in the outer compared to the inner SC layers. Moreover, dramatically lower activity was shown in the PE vs. PP samples. Furthermore, the enzyme activity has a positive correlation (r = 0.94 ± 0.03) with CE maturity, in particular hydrophobicity, and a negative correlation (r = -0.96 ± 0.01) with transepidermal water loss (TEWL). CONCLUSION: This novel enzyme assay revealed a lower 12R-LOX activity in tape strippings from PE cheek for the first time. This finding is in line with less mature CEs and higher TEWL compared to PP post-auricular samples. This study indicates a strong link between 12R-LOX activity and CE maturation and SC integrity.

CONTEXTE: Pendant le stade avancé de différenciation des kératinocytes, les cornéocytes acquièrent une solide structure protéines-lipides : l'enveloppe des cornéocytes (EC) composée de l'enveloppe protéinique des cornéocytes (EPC) interne et de l'enveloppe lipidique des cornéocytes (ELC) externe. L'hydrophobicité des EC dépend de la liaison covalente des linoléoyl-acyle-céramides par transglutaminases (TG). Ces céramides sont traités par un ensemble d'autres enzymes, y compris la 12R-lipoxygénase (12R-LOX), avant la liaison covalente des xhydroxycéramides à la surface de l'EPC pour former l'ELC. La force mécanique de l'EC est obtenue par la formation de liaisons isodipeptides par TG. L'augmentation de hydrophobicité et de la rigidité conduit à une maturation de l'EC qui soutient l'intégrité et la résistance mécanique de la couche cornée (stratum corneum, SC). OBJECTIFS: L'objectif de ce travail était de développer et de valider un test novateur de l'activité enzymatique pour le 12R-LOX par arrachage par bande sur une joue exposée à la lumière (photo-exposed, PE) et sur de la SC à l'arrière de l'oreille protégée de la lumière (photo-protected, PP) de volontaires sains chinois (n = 12; 25 ans ± 3 ans). RÉSULTATS: Un test de fluorescence a été développé avec de l'acide linoléique éthylique en tant que substrat et un anticorps polyclonal anti-12R-LOX en tant qu'inhibiteur. La spécificité a été démontrée par le manque d'effet d'un inhibiteur de LOX (ML351) et d'un anticorps anti-lipoxygénase 3 (eLOX3) de type épidermique sur l'activité du 12R-LOX acquise. Une réduction de l'activité du 12R-LOX a été observée dans les couches de SC externes par rapport aux couches internes. En outre, une activité considérablement plus faible a été démontrée dans les échantillons PE par rapport aux échantillons PP. De plus, l'activité enzymatique a une corrélation positive (r = 0,94 ± 0,03) avec la maturité de l'EC, en particulier l'hydrophobicité, et une corrélation négative (r = −0,96 ± 0,01) avec une perte d'eau transépidermique (transepidermal water los, TEWL). CONCLUSION: Ce dosage enzymatique novateur, à partir de tape stripping sur les joues exposée à la lumière, a révélé une activité 12RLOX plus faible pour la première fois. Cette découverte est cohérente avec des EC moins matures et une TEWL plus élevée par rapport aux échantillons PP de l'arrière de l'oreille. Cette étude indique un lien solide entre l'activité du 12R-LOX et la maturation de l'EC et l'intégrité de la SC.

Mass spectrometry-based proteomics reveals the distinct nature of the skin proteomes of photoaged compared to intrinsically aged skin.

OBJECTIVE: With increasing age, skin is subject to alterations in its organization, which impact on its function as well as having clinical consequences. Proteomics is a useful tool for non-targeted, semi-quantitative simultaneous investigation of high numbers of proteins. In the current study, we utilize proteomics to characterize and contrast age-associated differences in photoexposed and photoprotected skin, with a focus on the epidermis, dermal-epidermal junction and papillary dermis. METHODS: Skin biopsies from buttock (photoprotected) and forearm (photoexposed) of healthy volunteers (aged 18-30 or ≥65 years) were transversely sectioned from the stratum corneum to a depth of 250 µm. Following SDS-PAGE, each sample lane was segmented prior to analysis by liquid chromatography-mass spectrometry/mass spectrometry. Pathway analysis was carried out using Ingenuity IPA. RESULTS: Comparison of skin proteomes at buttock and forearm sites revealed differences in relative protein abundance. Ageing in skin on the photoexposed forearm resulted in 80% of the altered proteins being increased with age, in contrast to the photoprotected buttock where 74% of altered proteins with age were reduced. Functionally, age-altered proteins in the photoexposed forearm were associated with conferring structure, energy and metabolism. In the photoprotected buttock, proteins associated with gene expression, free-radical scavenging, protein synthesis and protein degradation were most frequently altered. CONCLUSION: This study highlights the necessity of not considering photoageing as an accelerated intrinsic ageing, but as a distinct physiological process.

OBJECTIF: Avec l'âge, la peau est sujette à des altérations dans son organisation, et outre le fait d'avoir des conséquences cliniques cela a un impact sur sa fonction. La protéomique est un outil utile pour l'évaluation non ciblée, semi-quantitative, simultanée d'un nombre élevé de protéines. Dans cette étude, nous utilisons la protéomique pour caractériser et comparer les différences associées à l'âge entre une peau photoexposée et une peau photoprotégée, avec une attention particulière sur l'épiderme, la jonction dermo-épidermique et le derme papillaire. MÉTHODES: Des biopsies de peau de la fesse (photoprotégée) et de l'avant-bras (photoexposée) de volontaires sains (âgés de 18 à 30 ans ou de ≥ 65 ans) ont été sectionnées transversalement depuis la couche cornée jusqu'à une profondeur de 250 µm. Suite à une électrophorèse SDS-PAGE, chaque échantillon a été segmenté avant l'analyse par chromatographie en phase liquide couplée à la spectrométrie de masse/spectrométrie de masse. Une analyse des voies de signalisation a été réalisée à l'aide d'Ingenuity IPA. RÉSULTATS: La comparaison des protéomes de la peau des sites des fesses et de l'avant-bras a révélé des différences dans l'abondance relative de protéines. Le vieillissement de la peau de l'avant-bras photoexposée montre une augmentation de 80% des protéines altérées avec l'âge, contrairement à la peau des fesses photoprotégée où une réduction de 74 % des protéines altérées avec l'âge a été mesurée. Sur le plan de la fonction, les protéines altérées par l'âge dans la peau de l'avant-bras photoexposée étaient associées à une structure, une énergie et un métabolisme. Dans la peau des fesses photoprotégée, les protéines associées à l'expression génique, la neutralisation des radicaux-libres, la synthèse des protéines et la dégradation des protéines étaient le plus fréquemment altérés. CONCLUSION: Cette étude souligne la nécessité de ne pas considérer le photovieillissement comme un vieillissement accéléré intrinsèque, mais comme un processus physiologique distinct.

Early-life regional and temporal variation in filaggrin-derived natural moisturizing factor, filaggrin-processing enzyme activity, corneocyte phenotypes and plasmin activity: implications for atopic dermatitis.

BACKGROUND: Filaggrin is central to the pathogenesis of atopic dermatitis (AD). The cheeks are a common initiation site of infantile AD. Regional and temporal expression of levels of filaggrin degradation products [natural moisturizing factors (NMFs)], activities of filaggrin-processing enzymes [bleomycin hydrolase (BH) and calpain-1 (C-1)] and plasmin, and corneocyte envelope (CE) maturity in early life are largely unknown. OBJECTIVES: We conducted a cross-sectional, observational study investigating regional and age-dependent variations in NMF levels, activity of proteases and CE maturity in stratum corneum (SC) from infants to determine whether these factors could explain the observed predilection sites for AD in early life. METHODS: We measured NMF using a tape-stripping method at seven sites in the SC of 129 children (aged < 12 months to 72 months) and in three sites in 56 neonates and infants (< 48 h to 3 months). In 37 of these neonates and infants, corneocyte size, maturity, BH, C-1 and plasmin activities were determined. RESULTS: NMF levels are low at birth and increase with age. Cheek SC, compared with elbow flexure and nasal tip, has the lowest NMF in the first year of life and is the slowest to reach stable levels. Cheek corneocytes remain immature. Plasmin, BH and C-1 activities are all elevated by 1 month of age in exposed cheek skin, but not in elbow skin. CONCLUSIONS: Regional and temporal differences in NMF levels, CE maturity and protease activities may explain the predilection for AD to affect the cheeks initially and are supportive of this site as key for allergen priming in early childhood. These observations will help design early intervention and treatment strategies for AD.

A new approach to assess the effect of photodamage on corneocyte envelope maturity using combined hydrophobicity and mechanical fragility assays.

BACKGROUND: The maturity of the corneocyte envelope (CE) provides information about the barrier functionality of the stratum corneum (SC). Corneocytes are enclosed by the CE, a protein-lipid matrix, contributing to mechanical resistance and hydrophobicity of the SC. OBJECTIVES: The aim of the work was to develop a novel and robust approach to characterize CE maturity based on rigidity, hydrophobicity and surface area. This offers an alternative approach to the Nile red staining and antigenicity of involucrin to characterize the CE. The photoexposed (PE) cheek and photoprotected (PP) post-auricular sites were selected for investigation. METHODS: Nine tape strips were obtained from the cheek and post-auricular sites of healthy Caucasians. CEs on the first and last tape strip were subjected to sonication to assess rigidity, and Nile red staining to determine hydrophobicity per unit surface area. In addition, the presence of involucrin and lipids was assessed to determine CE maturity by examination of the red/green pixel ratio, percentage of involucrin expressing CEs and alternatively the ratio of fluorescence density. RESULTS: The CE rigidity was lower in the deeper SC layers of the cheek, whereas post-auricular CEs were mechanically more resistant. Post-auricular CEs from the superficial SC had a larger surface area with a stronger fluorescence signal than those from the cheek. Interestingly, those CEs from the deeper SC layers had similar surface areas in both anatomical sites but were significantly different in hydrophobicity. These three parameters can be summarized as a relative CE maturity index that expresses CE maturity more precisely with a higher sensitivity than the conventional involucrin and Nile red staining approach. CEs of the cheek surface are more mature than CEs in the deeper SC layer, whereas CEs obtained from the post-auricular surface are more mature than those from the cheek surface. CONCLUSION: The combined method developed allows characterization of CE maturity based on hydrophobicity per unit surface area and rigidity rather than a simple ratio of lipid to involucrin. A more robust and sensitive measurement has therefore been developed addressing the limitations of earlier protocols.

The effect of photodamage on the female Caucasian facial stratum corneum corneome using mass spectrometry-based proteomics.

BACKGROUND: The effect of photodamage on facial stratum corneum (SC) is still poorly understood. OBJECTIVE: To describe the SC proteome from tape strippings of Caucasian SC from photoexposed cheek and photoprotected post-auricular (PA) site, a global analysis of photodamage on the skin will be developed leading to a better understanding of keratinocyte signalling pathways and identification of new molecular targets for the treatment of photoaged skin. METHODS: Female Caucasian subjects had nine consecutive tape strippings taken from their cheeks and PA site. Proteins were extracted and the trypsin-digested peptides were analysed by nanochromatography coupled to a high-resolution mass spectrometer. Data-dependent acquisition allowed protein identification that was processed by Paragon algorithm of Protein Pilot software. RESULTS: Changes in the levels of epidermal differentiation proteins were apparent indicating poor epidermal differentiation and SC maturation (keratins, cornified envelope (CE) proteins) on photoexposed cheeks. Differences in protease-anti-protease balance were observed for corneodesmolysis (favouring desquamation) and filaggrinolysis (favouring reduced filaggrin processing). 12R-LOX, a CE maturation enzyme, was reduced in photodamaged skin but not transglutaminases. Changes in signal keratinocyte transduction pathway markers were demonstrated especially by reduced levels of downstream signalling markers such as calreticulin (unfolded protein response; UPR) and increased level of stratifin (target of rapamycin; mTOR). Evidence for impaired proteostasis was apparent by reduced levels of a key proteasomal subunit (subunit beta type-6). Finally, key antioxidant proteins were upregulated except catalase. CONCLUSION: Clear examples of poor keratinocyte differentiation and associated metabolic and signalling pathways together with reduced SC maturation were identified in photodamaged facial SC. Corneocyte immaturity was evident with changes in CE proteins. Particularly, the reduction in 12R-LOX is a novel finding in photodamaged skin and supports the lack of SC maturation. Moreover, filaggrinolysis was reduced, whereas corneodesmolysis was enhanced. From our results, we propose that there is a poor cross-talk between the keratinocyte endoplasmic reticulum UPR, proteasome network and autophagy machinery that possibly leads to impaired keratinocyte proteostasis. Superimposed on these aberrations is an apparently enhanced mTOR pathway that also contributes to reduced SC formation and maturation. Our results clearly indicate a corneocyte scaffold disorder in photodamaged cheek SC.

Fractionated aliphatic alcohols as synthetic precursors of ultra long-chain monoacylglycerols for cosmetic applications.

BACKGROUND: Xerosis is an abnormally dry and flaky skin condition that is associated with a change in the packing behaviour of the lipid matrix in the stratum corneum (SC), the outermost layer of the skin. This skin condition can lead to an increase in transepidermal water loss (TEWL). As ultralong-chain fatty acids have a positive effect on maintaining the packing behaviour of the SC lipid matrix, a moisturizer which contains glycerides of ultralong-chain fatty acids could act as a semi-occlusive layer on the surface of the skin. This will lower the rate of water evaporation through the epidermis and consequently help prevent or improve skin xerosis. OBJECTIVE: To identify a novel source of ultralong-chain lipids and develop monoacylglycerols with mixed fatty acyl chain lengths that have occlusive properties superior to petrolatum. METHODS: Initially, Performacol 425, a mixture of very long-chain fatty alcohols, was fractionated using short path distillation to yield a fraction enriched with C22:0-C26:0 fatty alcohols. The fatty alcohol fraction was then oxidized using Jones reagent, and the resulting fatty acids were esterified with glycerol to yield the corresponding monoglycerides using Novozym 435. These were then evaluated using Fourier transform infrared spectroscopy, differential scanning calorimetry and water vapour transmission rate measurements. RESULTS: The monoacylglycerols enriched with C22:0-C26:0 displayed a melting point of 80°C and orthorhombic packing; packing behaviour mainly present in healthy SC. In addition, a phospholipid-structured emulsion containing 3% of the monoglycerides displayed occlusive properties superior to the vehicle containing 3% petrolatum jelly. CONCLUSIONS: Performacol 425 can be a potential source of fatty alcohols to synthesize monoacylglycerols that can improve the occlusive behaviour of phospholipid-structured emulsions.

The chemistry, function and (patho)physiology of stratum corneum barrier ceramides.

Research on understanding of the chemistry, function and (patho)physiology of stratum corneum (SC) lipids and especially ceramides has evolved over the last two decades. This has been made successful through the application of separation techniques that have become increasingly more sophisticated, and it has become increasingly evident that our understanding of these molecules remains in its infancy. Thirteen classes of ceramides with over 300 and possibly up to 1000 distinct ceramide species have been identified suggesting an exquisitely subtle relationship between the types of ceramides and physical and chemical behaviour. Nevertheless, research has demonstrated the importance of the correct SC lipid lamellar architecture with conformationally-ordered lipid bilayers, the presence of long-chain ceramides, as either free or covalently bound lipids, greater quantities of phytosphingosine-containing ceramides and a high SC lipid/protein ratio is essential for optimal barrier function. These features are known to change in a variety of physiological and pathophysiological conditions. Clearly, there is more to be learned but as we further decipher the complexity of SC lipids and understand their individual roles in the SC, we will learn how to better treat the disorders of cornification.

The effects of benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide), a dual plasmin and urokinase inhibitor, on facial skin barrier function in subjects with sensitive skin.

OBJECTIVE: The aim of this study was to optimize the synthesis of the plasmin and urokinase (uPA) inhibitor benzylsulfonyl-D-Ser-homoPhe-(4-amidino-benzylamide) (BSFAB), to characterize its activity and mechanism of action and to assess its use to improve stratum corneum (SC) barrier function. METHODS: Peptide coupling methods were used to synthesize BSFAB, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) together with 1 H- and 13 C-nuclear magnetic resonance spectroscopy (NMR) were applied to clarify its structure and determine its purity. Its binding mode was determined by docking studies to the catalytic domains of plasmin and uPA. Inhibition constants (Ki ) were determined by enzyme kinetic studies, and the effect of BSFAB on plasmin, uPA and transglutaminase 1 expression was evaluated in non-cytokine and cytokine-stimulated keratinocytes. A vehicle-controlled clinical study on SC barrier function was conducted on facial skin of subjects with self-perceived sensitive skin. RESULTS: BSFAB was synthesized with high purity (97.3%). In silico studies indicated that the amidine moiety of BSFAB was anchored in the S1 pocket of both enzymes by binding to Asp189, Ser190 and Gly219, whereas the backbone of the D-Ser residue makes an anti-parallel ß-sheet interaction with Gly216. BSFAB was shown to be an effective inhibitor of plasmin and uPA with Ki values of 29 and 25 nM, respectively. BSFAB also inhibited keratinocyte-secreted protease activities in basal (plasmin inhibition 37.7%, P < 0.05 and uPA inhibition 96.6%, P < 0.01) and cytokine-induced conditions (plasmin inhibition 41.1%, P < 0.05 and uPA inhibition 97.0%, P < 0.001) and stimulated the gene expression of transglutaminase 1 in cytokine-stimulated keratinocytes (approximately 4.5 times increased expression, P < 0.01). Clinically, BSFAB was shown to improve SC barrier integrity (P < 0.02 on day 29) and subjective improvements in the perception of healthy skin (P < 0.05 on day 28). CONCLUSION: BSFAB binds as a reversible competitive inhibitor to the active sites of plasmin and uPA. Additionally, BSFAB positively improved keratinocyte differentiation gene expression (transglutaminase 1). These effects were translated into improvements in SC barrier integrity clinically in subjects with dry and sensitive skin and improved their perception of having a healthy skin condition.

The rational design of biomimetic skin barrier lipid formulations using biophysical methods.

OBJECTIVE: The focus of this communication was to study phospholipid-structured emulsions whose phase behaviour is modified with monoalkyl fatty amphiphiles. Ideally, these systems would mimic key physical and structural attributes observed in human stratum corneum (SC) so that they better alleviate xerotic skin conditions. METHODS: Phosphatidylcholine-structured emulsions were prepared, and their phase behaviour modified with monoalkyl fatty amphiphiles. The effect of molecular volume, acyl chain length and head-group interactions was studied using a combination of physical methods. Water vapour transmission rate (WVTR) was used as a primary test to assess occlusive character. Changes in the vibrational modes observed in Fourier transform infrared (FTIR) spectroscopy and bilayer spacing measured by X-ray diffraction (XRD) were then applied to elucidate the lateral and lamellar microstructural characteristics in the systems. RESULTS: Water vapour transmission rate demonstrated that as the phosphatidylcholine acyl chain length increased from C14, to C18, to C22, there was a corresponding increase in occlusive character. The addition of monoalkyl fatty amphiphiles such as behenic acid, behenyl alcohol or cetostearyl alcohol to a base formulation incorporating dipalmitoyl and distearoylphosphatidylcholine (C18) was seen to further increase barrier characteristics of the emulsions. FTIR methods used to probe lipid-chain conformational ordering demonstrated that as phosphatidylcholine acyl chain lengths increased, there was a corresponding improvement in acyl chain ordering, with an increase in thermal transition temperatures. The addition of a monoalkyl fatty amphiphile resulted in conformational order and thermal transition temperature improvements trending towards those observed in stratum corneum. FTIR also demonstrated that systems containing behenic acid or behenyl alcohol exhibited features associated with orthorhombic character. X-ray diffraction data showed that addition of monoalkyl fatty amphiphile also resulted in thicker lamellar structures than when those agents are not present. CONCLUSION: The generalized approach described herein is shown to mechanistically describe the occlusive character of phospholipid-structured formulations in the presence of long-chain fatty acids or alcohols and that they exhibit characteristics mimicking those found in human SC lipids.

Effect of different alcohols on stratum corneum kallikrein 5 and phospholipase A2 together with epidermal keratinocytes and skin irritation.

OBJECTIVES: The aim of this exploratory study was to investigate the effect of ethanol, isopropanol and n-propanol on stratum corneum (SC) enzymes and keratinocytes in vitro together with their effects on skin condition and function. METHODS: Activities of kallikrein 5 (KLK5) and phospholipase A2 (PLA2) as well as keratinocyte metabolic activity, interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) were measured in vitro in the presence and absence of the different alcohols. We also measured transepidermal water loss (TEWL), skin capacitance, visual dryness and visual redness on the volar forearms of 25 Caucasian women following application of the alcohols 20 and 100 times per day over a period of 14 days in a clinical study. RESULTS: Reduced activities of KLK5 and PLA2 were observed in the presence of the alcohols. The greatest denaturing effect was always observed for n-propanol (P < 0.001), and in the case of PLA2, the effect of isopropanol was greater than ethanol (P < 0.001). Equally, ethanol had the mildest effects on keratinocyte metabolic activity and cytokine secretion (P < 0.001) and n-propanol always produced the most severe changes in normal and differentiated keratinocytes. These in vitro findings supported the clinical results where the major effects were on the induction of skin irritation (increased dropout rates) and ranked the intolerance of the different alcohols as follows: n-propanol > isopropanol > ethanol. At the high application frequencies, the effect of the different alcohols on transepidermal water loss (TEWL) and skin capacitance was similar, but at the low application frequencies, n-propanol had a significant effect on TEWL and capacitance values (P < 0.05). Equally, n-propanol and isopropanol produced significantly more skin redness at the low application frequencies. CONCLUSIONS: Clearly, isopropanol and n-propanol caused significant SC and keratinocyte perturbation in vitro together with damage to skin condition and function in vivo whereas ethanol did not. As a result, we show that ethanol-based sanitizers are better tolerated by skin, particularly in high-use settings, than other alcohols and should be the active ingredient of choice.