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.

Revealing the secret life of skin - with the microbiome you never walk alone.

The human skin microbiome has recently become a focus for both the dermatological and cosmetic fields. Understanding the skin microbiota, that is the collection of vital microorganisms living on our skin, and how to maintain its delicate balance is an essential step to gain insight into the mechanisms responsible for healthy skin and its appearance. Imbalances in the skin microbiota composition (dysbiosis) are associated with several skin conditions, either pathological such as eczema, acne, allergies or dandruff or non-pathological such as sensitive skin, irritated skin or dry skin. Therefore, the development of approaches which preserve or restore the natural, individual balance of the microbiota represents a novel target not only for dermatologists but also for skincare applications. This review gives an overview on the current knowledge on the skin microbiome, the currently available sampling and analysis techniques as well as a description of current approaches undertaken in the skincare segment to help restoring and balancing the structure and functionality of the skin microbiota.

Le microbiome de la peau humaine est récemment devenu un centre d'intérêt pour les domaines dermatologique et cosmétique. Comprendre le microbiote cutané, à savoir la collection de microorganismes vitaux vivant sur notre peau, et comment maintenir son équilibre délicat est une étape essentielle pour mieux comprendre les mécanismes responsables d'une peau saine et son apparence. Les déséquilibres dans la composition microbiotique de la peau (dysbiose) sont associés à plusieurs affections cutanées, soit pathologiques comme l'eczéma, l'acné, les allergies ou les pellicules, soit non pathologiques comme la peau sensible, irritée ou sèche. Par conséquent, le développement d'approches qui préservent ou restaurent l'équilibre naturel et individuel du microbiote représente une nouvelle cible non seulement pour les dermatologues mais aussi pour les experts en cosmétiques. Cette revue donne un aperçu des connaissances actuelles sur le microbiome cutané, les techniques d'échantillonnage et d'analyse actuellement disponibles ainsi qu'une description des approches actuelles entreprises dans le segment des soins de la peau pour aider à restaurer et équilibrer la structure et la fonctionnalité du microbiote de la peau.

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.

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.

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.

Novel approaches to characterize age-related remodelling of the dermal-epidermal junction in 2D, 3D and in vivo.

BACKGROUND/PURPOSE: The dermal-epidermal junction (DEJ) forms epidermal protrusions down into the dermis (rete ridges) and dermal projections up into the epidermis (dermal papillae). Usually visualized in two-dimensions (2D), our knowledge of how the DEJ changes with ageing is limited. We aimed to characterize how this structure exists in 3D and changes with age. METHODS: Photoprotected and photoexposed skin were imaged using reflectance confocal microscopy (RCM) in young and aged individuals. Biopsies of the imaged areas were processed for histological sectioning and for imaging using micro-computed X-ray tomography (microCT). RESULTS: Images obtained from RCM and microCT were used to 3D reconstruct the DEJ. DEJ heights obtained from microCT images showed strong correlation with histology-measured heights. We proposed a novel definition of rete ridges (RRm ) and dermal papillae (DPm ), which allowed easier automated measurement of reduced DPm and RRm volumes in aged skin from microCT reconstructions. An algorithm to map DPm connectivity showed reduced lengths of DPm branches with age. CONCLUSION: Three-dimensional images illustrated the complex topography of the DEJ and highlighted the distinct morphology of dermal papillae compared with rete ridges, which is not evident when evaluating 2D sections. Ex vivo imaging was more successful in differentiating DEJ architecture with respect to age.

A fundamental investigation into aspects of the physiology and biochemistry of the stratum corneum in subjects with sensitive skin.

BACKGROUND: Sensitive skin is a poorly understood skin condition. Defects in stratum corneum (SC) barrier function and/or extrasensory neuronal networks in the epidermis are believed to be involved in the problem. OBJECTIVES: This study aimed to unravel the relationships between bleomycin hydrolase (BH) and calpain-1 (C-1), pyrrolidone carboxylic acid (PCA) levels, corneocyte maturation, transglutaminase (TG) and plasmin activities on the cheeks of subjects with sensitive skin. METHODS: Forty-eight female Caucasian subjects, Fitzpatrick skin phototypes II-III, with self-perceived sensitive facial skin, were assessed and underwent a capsaicin reactivity test. Expert grading of skin condition was conducted as well as the measurement of transepidermal water loss (TEWL), skin capacitance, SC cohesion and SC integrity. BH, C-1 and plasmin activities were measured as well as PCA levels, plasmin and TG activity. Differential Nile red and involucrin immunostaining was performed to assess corneocyte maturation and size. RESULTS: About 52% of the subjects reacted to capsaicin. There were no significant differences between the capsaicin-sensitive and non-capsaicin-sensitive subjects with reference to skin grading, TEWL, skin capacitance and SC cohesion. PCA levels and BH activity were lowest in the capsaicin-sensitive panel (P < 0.05) and were correlated in non-capsaicin-sensitive subjects (r = 0.72). The activity of TG was significantly lower (48%) in the capsaicin-sensitive subjects (P < 0.001) and their corneocytes were less mature and smaller (P ≤ 0.05). SC was estimated to be thinner (6.87 ± 0.28 vs. 8.68 ± 0.26 µm; P = 0.001) in the capsaicin-sensitive subjects with a corresponding shorter SC path length (83.2 ± 4.4 µm and 113.1 ± 4.5 µm; P = 0.001). CONCLUSIONS: Despite the physiological similarities between the two groups of sensitive skin subjects, differences in their biochemistry were clearly evident. Lower levels of PCA, BH and TG activities together with a greater number of smaller and immature corneocytes indicate inferior SC maturation in the capsaicin-sensitive subjects. The reduced maturation of corneocytes and thinner SC likely contributes to a greater penetration of capsaicin and the associated increased skin sensitivity.

Variation in the activities of late stage filaggrin processing enzymes, calpain-1 and bleomycin hydrolase, together with pyrrolidone carboxylic acid levels, corneocyte phenotypes and plasmin activities in non-sun-exposed and sun-exposed facial stratum corneum of different ethnicities.

BACKGROUND: Knowledge of the ethnic differences and effects of photodamage on the relative amounts of natural moisturizing factor (NMF) together with filaggrin processing enzymes in facial stratum corneum is limited. Our aim was to characterize the activities of calpain-1 (C-1), bleomycin hydrolase (BH) and the levels of pyrrolidone carboxylic acid (PCA) as a marker for total NMF levels and to relate them to plasmin activities and corneocyte maturation. METHODS: Enzyme activities, PCA levels and corneocyte maturation were determined from facial tape strippings of photoexposed cheek and photoprotected post-auricular areas (PA) of healthy Caucasian (C), Black African (BA) and albino African (AA) female subjects living in South Africa. RESULTS: PCA concentration levels were of the order AA > BA > C subjects, and the highest activities of BH were present in the AA subjects. BH activities were greater on the photoexposed sites for the BA and C subjects, but they were only numerically elevated in the AA subjects. Photoprotected sites had an increase in C-1 activity in pigmented groups (C and BA), whereas in the AA subjects, the opposite was measured. Plasmin activities were greater on the cheek compared with the PA site for the AA and C subjects, but the activity was low in the BA subjects. In both test sites, the AA, but not the BA and C subjects, had smaller, parakeratotic and less mature corneocytes. CONCLUSION: Variation in PCA levels has been found for different ethnic groups in this study (AA > BA > C subjects). The values in the AA subjects are surprising as one might expect that the lack of pigmentation, and thereby increased photodamage, might lead to lower levels. Increased BH, but not C-1 activity, was observed in the AA subjects indicating that BH is associated with PCA production to a greater extent. Surprisingly, corneocyte maturation is still impaired with elevated PCA levels in AA subjects. The higher levels of plasmin and BH activities on the cheeks, especially for AA and C subjects, suggest that they can be used as markers for epidermal photodamage.

Variation in stratum corneum protein content as a function of anatomical site and ethnic group.

OBJECTIVES: Quantification of stratum corneum (SC) protein levels from tape strippings is frequently used to investigate skin conditions, to correct for amounts of SC protein removed in SC biomarker studies and to determine distribution of topically applied ingredients. In recent years, a rapid and convenient method for SC protein quantification from tape strippings has become available using infrared densitometry (IRD). However, standard curves have only been generated for Caucasian forearm and shoulder SC and have been assumed to be correct not only for facial SC but also for SC samples of other ethnic groups. The aim of this study was to investigate whether the use of IRD for SC protein measurement is valid for other body sites such as the cheek and for measuring SC protein content of darkly pigmented skin types. METHODS: Ten Caucasian and ten Black African female subjects with self-assessed normal skin participated in the study. Tape strippings were collected from two different body sites (forearm and cheek). First from the tape strippings, the SC optical absorption was determined densitometrically. This obtained absorption (%) was compared with absolute SC protein extracted from the same tapes using a colorimetric microbicinchoninic acid (µBCA) assay. RESULTS: Higher amounts of SC protein were removed from the forearm compared with the cheek (P < 0.01). The absolute SC protein concentration quantified by µBCA assay and the absorption of SC proteins by IRD followed a similar profile. There was no significant difference found between the two ethnicities in SC protein (P > 0.05). The overall coefficient of determination (R(2) ) shows a good fit to the regression line between the two methods in both sites (forearm = 0.82, cheek = 0.77). Also, both ethnicities showed good correlation (R(2) ≥ 0.69, P = 0.01). CONCLUSIONS: Facial SC is morphologically distinct from the forearm, as demonstrated by the differences in amounts of SC removed. Although the data distribution in different subject groups varied, the regression was always quite similar between the two body sites and both ethnic groups. Also, the correlations were similar to previously published data on other body sites. The resultant calibration curves can be used as a rapid indirect protein assessment of tape strippings from the cheek.

A novel continuous colour mapping approach for visualization of facial skin hydration and transepidermal water loss for four ethnic groups.

OBJECTIVES: The aim of this exploratory study was to develop a novel colour mapping approach to visualize and interpret the complexity of facial skin hydration and barrier properties of four ethnic groups (Caucasians, Indians, Chinese and Black Africans) living in Pretoria, South Africa. METHODS: We measured transepidermal water loss (TEWL) and skin capacitance on 30 pre-defined sites on the forehead, cheek, jaw and eye areas of sixteen women (four per ethnic group) and took digital images of their faces. Continuous colour maps were generated by interpolating between each measured value and superimposing the values on the digital images. RESULTS: The complexity of facial skin hydration and skin barrier properties is revealed by these measurements and visualized by the continuous colour maps of the digital images. Overall, the Caucasian subjects had the better barrier properties followed by the Black African subjects, Chinese subjects and Indian subjects. Nevertheless, the two more darkly pigmented ethnic groups had superior skin hydration properties. Subtle differences were seen when examining the different facial sites. CONCLUSIONS: There exists remarkable skin capacitance and TEWL gradients within short distances on selected areas of the face. These gradients are distinctive in the different ethnic groups. In contrast to other reports, we found that darkly pigmented skin does not always have a superior barrier function and differences in skin hydration values are complex on the different parts of the face among the different ethnic groups.

Facial skin pigmentation is not related to stratum corneum cohesion, basal transepidermal water loss, barrier integrity and barrier repair.

OBJECTIVE: Hypotheses have been developed for the evolutionary selection of skin pigmentation one of which relates to improved skin barrier function. The aim of this study was to compare facial skin condition on photoexposed (cheek) and photoprotected (post-auricular) sites of naturally pigmented subjects of different ethnicities (Fitzpatrick skin phototypes II/III and V/VI) and Albino African subjects to understand better the relationship between facial stratum corneum (SC) barrier function, skin surface pH and skin pigmentation. METHODS: Expert grading of skin conditions, capacitance, skin surface pH and skin barrier function measurements were performed. For the latter, transepidermal water loss (TEWL) measurements before (basal TEWL), after 3, 6 and 9 consecutive tape strippings (SC integrity) and 3.5 and 24 h post tape stripping (barrier recovery) were taken. Amounts of SC protein removed during stripping were estimated using infrared densitometry (SC cohesion). RESULTS: Firstly, correlation analysis of the biometric data of the Black African and Caucasian subjects showed there to be no relationship between skin surface pH and ITA° values nor pH and ITA° with basal TEWL. Neither skin surface pH nor ITA° correlated with SC integrity and barrier recovery measurements, but skin surface pH correlated with SC cohesion. ITA° values were correlated with skin hydration. Secondly, on comparing the three ethnic groups, severe skin photodamage was observed in the Albino African subjects and their SC was thicker. Whereas their basal TEWL was elevated, superior values for SC integrity and barrier recovery were measured. No differences in basal TEWL, SC integrity and barrier recovery were found between the other two subject groups. Equally, SC cohesion and skin surface pH values were similar among the three groups. CONCLUSION: There was no relationship between ITA° values and basal TEWL, SC integrity, SC cohesion and barrier recovery, but ITA° was correlated with skin hydration. Skin surface pH, irrespective of ITA° values, correlated with SC cohesion, indicating a greater intracorneal cohesion at lower pH values. Thus, pigmentation has no effect on SC barrier properties but was related to skin hydration. On comparing the three ethnic groups, Albino African SC was found to be superior to the Caucasian and Black African subjects in terms of SC integrity and barrier recovery but not basal TEWL. The Albino African subjects also have a thicker SC which contributes to their better SC integrity. No differences in skin barrier functionality or skin surface pH were observed for the other two groups. Skin hydration was, however, greatest in the Black African subjects. Our data support the evolutionary hypothesis that pigmentation protects the skin from UV irradiation and thereby the skin barrier but not the skin pigmentation-/pH-driven adaptive skin barrier hypothesis.

Increased mass levels of certain serine proteases in the stratum corneum in acute eczematous atopic skin.

Acute eczematous atopic dermatitis (AD) is associated with increases in stratum corneum (SC) serine protease activity. The purpose of this study was to examine whether the increased SC protease activities in acute eczematous atopic dermatitis were associated with increased mass levels of SC proteases. Six subjects with healthy skin and six patients with AD each with non-lesional skin or lesional acute eczematous skin had the mass levels of their extractable SC kallikreins (KLK), plasmin and urokinase quantified using Luminex multiplex bead-based assays from SC tape strippings. The mass levels of KLK5 and KLK14 together with urokinase were not elevated in the SC in atopic skin. However, the mass levels of KLK7 and KLK11 together with plasmin were greatly elevated compared with the extracts from the non-lesional and the healthy skin and correlated with the corresponding enzymatic activities.