Identification of a higher C-S lyase activity of Staphylococcus hominis in volunteers with unpleasant axillary odour.

OBJECTIVE: Excessive and unpleasant odours that emanate from the skin can induce bromhidrosis and substantially impair a person's quality of life. Enzymatic pathways generating unpleasant odours are well detailed, and among them, the C-S lyase is one of the enzymes involved in the conversion of non-volatile precursors into thiol-type malodorous volatile molecules such as 3M3SH. This study aimed to investigate the variation of axillary odour intensity correlated with Staphylococcus (S.) hominis C-S lyase activity within a group of volunteers after a physical activity. METHODS: First, a group of 24 volunteers from the same ethnicity with standardized hygienic and alimentary practices participated in a supervised indoor cycling activity. Following this session, worn T-shirts were recovered to enable the olfactory evaluation of axillary odours by qualified experts. To go further, the microbiota from the axillary zone of each volunteer was sampled and the bacterial relative abundance was investigated by using 16S rRNA metasequencing. Then, S. hominis isolates were obtained by culturomics from these microbiota samples and the C-S lyase activity was measured by spectrofluorometry in protein crude extracts. RESULTS: The evaluation of the odour intensity revealed that within the panel, two groups were significantly distinct. A non-odorous group and a malodorous one with volunteers having unpleasant odours. The 16S rRNA metasequencing reveals differences in bacterial communities between the two groups with a significant increase in the relative abundance of S. hominis in the malodorous group compared with the non-odorous one. The C-S lyase activities measured on S. hominis sampled on volunteers from the two groups demonstrate that for an equivalent quantity of protein, this enzymatic activity is significantly higher for the samples originating from the malodorous group. CONCLUSION: Hence, this study demonstrates that beyond the increase of S. hominis relative abundance, the C-S lyase enzymatic activity of this bacteria is also higher in volunteers with unpleasant axillary odours.

OBJECTIF: Les odeurs excessives et désagréables qui émanent de la peau peuvent induire une bromhidrose et altérer considérablement la qualité de vie d'une personne. Les voies enzymatiques générant des odeurs désagréables sont bien détaillées, et parmi elles, la C­S lyase est l'une des enzymes impliquées dans la conversion de précurseurs non volatils en molécules volatiles malodorantes de type thiols, telles que le 3M3SH. Cette étude avait pour objectif d'investiguer la variation de l'intensité de l'odeur axillaire corrélée à l'activité de la C­S lyase de Staphylococcus (S.) hominis d'un groupe de volontaires après la pratique d'une activité sportive. MÉTHODES: Un groupe de 24 volontaires de la même ethnie ayant des pratiques hygiéniques et alimentaires standardisées ont participé à une séance calibrée de vélo d'intérieur. Après cette activité, les t­shirts portés ont été récupérés pour permettre l'évaluation olfactive des odeurs axillaires par des experts qualifiés. Pour aller plus loin, le microbiote de la zone axillaire de chaque volontaire a été prélevé et l'abondance relative des bactéries a été étudiée par métaséquençage de l'ARNr 16S. Ensuite, des isolats de S. hominis ont été obtenus par culturomique à partir de ces échantillons de microbiote et l'activité de la C­S lyase a été mesurée par spectrofluorométrie dans des extraits protéiques totaux. RÉSULTATS: L'évaluation de l'intensité des odeurs a révélé qu'au sein du panel, deux groupes se distinguaient de manière significative. Un groupe non­odorant et un groupe malodorant avec des volontaires ayant des odeurs axillaires désagréables. Le métaséquençage de l'ARNr 16S révèle des différences dans les communautés bactériennes entre les deux groupes avec une augmentation significative de l'abondance relative de S. hominis dans le groupe malodorant par rapport au groupe non­odorant. L'activité enzymatique de la C­S lyase mesurée à partir d'isolats de S. hominis prélevés sur des volontaires des deux groupes démontrent que, pour une quantité équivalente de protéines, cette activité enzymatique est significativement plus élevée pour les échantillons provenant du groupe malodorant. CONCLUSION: Cette étude démontre qu'au­delà de l'augmentation de l'abondance relative de S. hominis, l'activité de la C­S lyase de cette bactérie est également plus élevée chez les volontaires présentant des odeurs axillaires désagréables.

Angiogenesis and full thickness wound repair in a cell sheet-based vascularized skin substitute.

Skin tissue engineering is undergoing tremendous expansion as a result from clinical needs, mandatory replacement of animal models and development of new technologies. Many approaches have been used to produce vascularized skin substitutes for grafting purposes showing the presence of capillary-like structures but with limited analysis of their in vitro maturation and plasticity. Such knowledge is however important for the development of tissue substitutes with improved implantation success as well as for validation of vascularization in vitro models, including as a readout in pharmacological analyses. For optimal interactions of cells with microenvironment and vasculature, we here used a cell sheet approach consisting in the sole production of matrix by the cells. In this context, we limited the density of endothelial cells seeded for self-assembly and rather relied on the stimulation of angiogenesis for the development of an extensive connected microvascular-like network. After detailed characterization of this network, we challenged its plasticity both during and after establishment of the skin substitute. We show that fine tuning of VEGF concentration and time of application differentially affects formation of capillary-like structures and their perivascular coverage. Furthermore, we performed a deep wound assay that displayed tissue repair and angiogenesis with unique characteristics of the physiological process. These studies demonstrate the importance of cell-derived microenvironment for the establishment of mature yet dynamic vascularized skin models allowing a wide range of pharmacological and basic investigations. STATEMENT OF SIGNIFICANCE: The significant advancements in organ-on-chips and tissue engineering call for more relevant models including microvascularization with remodeling potential. While vascularized skin substitutes have been developed for years, focus has primarily been on the impact of microvascularization on implantation rather than on its in vitro characterization. We here developed a cell sheet-based vascularized skin substitute relying on angiogenesis, i.e. growth of vessel-like structures within the 3D model, rather than solely on endothelial cell self-assembly. We then characterized :1/ vascularization after modulation of angiogenic factor VEGF during the substitute construction; -2/ angiogenesis associated to tissue repair after deep mechanical wounding. These studies establish a solid physiologically relevant model for further investigation of skin cell interactions and in vitro wound healing.

Type-2 immunity associated with type-1 related skin inflammatory diseases: friend or foe?

Chronic inflammatory skin diseases are multifactorial diseases that combine genetic predisposition, environmental triggers, and metabolic disturbances associated with abnormal immune responses. From an immunological perspective, the better understanding of their physiopathology has demonstrated a large complex network of immune cell subsets and related cytokines that interact with both epidermal and dermal cells. For example, in type-1-associated diseases such as alopecia areata, vitiligo, and localized scleroderma, recent evidence suggests the presence of a type-2 inflammation that is well known in atopic dermatitis. Whether this type-2 immune response has a protective or detrimental impact on the development and chronicity of these diseases remains to be fully elucidated, highlighting the need to better understand its involvement for the management of patients. This mini-review explores recent insights regarding the potential role of type-2-related immunity in alopecia areata, vitiligo, and localized scleroderma.

Commensal Cutibacterium acnes induce epidermal lipid synthesis important for skin barrier function.

Lipid synthesis is necessary for formation of epithelial barriers and homeostasis with external microbes. An analysis of the response of human keratinocytes to several different commensal bacteria on the skin revealed that Cutibacterium acnes induced a large increase in essential lipids including triglycerides, ceramides, cholesterol, and free fatty acids. A similar response occurred in mouse epidermis and in human skin affected with acne. Further analysis showed that this increase in lipids was mediated by short-chain fatty acids produced by Cutibacterium acnes and was dependent on increased expression of several lipid synthesis genes including glycerol-3-phosphate-acyltransferase-3. Inhibition or RNA silencing of peroxisome proliferator-activated receptor-α (PPARα), but not PPARß and PPARγ, blocked this response. The increase in keratinocyte lipid content improved innate barrier functions including antimicrobial activity, paracellular diffusion, and transepidermal water loss. These results reveal that metabolites from a common commensal bacterium have a previously unappreciated influence on the composition of epidermal lipids.

An in vitro autologous, vascularized, and immunocompetent Tissue Engineered Skin model obtained by the self-assembled approach.

A complete in vitro skin model, containing resident cell types is needed to understand physiology and to consider the role of immune and endothelial cells in dermal drug testing. In this study, a cell extraction technique was developed to isolate resident skin cells from the same human donor while preserving the immune and endothelial cells. Then those cells were used to reconstruct an autologous, vascularized, and immunocompetent Tissue-Engineered Skin model, aviTES. Phenotypic characterization of the viable cells was performed on freshly isolated cells and after thawing through flow cytometry. Dermal cell extracts were characterized as fibroblasts, endothelial and immune cells, and the average amount of each cell type represents 4, 0.5, and 1 million viable cells per g of the dermis, respectively. The 3D models, TES and aviTES, were characterized by a fully differentiated epidermis that showed an increase in the presence of Ki67+ cells in the basolateral layer of the aviTES model. Capillary-like network formation, through the self-assembly of endothelial cells, and the presence of functional immune cells were identified through immunofluorescence staining in aviTES. In addition, the aviTES model was immunocompetent, as evidenced by its capacity to increase the production of pro-inflammatory cytokines TNF-α, MIP-1α, and GM-CSF following LPS stimulation. This study describes an autologous skin model containing a functional resident skin immune system and a capillary network. It provides a relevant tool to study the contribution of the immune system to skin diseases and inflammatory responses and to investigate resident skin cell interactions and drug development. STATEMENT OF SIGNIFICANCE: There is an urgent need for a complete in vitro skin model containing the resident cell types to better understand the role of immune and endothelial cells in skin and to be able to use it for drug testing. Actual 3D models of human skin most often contain only fibroblasts and keratinocytes with a limited number of models containing endothelial cells or a limited variety of immune cells. This study describes an autologous skin model containing a functional resident skin immune system and a capillary network. It provides a relevant tool to study the contribution of the immune system to skin diseases and inflammatory responses and to investigate interactions between resident skin cell, improving our capacity to develop new drugs.

Papillary and reticular fibroblasts generate distinct microenvironments that differentially impact angiogenesis.

Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization corresponding to their specific functions. These characteristics are associated with gene expression patterns of fibroblasts freshly isolated from their native microenvironment. In order to assess the relevance of these fibroblast subpopulations in a tissue engineering context, we investigated their contribution to matrix production and vascularization using cell sheet culture conditions. We first performed RNA-seq differential expression analysis to determine whether several rounds of cell amplification and high-density culture affected their gene expression profile. Bioinformatics analysis revealed that expression of angiogenesis-related and matrisome gene signatures were maintained, resulting in papillary and reticular ECMs that differ in composition and structure. The impact of secreted or ECM-associated factors was then assessed using two independent 3D angiogenesis assays: -1/ a fibrin hydrogel-based assay allowing investigation of diffusible secreted factors, -2/ a scaffold-free cell-sheet based assay for investigation of fibroblast-produced microenvironment. These analyses revealed that papillary fibroblasts secrete highly angiogenic factors and produce a microenvironment characterised by ECM remodelling capacity and dense and branched microvascular network, whereas reticular fibroblasts produced more structural core components of the ECM associated with less branched and larger vessels. These features mimick the characteristics of both the ECM and the vasculature of dermis subcompartments. In addition to showing that skin fibroblast populations differentially regulate angiogenesis via both secreted and ECM factors, our work emphasizes the importance of papillary and reticular fibroblasts for engineering and modelling dermis microenvironment and vascularization. STATEMENT OF SIGNIFICANCE: Recent advances have brought to the forefront the central role of microenvironment and vascularization in tissue engineering for regenerative medicine and microtissue modelling. We have investigated the role of papillary and reticular fibroblast subpopulations using scaffold-free cell sheet culture. This approach provides differentiated cells conditions allowing the production of their own microenvironment. Analysis of gene expression profiles and characterisation of the matrix produced revealed strong and specific angiogenic properties that we functionally characterized using 3D angiogenesis models targeting the respective role of either secreted or matrix-bound factors. This study demonstrates the importance of cell-generated extracellular matrix and questions the importance of cell source and the relevance of hydrogels for developing physio-pathologically relevant tissue engineered substitutes.

Highlighting the hygroscopic capacities of apiogalacturonans.

To meet the needs of dehydrated skin, molecules with a high hygroscopic potential are necessary to hydrate it effectively and durably. In this context, we were interested in pectins, and more precisely in apiogalacturonans (AGA), a singular one that is currently only found in a few species of aquatic plants. As key structures in water regulation of these aquatic plants and thanks to their molecular composition and conformations, we hypothesized that they could have beneficial role for skin hydration. Spirodela polyrhiza is a duckweed known to be naturally rich in AGA. The aim of this study was to investigate the hygroscopic potential of AGA. Firstly, AGA models were built based on structural information obtained from previous experimental studies. Molecular dynamics (MD) simulations were performed, and the hygroscopic potential was predicted in silico by analyzing the frequency of interaction of water molecules with each AGA residue. Quantification of interactions identified the presence of 23 water molecules on average in contact with each residue of AGA. Secondly, the hygroscopic properties were investigated directly in vivo. Indeed, the water capture in the skin was measured in vivo by Raman microspectroscopy thanks to the deuterated water (D20) tracking. Investigations revealed that AGA significantly capture and retain more water in the epidermis and deeper than a placebo control. Not only do these original natural molecules interact with water molecules, but they capture and retain them efficiently in the skin.

Utilizing deep learning for dermal matrix quality assessment on in vivo line-field confocal optical coherence tomography images.

BACKGROUND: Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique providing non-invasive "optical biopsies" with an isotropic spatial resolution of ∼1 µm and deep penetration until the dermis. Analysis of obtained images is classically performed by experts, thus requiring long and fastidious training and giving operator-dependent results. In this study, the objective was to develop a new automated method to score the quality of the dermal matrix precisely, quickly, and directly from in vivo LC-OCT images. Once validated, this new automated method was applied to assess photo-aging-related changes in the quality of the dermal matrix. MATERIALS AND METHODS: LC-OCT measurements were conducted on the face of 57 healthy Caucasian volunteers. The quality of the dermal matrix was scored by experts trained to evaluate the fibers' state according to four grades. In parallel, these images were used to develop the deep learning model by adapting a MobileNetv3-Small architecture. Once validated, this model was applied to the study of dermal matrix changes on a panel of 36 healthy Caucasian females, divided into three groups according to their age and photo-exposition. RESULTS: The deep learning model was trained and tested on a set of 15 993 images. Calculated on the test data set, the accuracy score was 0.83. As expected, when applied to different volunteer groups, the model shows greater and deeper alteration of the dermal matrix for old and photoexposed subjects. CONCLUSIONS: In conclusion, we have developed a new method that automatically scores the quality of the dermal matrix on in vivo LC-OCT images. This accurate model could be used for further investigations, both in the dermatological and cosmetic fields.

Polyphenol Composition and Antioxidant Activity of Tapirira guianensis Aubl. (Anarcadiaceae) Leaves.

Tapirira guianensis (Anacardiaceae) is a natural resource from the Amazonian Forest and is locally known in French Guiana as "loussé" (creole), "tata pilili" (wayãpi), or "ara" (palikur). The tree is used by indigenous populations for medicinal purposes. To increase the potential of this tree for cosmetic, agro-food, or pharmaceutical uses, extracts were obtained through ultrasound-assisted extraction (UAE) from T. guianensis leaves using various extraction solvents such as water, methanol, and methanol-water (85/15; v/v). Chemical (DPPH, TEAC, ORAC) tests were applied to assess the anti-radical potential of these extracts. The polyphenol contents were determined by spectrophotometric (UV/Visible) and by means of chromatographic (UPLC-DAD-ESI-IT-MSn) methods. Tapirira guianensis leaf hydromethanolic extract produced the highest polyphenol content and exhibited antiradical activities in chemical assays (DPPH, TEAC, and ORAC) similar to (or higher than) those of a well-known antiradical plant, green tea. In T. guianensis, two classes of polyphenols were evidenced: (1) galloylquinic acids (identified for the first time in the studied species) and (2) flavonols and flavanols (present in small amounts). Flavonols seemed to play a major role in the antioxidant activity of DPPH. These findings provide a rationale for the use of T. guianensis in traditional medicine and to pave the way for seeking new biological properties involving this Amazonian tree.

Investigation of the molecular signature of greying hair shafts.

OBJECTIVE: Hair greying (i.e. canitie) is a physiological process occurring with the loss of melanin production and deposition within the hair shafts. Many studies reported the oxidation as the main biological process underlying this defect of pigmentation. Even though the overall appearance and biomechanical properties of hairs are reported to be altered with greying, there is a lack of information about molecular modifications occurring in grey hair shafts. The aim of this study was thus to investigate the molecular signature and associated changes occurring in greying hair shafts by confocal Raman microspectroscopy. METHODS: This study was conducted on pigmented, intermediate (i.e. grey) and unpigmented hairs taken from 29 volunteers. Confocal Raman microspectroscopy measurements were acquired directly on hair shafts. RESULTS: Automatic classification of Raman spectra revealed 5 groups displaying significant differences. Hence, the analysis of the molecular signature highlighted the existence of 3 sub-groups within grey hair: light, medium and dark intermediate. Among molecular markers altered in the course of greying, this study identified for the first time a gradual modification of lipid conformation (trans/gauche ratio) and protein secondary structure (α-helix/ß-sheet ratio), referring respectively to an alteration of barrier function and biomechanical properties of greying hair. CONCLUSION: This study thus reports for the first time a highly specific molecular signature as well as molecular modifications within grey hair shaft.

OBJECTIF: Le grisonnement du cheveu (i.e. canitie) est un processus physiologique correspondant à l'altération de la production et du dépôt des pigments de mélanine au sein de la tige pilaire. De nombreuses études identifient l'oxydation en tant que principal phénomène à l'origine de ce défaut de pigmentation. L'apparence globale et les propriétés biomécaniques des cheveux grisonnants sont également rapportées comme étant altérées. Cependant, il existe un manque d'information concernant les modifications moléculaires ayant lieu dans la tige pilaire grisonnante. Le but de cette étude était donc d'investiguer par microspectroscopie confocale Raman la signature moléculaire de la tige pilaire grisonnante ainsi que les changements biologiques associés. MÉTHODES: Cette étude a été réalisée sur des cheveux pigmentés, intermédiaires (i.e. gris) et non pigmentés, prélevés sur 29 volontaires. Les mesures par microspectroscopie Raman confocale ont directement été acquises sur la tige pilaire. RÉSULTATS: Une classification automatique des spectres Raman a permis de révéler 5 groupes présentant des différences significatives. Ainsi, l'analyse de la signature moléculaire spectrale identifie 3 sous-groupes au sein des cheveux gris : intermédiaires clairs, moyens et foncés. Parmi les marqueurs moléculaires altérés au cours du grisonnement, cette étude identifie pour la première fois une modification graduelle de la conformation des lipides (ratio trans /gauche) et de la structure secondaire des protéines (ratio hélice α/feuillets ß). Ces marqueurs correspondent respectivement à l'altération de la fonction barrière et des propriétés biomécaniques des cheveux gris. CONCLUSION: Cette étude met en évidence pour la première fois une signature moléculaire extrêmement précise ainsi que des modifications moléculaires en lien avec le grisonnement de la tige pilaire.

Development and characterization of a 3D in vitro model mimicking acneic skin.

Acne is an inflammatory skin disease of the pilosebaceous unit, involving four essential factors: hyperseborrhoea combined to a modification of sebum composition, colonization by Cutibacterium (C.) acnes, hyperkeratinization and secreted inflammation. Understanding and mimicking compromised skin is essential to further develop appropriate therapeutic solutions. This study aimed to develop new in vitro 3D models mimicking acneic skin, by combining two main factors involved in the physiopathology, namely, altered sebum composition and C. acnes invasion. Normal human keratinocytes were first used to generate reconstructed human epidermis (RHE) that were then left untreated (control) or treated topically with a combination of both peroxidized squalene and C. acnes cultures. Once validated, this model considered relevant to mimic acneic skin, was further improved by using different phylotypes of C. acnes strains specifically isolated from healthy and acneic patients. While both phylotypes IB and II did not significantly alter RHE, C. acnes IA1 strains induce major acneic skin hallmarks such as hyperkeratinization, secreted inflammation and altered barrier function. Interestingly, these results are obtained independently of the origin of IA1 phylotypes (acneic vs. healthy patient), thus suggesting a role of the ecosystem in controlling C. acnes virulence in healthy skin. In conclusion, by combining two major factors involved in the physiopathology of acne, we (1) succeeded to design in vitro 3D models mimicking this skin disorder and (2) highlighted how C. acnes phylotypes can have an impact on epidermal physiology. These relevant models will be suitable for the substantiation of therapeutic molecules dedicated to acne treatment.

Comparison of line-field confocal optical coherence tomography images with histological sections: Validation of a new method for in vivo and non-invasive quantification of superficial dermis thickness.

BACKGROUND: Line-field confocal optical coherence tomography (LC-OCT) is an imaging technique providing "optical biopsies" of the skin in real time and non-invasively. At a center optical wavelength of 1.3 µm, this innovative technology can be applied to dermo-cosmetic product development due to both high image resolution (~2 µm) and sufficient penetration (~0.5 mm). Nevertheless, the precise dermal area analyzed with LC-OCT has never been identified. In this study, the objective was to compare LC-OCT images with histological sections of the same area, in order to validate a new method for in vivo and non-invasive quantification of superficial dermis thickness. Once validated, this standardized and quantitative method was used to assess age-related changes of the superficial dermis. MATERIALS AND METHODS: Ex vivo LC-OCT acquisitions and hematoxylin-eosin-safran staining were performed on a panel of four healthy Caucasian female volunteers. In vivo LC-OCT study of skin aging was performed on a panel of 37 healthy Caucasian female divided into five different age-groups. RESULTS: Comparison with histological sections revealed that LC-OCT images allow the visualization and the quantification of the superficial portion of papillary dermis. Applied to different age-group of volunteers, LC-OCT images show a constant decrease in this superficial dermis thickness with age. CONCLUSIONS: In conclusion, we have introduced LC-OCT as a novel technique for in vivo and non-invasive evaluation of superficial dermis thickness. This approach could be used in the future to demonstrate visually and quantitatively the capacity of a dermo-cosmetic active ingredient to renormalize the structural properties of the dermis.

Extracellular Vesicles from Activated Dermal Fibroblasts Stimulate Hair Follicle Growth Through Dermal Papilla-Secreted Norrin.

Dermal papilla cells (DPCs) play a pivotal role in the regulation of hair follicle (HF) growth, formation, and cycling, mainly through paracrine mechanisms. In the last decade, extracellular vesicles (EVs) have been recognized as a new paracrine mechanism that can modify the physiological state of recipient cells by transferring biological material. Herein, we investigated the effect of EVs isolated from stimulated human dermal fibroblasts (DFs) on DPC activation and HF growth. We found that these EVs (st-EVs) enhanced HF growth ex vivo. Comparative transcriptomic analysis on DPCs identified specific activation of the NDP gene, encoding the non-Wnt ligand Norrin. We found that Norrin was secreted by st-EVs-stimulated DPCs activating in a noncell autonomous manner ß-catenin pathway in follicular keratinocytes (human HF keratinocyte [HHFK]) and hair growth ex vivo. Although Norrin-specific receptor Frizzled4 was barely detected in HHFK, we found its presence in DF-EVs. Accordingly, DF-EVs provided Frizzled4 to potentiate Norrin effects ex vivo. Our study identifies DF-EVs as efficient activators of DPCs and Norrin as a novel modulatory player in HF physiopathology. Stem Cells 2019;37:1166-1175.

Clinical Efficacy of Oligofructans from Ophiopogon japonicus in Reducing Atopic Dermatitis Flare-ups in Caucasian Patients.

Atopic dermatitis is a chronic relapsing inflammatory skin disease affecting 15-20% children and 2-10% adults worldwide. Topical treatments include corticosteroids and calcineurin inhibitors, despite frequently observed adverse events such as skin atrophy, itching and burning sensations. Good alternatives that can prolong disease relief in between flare-ups are therefore needed. We conducted a randomized, single-blind, placebo-controlled, multicenter clinical trial in a Caucasian cohort of 90 children and 144 adults with mild-to-moderate atopic dermatitis that applied tested products twice daily for 60 days. A natural active from Ophiopogon japonicus, that improves atopic dermatitis symptoms in vivo, was successful in reducing the SCORing of Atopic Dermatitis (SCORAD), including erythema, pruritus and body surface area in both cohorts. The active also improved patient's quality of life and significantly reduced the number of patients relapsing compared to placebo. We conclude that this treatment could be an effective solution to help control the disease in between flare-ups.

Tissue microenvironment initiates an immune response to structural components of Staphylococcus aureus.

Cell-to-cell communication in skin participates to the maintenance of homeostatic responses to foreign substances. Certain strains of Staphylococcus (S) aureus are vicious pathogens that cause deleterious effects in host cells and tissues. Both secreted toxins and structural components of S. aureus trigger an immune response, though how S. aureus stimulates host immune responses is poorly understood. We explored here how keratinocytes and fibroblasts initiate the first steps of an immune response by activating dendritic cells (DCs) through recognition of structural components of S. aureus. We treated monocyte-derived Langerhans cells (moLCs) and monocyte-derived DCs (moDCs) with conditioned media from keratinocytes (K-CM) and fibroblasts (F-CM) treated with heat-killed S. aureus (HKSA) respectively, or directly with HKSA. Immune and inflammatory responses from keratinocytes, fibroblasts, moLCs and moDCs were assessed by analysis of cell surface markers and cytokine production using flow cytometry, real-time PCR and ELISA assays. K-CM and F-CM increased the expression of CD86 and HLA-DR on moLCs and moDCs, in association with a specific cytokine profile. K-CM upregulated TNFA, IL-1B and GM-CSF mRNA expression in moLCs, while F-CM upregulated IL-12 and downregulated TNFA and TGFB mRNA expression in moDCs. Additionally, F-CM attenuated the induction of an inflammatory profile in monocytes. The recognition of structural components from S. aureus by cutaneous microenvironment induces the activation and the expression of specific cytokines from LCs and DCs.

Effect of cross-linking on the physicochemical and in vitro properties of pullulan/dextran microbeads.

Hydrogels are very promising for tissue engineering as they provide scaffolds and a suitable microenvironment to control cell behavior and tissue regeneration. We used a patented method to obtain beads of pullulan/dextran cross-linked with sodium trimetaphosphate (STMP), that were already described for in vivo bone repair. The aim of this study was to provide a comparative analysis of microbeads made of polysaccharides prepared using three different STMP feeding ratio of 1.5, 2.25 or 3 % w/w. The morphology, swelling and biodegradability of these structures were assessed. Mesenchymal stem cells were also seeded to evaluate the cell organization onto the beads. We found that the amount of phosphorus resulting from the cross-linking was proportional to the introduced STMP concentration. An increase of cross-linking decreased the in vitro enzymatic degradability, and also decreased the swelling in PBS or water. The microstructures observed by SEM and confocal microscopy indicated that homogeneous spherical microbeads were obtained, except for the lower cross-linking ratio where the shapes were altered. Beads hydrated in PBS exhibited a mean diameter ranging from 400 to 550 µm with the decrease of STMP ratio. Cells adhered to the surface of microbeads even in the absence of protein coating. Cell viability studies revealed an increase in cell numbers over two weeks for the highest cross-linked beads, whereas the two lowest STMP concentrations induced a decrease of cell viability. Overall, this study demonstrated that pullulan/dextran hydrogels can be designed as microbeads with adjustable physicochemical and biological properties to fulfill requirements for tissue engineering approaches.

Atopic skin: In vivo Raman identification of global molecular signature, a comparative study with healthy skin.

Atopic dermatitis (AD) is the most common skin inflammatory disease, affecting up to 3% of adults and 20% of children. Skin barrier impairment is thought to be the primary factor in this disease. Currently, there is no method proposed to monitor non-invasively the different molecular disorders involved in the upper layer of AD skin. Raman microspectroscopy has proved to be a powerful tool to characterize some AD molecular descriptors such as lipid content, global hydration level, filaggrin and its derivatives. Our investigations aimed to extend the use of in vivo Raman microspectroscopy as a rapid and non-invasive diagnostic technique for lipid conformation and organization, protein secondary structure and bound water content analysis in atopic skin. Our approach was based on the analysis of Raman data collected on the stratum corneum (SC) of 11 healthy and 10 mild-to-moderate atopic patients. Atopic skin revealed a modification of lipid organization and conformation in addition to the decrease of the lipid-to-protein ratio. This study also highlighted a reduction of the bound water and an increase in protein organized secondary structure in atopic skin. All these descriptors worsen the barrier function, state and appearance of the skin in AD. This precise and relevant information will allow an in vivo follow-up of the pathology and a better evaluation of the pharmacological activity of therapeutic molecules for the treatment of AD.

Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Keratinocytes.

UV irradiation is a major environmental factor causing skin dryness, aging and cancer. UVB in particular triggers cumulative DNA damage, oxidative stress and mitochondrial dysfunction. The objective of our study was to provide both qualitative and quantitative analysis of how mitochondria respond to UVB irradiation in normal human epidermal keratinocytes (NHEK) of healthy donors, with the rationale that monitoring mitochondrial shape will give an indication of cell population fitness and enable the screening of bioactive agents with UVB-protective properties. Our results show that NHEK undergo dose-dependent mitochondrial fragmentation after exposure to UVB. In order to obtain a quantitative measure of this phenomenon, we implemented a novel tool for automated quantification of mitochondrial morphology in live cells based on confocal microscopy and computational calculations of mitochondrial shape descriptors. This method was used to substantiate the effects on mitochondrial morphology of UVB irradiation and of knocking-down the mitochondrial fission-mediating GTPase Dynamin-related protein 1 (DRP1). Our data further indicate that all the major mitochondrial dynamic proteins are expressed in NHEK but that their level changes were stronger after mitochondrial uncoupler treatment than following UVB irradiation or DRP1 knock-down. Our system and procedures might be of interest for the identification of cosmetic or dermatologic UVB-protective agents.

Autophagy in human keratinocytes: an early step of the differentiation?

Studies have established that autophagy constitutes an efficient process to recycle cellular components and certain proteins. The phenomenon was demonstrated primarily in response to nutrient starvation, and there are increasing evidences that it is implied in differentiation. Keratinocyte differentiation was going along an activation of lysosomal enzymes and organelle clearance, and terminal steps are sometimes described as a specialized form of cell death leading to corneocytes. We examined whether initiation of the process in human keratinocyte HaCaT involves autophagy. The KSFM™ culture medium was substituted by M199, which contains a low glucose concentration but a high calcium level (known to induce differentiation). Metabolic stress reduced enhanced cell number in G(1) phase, without apoptotic features (ΔΨmt and membrane integrity are unchanged). Morphological changes were associated with a lower integrin ß1 expression and modifications of protein levels involved in keratinocyte differentiation (involucrin, keratin K10 and ΔNp63α). Whereas autophagic signalling was supported by SIRT1 and pAMPK (T172) increase according to time kinetic, which led to the disappearance of mTOR phosphorylated on S2448 residue. The significant Bcl-X(L) level reduction with stress promoted autophagy, by the release of Beclin-1, whereas ATG5-ATG12 and LC3-II that are involved in autophagosome formation were enhanced significantly. Then, the level of lysosomal protein cathepsin B rose to execute autophagy. Kinetic studies established that autophagy would constitute an early signalling process required for keratinocyte commitment in differentiation pathway.