Skin biological responses to urban pollution in an ex vivo model.

The skin epidermis is continuously exposed to external aggressions, including environmental pollution. The cosmetic industry must be able to offer dedicated products to fight the effects of pollutants on the skin. We set up an experimental model that exposed skin explants maintained in culture to a pollutant mixture. This mixture P representing urban pollution was designed on the basis of the French organization 'Air Parif' database. A chamber, called Pollubox®, was built to allow a controlled nebulization of P on the cultured human skin explants. We investigated ultrastructural morphology by transmission electron microscopy of high pressure frozen skin explants. A global transcriptomic analysis indicated that the pollutant mixture was able to induce relevant xenobiotic and antioxidant responses. Modulated detoxifying genes were further investigated by laser micro-dissection coupled to qPCR, and immunochemistry. Both approaches showed that P exposure correlated with overexpression of detoxifying genes and provoked skin physiological alterations down to the stratum basale. The model developed herein might be an efficient tool to study the effects of pollutants on skin as well as a powerful testing method to evaluate the efficacy of cosmetic products against pollution.

Vitamin C-squalene bioconjugate promotes epidermal thickening and collagen production in human skin.

Vitamin C (Vit C) benefits to human skin physiology notably by stimulating the biosynthesis of collagen. The main cutaneous collagens are types I and III, which are less synthesized with aging. Vit C is one of the main promotors of collagen formation but it poorly bypasses the epidermis stratum corneum barrier. To address this challenge, we developed a lipophilic version of Vit C for improving skin diffusion and delivery. Vit C was covalently conjugated to squalene (SQ), a natural lipid of the skin, forming a novel Vit C-SQ derivative suitable for cream formulation. Its biological activity was investigated on human whole skin explants in an ex vivo model, through histology and protein and gene expression analyses. Results were compared to Vit C coupled to the reference lipophilic compound palmitic acid, (Vit C-Palmitate). It was observed that Vit C-SQ significantly increased epidermal thickness and preferentially favored collagen III production in human skin after application for 10 days. It also promoted glycosaminoglycans production in a higher extent comparatively to Vit C-Palmitate and free Vit C. Microdissection of the explants to separate dermis and epidermis allowed to measure higher transcriptional effects either in epidermis or in dermis. Among the formulations studied, the strongest effects were observed with Vit C-SQ.

Efficacy of an agonist of α-MSH, the palmitoyl tetrapeptide-20, in hair pigmentation.

OBJECTIVE: Hair greying (i.e., canities) is a component of chronological ageing and occurs regardless of gender or ethnicity. Canities is directly linked to the loss of melanin and increase in oxidative stress in the hair follicle and shaft. To promote hair pigmentation and reduce the hair greying process, an agonist of α-melanocyte-stimulating hormone (α-MSH), a biomimetic peptide (palmitoyl tetrapeptide-20; PTP20) was developed. The aim of this study was to describe the effects of the designed peptide on hair greying. METHODS: Effect of the PTP20 on the enzymatic activity of catalase and the production of H2 O2 by Human Follicle Dermal Papilla Cells (HFDPC) was evaluated. Influence of PTP20 on the expression of melanocortin receptor-1 (MC1-R) and the production of melanin were investigated. Enzymatic activity of sirtuin 1 (SIRT1) after treatment with PTP20 was also determined. Ex vivo studies using human micro-dissected hairs allowed to visualize the effect of PTP20 on the expression in hair follicle of catalase, TRP-1, TRP-2, Melan-A, ASIP, and MC1-R. These investigations were completed by a clinical study on 15 human male volunteers suffering from premature canities. RESULTS: The in vitro and ex vivo studies revealed the capacity of the examined PTP20 peptide to enhance the expression of catalase and to decrease (30%) the intracellular level of H2 O2 . Moreover, PTP20 was shown to activate in vitro and ex vivo the melanogenesis process. In fact, an increase in the production of melanin was shown to be correlated with elevated expression of MC1-R, TRP-1, and Melan-A, and with the reduction in ASIP expression. A modulation on TRP-2 was also observed. The pivotal role of MC1-R was confirmed on protein expression analysed on volunteer's plucked hairs after 3 months of the daily application of lotion containing 10 ppm of PTP20 peptide. CONCLUSION: The current findings demonstrate the ability of the biomimetic PTP20 peptide to preserve the function of follicular melanocytes. The present results suggest potential cosmetic application of this newly designed agonist of α-MSH to promote hair pigmentation and thus, reduce the hair greying process.

Study of gene expression alteration in male androgenetic alopecia: evidence of predominant molecular signalling pathways.

BACKGROUND: Male androgenetic alopecia (AGA) is the most common form of hair loss in men. It is characterized by a distinct pattern of progressive hair loss starting from the frontal area and the vertex of the scalp. Although several genetic risk loci have been identified, relevant genes for AGA remain to be defined. OBJECTIVES: To identify biomarkers associated with AGA. METHODS: Molecular biomarkers associated with premature AGA were identified through gene expression analysis using cDNA generated from scalp vertex biopsies of hairless or bald men with premature AGA, and healthy volunteers. RESULTS: This monocentric study reveals that genes encoding mast cell granule enzymes, inflammatory mediators and immunoglobulin-associated immune mediators were significantly overexpressed in AGA. In contrast, underexpressed genes appear to be associated with the Wnt/ß-catenin and bone morphogenic protein/transforming growth factor-ß signalling pathways. Although involvement of these pathways in hair follicle regeneration is well described, functional interpretation of the transcriptomic data highlights different events that account for their inhibition. In particular, one of these events depends on the dysregulated expression of proopiomelanocortin, as confirmed by polymerase chain reaction and immunohistochemistry. In addition, lower expression of CYP27B1 in patients with AGA supports the notion that changes in vitamin D metabolism contributes to hair loss. CONCLUSIONS: This study provides compelling evidence for distinct molecular events contributing to alopecia that may pave the way for new therapeutic approaches.

The regulatory role of the tetrapeptide AcSDKP in skin and hair physiology and the prevention of ageing effects in these tissues--a potential cosmetic role.

The naturally occurring tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) recognized as a potent angiogenic factor was shown recently to contribute to the repair of cutaneous injuries. In the current article, we report the ability of AcSDKP to exert a beneficial effect on normal healthy skin and scalp and to compensate for the ageing process. In vitro AcSDKP at 10⁻¹¹-10⁻7 M significantly stimulates the growth of human keratinocytes, fibroblasts and follicle dermal papilla cells. Moreover, it enhances the growth of human epidermal keratinocyte progenitor and stem cells as shown in a clonogenic survival assay. Topical treatment of ex vivo cultured skin explants with 10⁻5 M AcSDKP increases the thickness of the epidermis and upregulates the synthesis of keratins 14 and 19, fibronectin, collagen III and IV as well as the glycoaminoglycans (GAGs). In the ex vivo-cultured hair follicles, AcSDKP promotes hair shaft elongation and induces morphological and molecular modifications matching the criteria of hair growth. Furthermore, AcSDKP at 10⁻¹¹-10⁻7 M was shown to improve epidermal barrier, stimulating expression of three protein components of tight junctions (claudin-1, occludin, ZO-1) playing an important role in connecting neighbouring cells. This tetrapeptide exercises also activation of SIRT1 implicated in the control of cell longevity. Indeed, a two-fold increase in the synthesis of SIRT1 by cultured keratinocytes was observed in the presence of 10⁻¹¹-10⁻7 M AcSDKP. In conclusion, these findings provide convincing evidence of the regulatory role of AcSDKP in skin and hair physiology and suggest a cosmetic use of this natural tetrapeptide to prevent skin ageing and hair loss and to promote the cutaneous regeneration and hair growth.

Glycation induction and antiglycation activity of skin care ingredients on living human skin explants.

Glycation is an ageing reaction of naturally occurring sugars with dermal proteins, whose clinical signs may appear in vivo around age 30, and increases steadily/regularly with age. The suppleness of the dermis is affected by the formation of bridges between proteins and sugars (Maillard's reaction). The residues formed (Amadori products, Advanced Glycation End products) as well as the proteins they alter, can be visualized by specific immunostainings. Induced in a few days on living skin explants by methylglyoxal, glycation can be prevented by the application of aminoguanidine HCl, the reference anti-glycation molecule. This model enabled to highlight the anti-glycation activity of substances of vegetal origin such as puerarin and chlorogenic acid.

Cocoa polyphenols and their influence on parameters involved in ex vivo skin restructuring.

Polyphenols in general are compounds that are known to promote health and have a preventive effect against various chronic diseases. The influence of cocoa polyphenols on skin, however, has scarcely been studied from a histological point of view. The aim of this study is to assess the influence of cocoa polyphenols on several indicators of skin elasticity and skin tonus, namely, glycosaminoglycans and collagen I, III and IV. This was carried out by using a model of ex vivo human skin explants maintained in survival, on which a cocoa polyphenol extract was applied. After processing by standard histological techniques (fixation, paraffin embedding, sectioning, staining, immunostaining and microscopical observation), the influence of cocoa polyphenols on the evaluated parameters was quantified by image analysis. The results obtained show that cocoa polyphenols exhibit a positive action on the parameters assessed, and the dose at which they improve the most parameters associated with skin tonus and elasticity was determined. Their activity was compared with a commercially available product, and the results obtained show that their efficacy is equivalent. Moreover, an enhancing effect of cocoa butter on activity of cocoa polyphenol was highlighted. Now that the properties of cocoa polyphenols on ex vivo skin restructuring parameters have been assessed, the next step could include their evaluation in vivo.

Original semiologic standardized evaluation of stratum corneum hydration by Diagnoskin stripping sample.

In a normal and healthy skin, the regular elimination of the superficial corneocytes, called desquamation, is a fundamental physiologic process intended to protect the barrier function of the skin. This invisible loss of corneocytes, individually or in small groups, is incessantly compensated by the divisions of the proliferative layer and the upward cellular maturation in order to maintain the harmonious renewal of the epidermis and the integrity of the stratum corneum. The harmony of this desquamation process is intimately conditioned by a sufficient hydration of the stratum corneum: (i) an abnormal desquamation leads to a disruption of the water barrier function and consequently to a dehydration tendency of the stratum corneum, and (ii) a cutaneous dryness (whatever the cause) is able to disturb the desquamation process. Protecting the water content of the stratum corneum has always been a major preoccupation of the cosmetic industry scientists. Consequently, the moisturizing properties of a cosmetic product are objectively measured by various explorations directly targeted on the hydration (corneometry) and on the level of the water barrier function (transepidermal water loss (TEWL) measurements), which depends directly on the skin hydration state. This intimate linkage of the desquamation process and the water content of the stratum corneum enable us to suggest an indirect assessment of the hydration from a direct study of the desquamation by examining a skin-stripping sample (D-Squames) by an optical microscope (linked to a computer). We will describe this already known technique and mainly its new and unpublished semiologic exploitation, named Diagnoskin, whose advantages are its simplicity and its reproducibility particularly interesting in the case of sequential appraisal of dermatologic or cosmetic treatments.

Inhibition of human neutrophil elastase by wheat ceramides.

Synopsis Ceramides, composed of sphingosine N-acyl linked to fatty acids have become widely used in cosmetology. They play several physiological roles in the regulation of skin barrier function. Human neutrophil elastase (HNE) can be inhibited by long-chain fatty acids and their derivatives; it was therefore postulated that plant ceramides could be inhibitors of HNE. Ceramides were extracted from wheat, isolated and characterized. The main fatty acids were 16:0, 18:1, 18:2 and the sphingoid moiety was phytosphingosine. Concentrations necessary to reach 50% inhibition of HNE were, respectively, 33 and 41 mug ml(-1) for non-glycosyl ceramides (CER) and glycosyl ceramides (gly-CER) when using a synthetic specific substrate. Similar extents of inhibition were obtained using a physiological substrate, insoluble elastin. Ex vivo studies showed that CER protected human skin elastic fibres against HNE degradation. Ceramides, being natural non-toxic substances, besides their role in cosmetics, could be of pharmacological interest in dermal inflammatory disorders.

Plasma concentrations of beta-carotene, vitamin A and vitamin E after beta-carotene and vitamin E intake.

We have studied the metabolism (absorption) of beta-carotene and vitamin E by assigning eleven volunteers to receive daily two capsules of OENOBIOL, each containing 15 mg of beta-carotene and 15 mg of vitamin E, over 60 days. The beta-carotene, vitamin E and vitamin A plasma levels were then determined using new methods developed in our laboratory. After two months, the actively treated group's median beta-carotene and vitamin E levels were significantly higher than those of a control group. However, no significant change between treated and control groups in the mean of vitamin A (retinol) plasma levels were observed. Treatment with beta-carotene, a vitamin A precursor, does not significantly modify the vitamin A levels. This conclusion had already been observed and it is assumed that a plasma level of beta-carotene equal or higher than 0.3 mg/L reflects a nutritional intake of provitamins sufficient to support homeostasis of retinol (Brubacher et al., 1982).

[Analytical study of the reciprocal influence of amino acids in mixtures on their determination]. / Etude analytique de l'influence réciproque des acides aminés en mélange sur leur dosage cuprimétrique.

The behaviour of divers mixtures of amino acids in alkaline solution with copper II ions, determined with the help of the potential measurement taken by a copper electrode, was compared with that of a solution of isolated amino acids. The influence of the simultaneous presence of several of these compounds on the stoichiometry of the complexation was studied and discussed.

[Potentiometric determination of amino acids with copper II, and with a copper indication electrode]. / Dosage potentiométrique des acides aminés par le cuivre II, à l'aide d'une électrode indicatrice de cuivre.

The measurement potentiometrically with a copper electrod by the addition of a solution of copper sulfate, to an alcalin solutions of amino acids allows to establish an exact and simple and fast method. The nature of the tested amino acid can intervene in the quantitative composition of the formed complex.