Positive and ecobiological contribution in skin photoprotection of ectoine and mannitol combined in vivo with UV filters.

BACKGROUND: Chronic exposure to ultraviolet (UV) irradiation causes immunosuppression, photoaging, and carcinogenesis by induction of a cascade of skin damages. Sunscreens currently on the market are not absorbing UV rays uniformly throughout the full UV range, high sun protection factor (SPF) sunscreens absorb most of UVB rays but are less effective in absorbing the UVA part of the spectrum. In the context, one approach could consist of preserving the skin natural resources and mechanisms, which is the foundation of the ecobiological approach, by combing UV filters and antioxidants to enhance their photoprotective effect. METHODS: First, the photoprotection properties of ectoine and mannitol association were characterized by the quantification of glutathione, reactive oxygen species, and double-stranded DNA breaks and by the epidermal Langerhans cells functionality. Second, the protection of squalene oxidation, catalase activity, and trans-urocanic acid (UCA) by the ectoine and mannitol association combined or not with SPF30 UV filters was assessed in vivo via non-invasive skin samplings in 10 subjects on irradiated areas. RESULTS: Using in vitro irradiated skin cell models, we demonstrated that this association significantly preserved intracellular glutathione levels, reduced DNA strand breaks induced by oxidative stress, and maintained Langerhans cell functionality. In vivo this association combined with UV filters presented significantly higher protection of three natural defense systems altered by UV compared to UV filters alone: squalene oxidation, catalase activity, and preservation of trans-UCA. CONCLUSION: This study demonstrates the ecobiological potential of combining UV filters with biological protection to increase skin photoprotection provided by specific active ingredients with antioxidative and immunosuppressive properties.

Impact of Leave-on Skin Care Products on the Preservation of Skin Microbiome: An Exploration of Ecobiological Approach.

Purpose: Skincare products are used daily to maintain a healthy skin, although their skin microbiome impact is still poorly known. Preserving the natural resources and mechanisms of the skin ecosystem is essential, and a novel approach based on these premises, called ecobiology, has recently emerged in skincare. We evaluated the impact on the skin microbiome of three types of leave-on face skincare products: a hydrophilic solution, a micellar solution, and an oil-in-water emulsion. Patients and Methods: Samples for microbial profiling were obtained from 20 Caucasian females twenty-four hours and four days following daily application of the skincare products and compared to an untreated area. The bacterial diversity and the abundance of the skin microbiome were analyzed by 16S rRNA gene sequencing using an Illumina MiSeq platform. Results: Our results confirmed the skin microbiome diversity and the prevalence of Cutibacterium spp. and Staphylococcus spp. at sebaceous sites. The bacterial diversity and abundance were not affected by the products, and no dissimilarities versus the control nor between each product were noted at both times. Conclusion: These preliminary results demonstrate for the first time that three types of leave-on face skincare products have no impact on the human skin microbiome and can be considered to be "microbiome friendly".

Inhibition of thymic stromal lymphopoietin production to improve pruritus and quality of life in infants and children with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory pruritic chronic dermatosis involving the alarmin thymic stromal lymphopoietin (TSLP), which is directly implicated in AD pruritus. AIMS: To evaluate the efficacy of Tambourissa trichophylla leaf extract (TTLE) titrated in polyphenols and 18ß-glycyrrhetinic acid (GA) in vitro and in vivo for AD pruritus. PATIENTS/METHODS: Initially, in vitro assessment of TSLP production in keratinocytes was undertaken. In normal human keratinocytes in vitro, TSLP was induced by polyinosinic:polycytidylic acid (Poly:IC), tumor necrosis factor (TNF)-α, and interleukin (IL)-4 and then quantified by ELISA in supernatants. Some cells were pretreated with TTLE and/or GA. Thereafter, an in vivo clinical study was performed including 48 infants and children with mild to severe AD flare-ups, some of which were treated with topical corticosteroids. A topical spray containing TTLE and GA was applied. After 21 days of topical spray application, pruritus, sleeplessness, the SCORing Atopic Dermatitis (SCORAD) index, the Infant's Dermatitis Quality of Life index (IDQOL), and the Dermatitis Family Impact Questionnaire (DFIQ) were assessed. RESULTS: Thymic stromal lymphopoietin secretion was inhibited significantly in an AD environment by TTLE and GA by up to 57.2% and 73.3%, respectively. The use of the topical spray induced a significant reduction in pruritus and sleeplessness scores, as well as the SCORAD, IDQOL, and DFIQ indexes in the total group. Similar results were observed in patient subgroups with or without topical corticosteroid treatment. CONCLUSIONS: A topical spray containing TTLE and GA, which inhibit TSLP secretion, efficiently decreases AD pruritus and improves the quality of life of AD patients.

Modulation of transepithelial electric resistance (TEER) in reconstructed human epidermis by excipients known to permeate intestinal tight junctions.

Several excipients are commonly used to enhance the drug absorption through simple epithelia of the digestive tract. They permeate the paracellular barrier constituted by tight junctions (TJs). We compared the effects of two excipients, sodium caprate (C10) and a self-emulsifying excipient Labrasol composed of a mixture of caprylocaproyl polyoxyl-8 glycerides, both applied to emerged reconstructed human epidermis either 'systemically', that is by addition to the culture medium, or topically. During the 'systemic' application, which produced cytoplasmic translocation of occludin and leakage of the biotin marker into the lower stratum corneum, the decrease in the trans-epithelial electrical resistance (TEER) was less abrupt with Labrasol when compared with C10, even though both excipients produced comparable final effects over time. With topical Labrasol, a significant TEER decrease was obtained with 5 times the 'systemic' concentrations. Topical application of C10 also resulted in the loss of the barrier function measured with TEER but had dramatic deleterious effects on the tissue morphology observed with light and electron microscopy. Our study demonstrates the potential value of Labrasol as an enhancer of bioavailability of molecules applied through the transcutaneous route. Our results suggest modulation of the epidermal TJs by both compounds. Even though the C10 action was at least partly due to overall cell damage and despite the fact that the decrease in TEER after topical application was apparently related to the permeabilization of the primary barrier of the stratum corneum in the first place.

Analysis of epidermal lipids in normal and atopic dogs, before and after administration of an oral omega-6/omega-3 fatty acid feed supplement. A pilot study.

Alterations of the lipid expression in the skin of human and canine atopic subjects may be one of the key factors in the disease development. We have analyzed the ultrastructure of the clinically uninvolved skin of atopic dogs and compared it with the lipid composition of their tape-stripped stratum corneum (SC). The effect of a 2 month treatment of atopic dogs by food supplementation with a mixture of essential fatty acids was evaluated on skin samples taken before and after the treatment period. Electron microscopy revealed that the non-lesional skin of atopic dogs exhibited an abnormal and largely incomplete structure of the lamellar lipids with little cohesion between the corneocyte strata. The SC of atopic dogs was characterized by a significant decrease in the lipid content when compared to the healthy controls. Following oral supplementation with the mixture of essential fatty acids, the overall lipid content of the SC markedly increased. This feature was observed both with the free and, most importantly, with the protein-bound lipids (cholesterol, fatty acids and ceramides), the latter constituting the corneocyte-bound scaffold for ordinate organisation of the extracellular lipid bi-layers. Indeed, the semi-quantitative electron microscopy study revealed that the treatment resulted in a significantly improved organization of the lamellar lipids in the lower SC, comparable to that of the healthy dogs. Our results indicate the potential interest of long-term alimentary supplementation with omega-6 and omega-3 essential fatty acids in canine atopic dermatitis.

Development and in vitro assay of oxidative stress modifying formulations for wound healing promotion.

Often presented as metabolism byproducts, reactive oxygen species are linked to detrimental effects such as chronic wound, mutagenesis, cancer and skin ageing. However, recent in vitro and in vivo observations suggest that ROS, and mainly hydrogen peroxide, interfere with cell signaling acting like second messenger and inducing adaptive responses. This is particularly observed in skin wound healing where cells are exposed to H2O2 following injury. In this study, we developed and characterized an innovative formulation producing H2O2 at low concentrations, in order to mimic physiological inflammation phase. Then, this pro-oxidative formulation (CAM-GOx) was assayed in vitro on keratinocytes cell culture, compared to the blank formulation (CAM) and the anti-oxidative formulation (CAM-CAT) to assess whether oxidative stress was implied or not in cellular responses.

Compartmentalization of the human stratum corneum by persistent tight junction-like structures.

Several tight junction (TJ) proteins were detected in the living layers of adult human epidermis, and TJ-like membrane ridges were observed at the top of the stratum granulosum (SG) in freeze-fracture studies. We applied standard and immunoelectron microscopy to look for TJ-derived structures in the stratum corneum (SC) of human adult epidermis and in cornified envelopes purified from the plantar SC. Besides confirming claudin-1 labelling in the proximity of SG desmosomes, we also observed immunolocalization near corneodesmosomes in the lower SC. In addition, TJ proteins were consistently detected in the purified cornified envelopes. Lateral but not horizontal walls of the corneocytes showed frequent points of molecular fusion between lipid envelopes. These structural associations were very frequently localized at the top of the lateral corneocyte membranes, thus sealing the extremities of lateral intercorneocyte spaces. We propose that TJ-like structures persist in the SC and contribute to the reinforcement of lateral contacts and to the formation of membrane interdigitations between corneocytes. Their presence could contribute to subdivision of the extracellular spaces of SC into consecutive individualized compartments. Intercellular lipids, enzymes and other (glyco)protein content could thus evolve in the keratinized epidermal layer at different paces, as preprogrammed in the underlying living cells and influenced by the environment, e.g. humidity. Such situation might explain differences in the degradation rates between the 'peripheral' and the 'non-peripheral' corneodesmosomes observed during physiological desquamation, as previously suggested by us and others.

Human epidermal desmosome-enriched tissue fractions for analytical and prospective studies.

Here, we report a method, adapted to the human epidermis, allowing isolation of desmosomes in small tissue fractions. The methods previously developed for animal skin did not work efficiently with human tissue. Enrichment of desmosomes was performed by the association of two incubation steps in acidic solutions containing detergent NP-40 at two different concentrations followed by a sonication step. The suspension was centrifuged twice: first to remove the heavy cell fragments and then at 16000 g on a discontinuous sucrose gradient. A desmosome-enriched fraction (DsF) was collected at the 30-50% sucrose interface. We demonstrate by immunoelectron microscopy and by western blotting that the central part of the desmosome structure is preserved as well as the antigenicity of its components. Our approach, allowing a significant enrichment of the cell fractions containing desmosomes, can be used to immunize animals and create new antibodies directed against desmosomal components. Using this strategy, new and so far poorly studied molecules incorporated into the desmosome cores could be targeted more easily.

Internalisation of hybrid titanium dioxide/para-amino benzoic acid nanoparticles in human dendritic cells did not induce toxicity and changes in their functions.

Nanoparticles (NPs) have been reported to penetrate into human skin through lesional skin or follicular structures. Therefore, their ability to interact with dendritic cell (DC) was investigated using DCs generated from monocytes (mono-DCs). Hybrid titanium dioxide/para-amino benzoic acid (TiO(2)/PABA) NPs did not induce any cell toxicity. NPs were internalised into DCs through macropinocytosis and not by a receptor-mediated mechanism. Confocal microscopy showed that NPs were not detected in the nucleus. These data are confirmed by electronic microscopy which demonstrated that hybrid NPs were rapidly in contact with cellular membrane and localised into cytoplasmic vesicles without colocalisation with clathrin-coated vesicles. Hybrid NPs did not induce CD86 or HLA-DR overexpression or cytokine secretion (IL-8 and TNF-α) indicating no DC activation. Internalisation of hybrid NPs did not modify DC response towards sensitisers such as nickel and thimerosal or LPS used as positive controls. Moreover, hybrid NPs did not induce any oxidative stress implicated in DC activation process. After mono-DC irradiation by ultraviolet A (UVA), hybrid NP-treated cells did not produce UVA-induced reactive oxygen species (ROS) and exhibited a better cell viability compared with UVA-irradiated control cells, suggesting a protecting effect of hybrid TiO(2)/PABA NPs against UVA-induced ROS.