BackgroundLoss-of-function (LOF) mutations in the filaggrin gene (FLG) are a well-replicated risk factor for atopic dermatitis (AD) and are known to cause an epidermal barrier defect. The nature of this barrier defect is not fully understood. Patients with AD with FLG LOF mutations are known to have more persistent disease, more severe disease, and greater risk of food allergies and eczema herpeticum. Abnormalities in corneocyte morphology have been observed in patients with AD, including prominent villus-like projections (VP); however, these ultrastructural features have not been systematically studied in patients with AD in relation to FLG genotype and acute and convalescent status.ObjectiveWe sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD.MethodsWe studied 15 children at first presentation of AD and after 6 weeks of standard therapy. We applied atomic force microscopy to study corneocyte conformation in patients with AD stratified by FLG status and NMF level. By using a new quantitative methodology, the number of VPs per investigated corneocyte area was assessed and expressed as the Dermal Texture Index score. Corneocytes were also labeled with an anti-corneodesmosin antibody and visualized with scanning electron microscopy.ResultsWe found a strong correlation between NMF levels and Dermal Texture Index scores in both acute and convalescent states (respective r = −0.80 and −0.75, P < .001 and P = .002). Most, but not all, VPs showed the presence of corneodesmosin abundantly all over the cell surface in homozygous/compound heterozygous FLG patients and, to a lesser extent, in heterozygous and wild-type patients.ConclusionsNMF levels are highly correlated with corneocyte morphology in patients with AD. These corneocyte conformational changes shed further insight into the filaggrin-deficient phenotype and help explain the barrier defect in patients with AD with FLG LOF mutations.
(1) Background: Skin undergoes constant changes, providing capabilities to repair and renovate its constituents once damaged and a fundamental shield to contrast environmental stress. Nevertheless, environmental stressors may overcome the skin’s protective potential inducing premature aging and accelerating the appearance of anaesthetic age-related skin aspects. Ultraviolet radiation (UVR) and pollutants (particulate matters, PAHs) contribute to skin aging and functional decline inducing harmful oxidative modifications of macromolecules and stress-related skin disorders. Innovative approaches to preserve skin are needed. (2) Methods: Skin keratinocytes were treated (or not) with a combination of ingredients (Lactobacillus plantarum extract, Withania somnifera root extract and Terminalia ferdinandiana fruit extract; “MIX”) in the presence or absence of stress (oxidative stress or pollution). The effects of the MIX adaptogen technology on (a) cellular resilience, (b) the regulation of cellular functions and (c) regeneration of skin were disclosed through expression proteomics and bioinformatics analyses first, and then through focused evaluations of protein carbonylation as a hallmark of oxidative stress’ deleterious impact and mitochondrial activity. (3) Results: The deleterious impact of stressors was evidenced, as well as the beneficial effects of the MIX through (a) mitochondrial activity preservation, (b) the “vigilance” of the NRF2 pathway activation, (c) NADPH production and protein homeostasis improvements, (d) preserving skin regeneration function and I the contrasting stress-induced oxidation (carbonylation) of mitochondrial and nuclear proteins. (4) Conclusions: The effects of the MIX on increasing cell adaptability and resilience under stress suggested a beneficial contribution in precision cosmetics and healthy human skin by acting as an adaptogen, an innovative approach that may be employed to improve resistance to harmful stress with a potential favourable impact on skin homeostasis.
To the editor Ex vivo and in vitro skin permeation tests are extensively used to evaluate transcutaneous penetration of drugs. However, surprisingly little attention is paid to the tissue morphology during such studies. We want to strike a cautionary note, important for the interpretation of experiments predicting transcutaneous drug absorption in vivo. Transcutaneous diffusion devices, called Franz cells, are most frequently used. Excised skin is positioned between the 'donor' and 'receiver' compartments of the device. The studied molecule applied to the 'donor' chamber must cross the skin barrier to be subsequently quantified in the 'receiver' fluid. Because of its availability in large quantities and its similarity to human skin, pig skin is frequently used for the tests 1. In accordance with the international recommendations, skin samples are stored at −20°C and thawed before experiments (S1). However, contradictory reports exist in the literature concerning the validity of the procedure 2, 3. Most of the studies focus on the permeation results alone (S2, S3). Results of the functional assays demonstrated that permeation coefficient of one or four times frozen skin increased 4 and 5 fold, respectively, compared to the fresh skin (Fig. 1). Ruthenium tetroxide postfixation of specimens was used to explore ultrastructure of the four differently prepared skin substrates (n = 5, per group). This method visualized the inter-corneocyte lipids and their lamellar organization in intact SC (Fig. 2a). Interestingly, strongly stained zones within the lipid matrix were noticed. They correspond to the already described hydrophilic lacunae that participate in skin hydration and potentially contain enzymes and their natural inhibitors, essential for the regulation of epidermal desquamation 4. After freezing/thawing cycles, these hydrophilic poaches were found enlarged in all the specimens examined, creating holes within the SC lipid matrix. However, the lamellar organization of intercellular lipids was preserved (Fig. 2b). Freezing, and the related ice crystal formation, harmed the ultrastructure of all water-containing elements within the SC. Void spaces were thus observed within the keratin matrix of corneocytes and, to a lesser extent, in corneodesmosomes (Fig. 2c). The secondary barrier of epidermal tight junctions (TJ) was evaluated using lanthanum infiltration assay. In untreated skin, lanthanum salts added to biopsy fragments during their postfixation with osmium tetroxide were stopped at the stratum granulosum (SG) level, where functional TJ are present 5 (Fig. 2d). However, in frozen skin, lanthanum was detected dispersed between the lower SC cells, with no accumulation between the SG keratinocytes, indicating the complete loss of TJ functionality (Fig. 2e). Because cornified cell envelopes are impermeable to most diffusing substances, the main penetration route through the SC remains the intercellular one 6. Elimination of the majority of intercellular lipids by solvents resulted in enlargement of the intercorneocyte spaces (Fig. 2f). We propose that morphological changes within the extracellular space of the SC caused by freezing are responsible for the increase of the skin permeability to hydrophilic and amphiphilic molecules. Our findings confirm the suggestion that cracks in the lipid bilayers and degradation of SC cohesion could increase the permeation of hydrophilic molecules 7. In the case of hydrophobic compounds, we suppose that the permeation fluxes observed in fresh and frozen skin should be less divergent. In fact, lipophilic molecules would not pass thought the enlarged hydrophilic pathway caused by freezing but by the preserved multilayered lipids that do not retain water (8, S7). Whether partitioning of lipophilic molecules from the SC to the underlying living epidermal layers could be influenced by freezing due to the disorganization of the TJ barrier remains to be investigated. Ex vivo permeation tests performed on frozen porcine skin are useful for development of drugs and their galenic formulations 9. However, data obtained with frozen skin substrates are not directly transposable to the in vivo situation because skin freezing for storage is detrimental to the quality of the skin barrier. Conservation of skin substrates appears to be an important factor influencing results of permeation, and it should be taken into account during predictive studies on percutaneous drug absorption. The authors state no conflict of interest. RA, LR, MH, designed the study and wrote the manuscript; NZ – an ERASMUS student from Ghent University – performed the experiments with LR, RA, EG and MH; FP and EG contributed to analysis and discussion of results. Data S1. Materials and methods. Data S2. Supplementary References. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
As odd as it may seem at first glance, minerals, it is what we are all about…or nearly. Although life on Earth is carbon-based, several other elements present in the planet’s crust are involved in and often indispensable for functioning of living organisms. Many ions are essential, and others show supportive and accessory qualities. They are operative in the skin, supporting specific processes related to the particular situation of this organ at the interface with the environment. Skin bioenergetics, redox balance, epidermal barrier function, and dermal remodeling are amongst crucial activities guided by or taking advantage of mineral elements. Skin regenerative processes and skin ageing can be positively impacted by adequate accessibility, distribution, and balance of inorganic ions.
