Marek Haftek

Effects of a Topically Applied Preparation of Epidermal Lipids on the Stratum Corneum Barrier of Atopic Dogs

Canine atopic dermatitis (AD) is characterized ultrastructurally by disorganization of the lamellar lipids (LLs) in the stratum corneum (SC), similar to that seen in the human disease. This study, based on the examination of biopsy samples, was designed to investigate the expression of canine epidermal lipids and to evaluate quantitatively, by means of electron microscopy and ruthenium tetroxide post-fixation, the effect of a new topical skin lipid complex (SLC) on the structural deficit in the skin of five dogs with AD. The non-lesional skin of atopic dogs differed from the skin of healthy dogs in that the LLs were reduced in number and highly disorganized. After repeated applications of SLC to the non-lesional skin of dogs with AD, numerous LLs were observed in the deepest part of the SC, occupying 74% of the inter-corneocyte space, while they accounted for only 31.8% of the inter-corneocyte space in comparable biopsy samples from untreated (control) skin of the same dogs. In contrast, the LLs filled 89.5% of the deepest inter-corneocyte spaces in the SC of healthy dogs. Many keratinosomes were observed at the interface between living epidermis and SC after treatment of non-lesional AD skin. Stacks of short LL discs represented 57.6% of the total LLs found in the newly formed SC compactum in the treated atopic dogs. It is suggested that the treatment with SLC stimulated the production and secretion of endogenous SC lipids, contributing to the formation of an improved epidermal barrier.

PAR2 absence completely rescues inflammation and ichthyosis caused by altered CAP1/Prss8 expression in mouse skin

Altered serine protease activity is associated with skin disorders in humans and in mice. The serine protease channel-activating protease-1 (CAP1; also termed protease serine S1 family member 8 (Prss8)) is important for epidermal homeostasis and is thus indispensable for postnatal survival in mice, but its roles and effectors in skin pathology are poorly defined. In this paper, we report that transgenic expression in mouse skin of either CAP1/Prss8 (K14-CAP1/Prss8) or protease-activated receptor-2 (PAR2; Grhl3PAR2/+), one candidate downstream target, causes epidermal hyperplasia, ichthyosis and itching. K14-CAP1/Prss8 ectopic expression impairs epidermal barrier function and causes skin inflammation characterized by an increase in thymic stromal lymphopoietin levels and immune cell infiltrations. Strikingly, both gross and functional K14-CAP1/Prss8-induced phenotypes are completely negated when superimposed on a PAR2-null background, establishing PAR2 as a pivotal mediator of pathogenesis. Our data provide genetic evidence for PAR2 as a downstream effector of CAP1/Prss8 in a signalling cascade that may provide novel therapeutic targets for ichthyoses, pruritus and inflammatory skin diseases.

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

Abstract:u2002 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.

CD44 regulates tight-junction assembly and barrier function.

Upon barrier disturbance, adult CD44 knockout (KO) mice show delayed recovery of epidermal barrier function. This correlates with the loss of apical polarization of lamellar body (LB) secretion. As tight junctions (TJs) are crucial for barrier function and regulate polarized targeting of vesicles, we hypothesized that CD44 regulates TJs and associated cell polarity complexes, which in turn contributes to altered skin barrier function in CD44 KO mice. We show a delay in embryonic barrier formation associated with a loss of apical LB localization in CD44 KO mice, which correlates with alterations in TJ proteins and Par3. Simultaneously, the activity of Rac1, a major regulator of TJ barrier function, was reduced. Importantly, normalization of barrier function at E18.5 coincided with the recovery of these proteins. Tape-stripping experiments revealed that the loss of CD44 also affected TJ proteins upon induced disturbance of the barrier in adult mice. In CD44 KO keratinocytes, cell polarization and TJ barrier function were impaired. An alteration of differentiation markers was also observed, but was less pronounced than alterations of TJ proteins. Taken together, the results reveal an important function for CD44 in the assembly and function of TJs, suggesting their involvement in the skin barrier phenotype of CD44 KO mice.

Characterization of early events involved in human dendritic cell maturation induced by sensitizers : Cross talk between MAPK signalling pathways

Dendritic cells (DCs), efficient-antigen presenting cells play an important role in initiating and regulating immune responses. DC maturation following exposure to nickel or DNCB induced an up-regulation of phenotypic markers and inflammatory cytokine secretion. Early intracellular mechanisms involved in DC maturation required to be precise. To address this purpose, DCs derived from human monocytes were treated with sensitizers (nickel, DNCB or thimerosal) in comparison with an irritant (SDS). Our data confirming the up-regulation of CD86, CD54 and cytokine secretion (IL-8 and TNFalpha) induced by sensitizers but not by SDS, signalling transduction involved in DC maturation was investigated using these chemicals. Kinase activity measurement was assessed using two new sensitive procedures (Facetrade mark and CBA) requiring few cells. SDS did not induce changes in signalling pathways whereas NiSO(4), DNCB and thimerosal markedly activated p38 MAPK and JNK, in contrast Erk1/2 phosphorylation was completely inhibited by DNCB or thimerosal and only activated by nickel. A pre-treatment with p38 MAPK inhibitor (SB203580) suppressed Erk1/2 inhibition induced by DNCB or thimerosal demonstrating a direct interaction between p38 MAPK and Erk1/2. A pre-treatment with an antioxidant, N-acetyl-L-cysteine (NAC) markedly reduced Erk1/2 inhibition and p38 MAPK phosphorylation induced by DNCB and thimerosal, suggesting a direct activation of p38 MAPK via an oxidative stress and a regulation of MAPK signalling pathways depending on chemicals. Because of a high sensitivity of kinase activity measurements, these procedures will be suitable for weak or moderate sensitizer screening.

Clinical, biometric and structural evaluation of the long‐term effects of a topical treatment with ascorbic acid and madecassoside in photoaged human skin

Abstract:u2002 Skin ageing is a complex process determined by the genetic endowment of individual and environmental factors, such as sun exposure. The effects of skin ageing are mostly encountered in the superficial dermis and in the epidermis. We have previously demonstrated in vivo the beneficial effect of a topically applied formula of 5% vitamin C in the treatment of skin ageing. Another active compound, madecassoside extracted from Centella asiatica, known to induce collagen expression and/or to modulate inflammatory mediators, might thus prevent and correct some signs of ageing. A randomized double‐blind study was carried out on photoaged skin of 20 female volunteers to investigate the effects of topically applied 5% vitamin C and 0.1% madecassoside on the clinical, biophysical and structural skin properties. After 6u2003months of treatment, we observed a significant improvement of the clinical score for deep and superficial wrinkles, suppleness, firmness, roughness and skin hydration. These results were corroborated by measurements of skin elasticity and semi‐quantitative histological assessment of the elastic fibre network in the papillary dermis. Two‐thirds of the subjects showed an improvement. The re‐appearance of a normally structured elastic fibre network was observed. Our results revealed a functional and structural remodelling of chronically sun‐damaged skin.

The lipid alterations in the stratum corneum of dogs with atopic dermatitis are alleviated by topical application of a sphingolipid-containing emulsion

Background.u2002 Atopic dermatitis (AD) results from an altered skin barrier associated with defects in the lipid composition of the skin. Dogs with AD present similar clinical symptoms to humans, and may be a useful model for investigations into AD.

Filaggrin breakdown products determine corneocyte conformation in patients with atopic dermatitis.

Background Loss-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. Objective We sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD. Methods We 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. Results We 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. Conclusions NMF 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.

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 2xa0month 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.

Atopic dermatitis in dogs is associated with a high heterogeneity in the distribution of protein-bound lipids within the stratum corneum

The stratum corneum (SC) was taken from five atopic dogs by tape stripping (12 strips) of non-lesional areas of the abdomen. The free and protein-bound lipids were extracted and analyzed by thin-layer chromatography after fractionation on aminopropyl-bonded silica gel columns. A very frequent feature was the heterogeneity in the lipid content of consecutive layers. This was even more accentuated for the covalently bound lipids, with variations from one layer to another in the concentrations of cholesterol, omega hydroxylated ceramides and omega hydroxylated long-chain fatty acids. Among the free lipids, large amounts of glucosylceramides were present in canine atopic SC although they are nearly absent from the SC of normal dogs. A heterogeneous distribution of lipids was seen in canine atopic SC. These results suggest that strikingly deep variations occur in the lipid metabolism of keratinocytes in the skin of atopic dogs. In order to gain insight into this phenomenon, further studies should be focused on the activity of enzymes involved in both biosynthetic and catabolic processes.