Analysis of epidermal genes, proteins and lipids is important in the research and diagnosis of skin diseases. Although punch biopsy is the first-choice technique for the skin sampling, it is unnecessarily invasive for obtaining a sample just for the epidermal analysis. Here we compare two less invasive methods, suction blistering (SB) and tape stripping (TS), for the analysis of selected epidermal genes (quantitative real-time reverse transcription PCR, qRT-PCR), proteins (western blotting, WB), and lipids in ten healthy volunteers. TS provided significantly less material than SB and no viable epidermal layers could be obtained according to the reflectance confocal microscopy. Consistently, only the SC protein filaggrin and housekeeping GAPDH together with FLG and RPL13A mRNA were detected by TS. In the SB samples, WB and qRT-PCR could easily detect all the selected proteins (claudin-1, occludin, filaggrin, laminin and GAPDH) and genes (CLDN1, OCLN, FLG, LAMA3 and RPL13A), respectively. A single SB sample further provided enough of material for immunohistochemistry and lipid analyses, which was not feasible with the TS samples. Immunohistochemistry of the SB samples showed intact epidermal structure and a characteristic expression of claudin-1. Infrared spectroscopy showed well-ordered lipids with both orthorhombic and hexagonal packing and high-performance thin layer chromatography confirmed all lipid classes (including ceramide subclasses) in correct proportions. Taken together, SB represents a reliable sampling technique that can be utilized for multipurpose epidermal analyses in various studies.

Irritant damage to the permeability barrier elicits a cascade of responses that could be investigated by minimally invasive in vivo methods providing different information on biochemical and functional levels. Recent studies have shown that parallel to impairment of the barrier function and reduction of the natural moisturising factor (NMF), irritants such as the anionic detergent sodium lauryl sulfate (SLS) influence the corneocyte surface topography, investigated by atomic force microscopy and expressed by the Dermal Texture Index (DTI). To extend these findings, we investigated the early and late effects of different water soluble irritants on the barrier function, DTI, NMF and primary cytokine levels in healthy volunteers exposed to 60% N-propanol, 0.5% SLS, 0.15% sodium hydroxide, 2.0% acetic acid and occlusion with water in a controlled tandem repeated irritation test (TRIT) over 96 h. The irritant response was assessed by measuring erythema, transepidermal water loss (TEWL) and capacitance at baseline, 24 h and 96 h later; the cytokine levels, NMF and DTI were assessed in tape strips, collected at the same time points from the irritant-exposed and non-exposed sites. The magnitude of effects exerted by the irritants on the barrier integrity and properties, primary cytokine levels and DTI, found in the study, differed significantly, based on the chemical characteristics. The changes in DTI correlated with the NMF levels while being less influenced by inflammation. Though the observed differences may be influenced by the applied irritant concentration, our results confirm the need for a multi-parametric approach in the characterization of the irritant-specific barrier response and identification of sensitive outcome parameters for studying skin irritation in vivo.

Filaggrin (FLG) loss-of-function mutations and the T helper 2 (Th2) dominated cytokine milieu are assumed to cause an impaired skin barrier function in atopic dermatitis (AD), but this presumed mechanism is still largely hypothetical. Previous studies have used in vitro skin equivalents to provide experimental evidence for the role of FLG deficiency but different experimental setups and incomplete knockdown approaches make these data difficult to compare and interpret. Using 3D epidermal equivalents, we here addressed the question if FLG deficiency alters skin barrier function. We excluded interplay of FLG mutations with other AD-typical concomitant factors like inflammation or altered microbiome. We therefore used keratinocytes of ichthyosis vulgaris patients that innately carry homozygous FLG null mutations. This approach uses genetically defined cells without detectable filaggrin protein and avoids potential off-target effects of knockdown approaches. FLG did not alter constitutive or Th2-cytokine dependent expression of differentiation-associated proteins. We observed a decrease of tight junction protein expression, which, however, did not lead to alteration of the outside-in nor did it affect inside-out stratum corneum barrier as measured by permeability for low molecular weight tracers (Lucifer Yellow or biotin-SH). Although these findings do not completely rule out alterations in epidermal permeability for molecules with other biophysical properties, our study contradicts previous work that suggests an increased permeability for low molecular weight polar solutes in FLG deficient epidermis.

L-carnitine is required for transport of long chain fatty acids to the mitochondria. Therefore, L-carnitine is an important component of cell energy metabolism and its defi ciency or imbalance causes functional diseases in organism. We aimed to investigate the role of L-carnitine and its transporter SLC22A5 (Solute Carrier Family 22 Member 5) in energy metabolism of skin cells and its connection to skin ageing. Results:Potential marker of skin ageing: Microarray detected 2.57-fold decrease in SLC22A5 gene expression in the old keratinocytes compared to the young. No signifi cant change of SLC22A5 expression was found in old fi broblasts compare to the young. The results were confi rmed by qRT-PCR.Eff ect of L-carnitine treatment on skin cells in vitro: In vitro, signifi cant increase of ATP level was observed in dermal fi broblasts (NHDF) treated by L-carnitine (Fig. 1). Keratinocyte cell line showed also elevation of ATP, however not signifi cant. Increase of ATP refers to support of cell energy metabolism.