表皮角质层屏障功能与特应性皮炎相关研究进展

 皮肤持续暴露于外界病原体,其屏障功能对皮肤的稳态至关重要。既往研究表明,屏障功能的紊乱和缺陷是特应性皮炎重要的发病机制之一。本文从角质层屏障以下几个方面进行概述：①丝聚蛋白减少;②脂质改变;③角质化包膜形成异常;④角质细胞的脱落失衡;⑤皮肤微生物区的变化,总结了表皮角质层是如何形成的,并回顾了它与特应性皮炎病理机制的联系。     

Estimation of the relative stratum corneum amount removed by tape stripping

BACKGROUND/PURPOSE: The tape stripping procedure is a suitable minimal invasive tool to study, e.g. the penetration and dermatopharmacokinetics of topically applied substances. In the present study, this procedure was used to remove the stratum corneum (SC) completely and to study the penetration of the UVA filter substance butyl methoxydibenzoylmethane after application in two different vehicles. METHODS: The amount of corneocytes removed by each tape strip from the flexor forearm of human volunteers was determined via their pseudo-absorption. In a second part, the penetration profiles of a UVA filter substance applied in two different vehicles were determined following the developed standard protocol using the tape stripping procedure in combination with UV/VIS spectroscopy. RESULTS: The amount of corneocytes removed by each tape strip was related to the number of tape strips used for removal. Mean values with a deviation of less than 20% concerning the relative amount of SC removed by a constant number of tape strips were obtained. For instance, a relative amount of 66 +/- 12% was removed with the first 20 tape strips, while nearly the complete SC (95 +/- 3%) was removed using 50 tape strips. In addition, these results were used to estimate the relative SC amounts removed, studying the penetration of the UVA filter substance after application in two different vehicles. No significant differences between the distributions of the UV filter substance applied in both emulsions were obtained (P > 0.05). CONCLUSIONS: The reported procedure for the estimation of the removed SC amount provides the possibility to avoid the complete removal of the SC and to compare the penetration characteristics obtained for different volunteers and different products in relation to the relative horny layer profile.     

Physical and physiological effects of stratum corneum tape stripping

Background/aims: Tape stripping of human stratum corneum has been performed to measure stratum corneum mass, barrier function, drug reservoir and percutaneous penetration. However, the technique itself requires further development to facilitate interpretation.
Methods: In this study we quantified stratum corneum (SC) tape stripping and water kinetic parameters utilizing three types of adhesive tapes, in an in vivo randomized clinical trial. Stratum corneum was tape stripped, and the mass of SC removed by each tape was quantified utilizing a protein assay. Transepidermal water loss (TEWL) was measured and barrier disruption and SC water kinetics calculated. Three commonly utilized acrylate adhesive tapes were utilized and a comparison made between them.
Results: Each type of tape successfully stripped the stratum corneum, but the rayon tape did not induce SC barrier disruption. Neither the type of tape nor the site stripped significantly influenced the mass of SC removed. Water kinetic parameters did not differ significantly for the tapes that did induce barrier disruption. Individual variation in barrier disruption to water following tape stripping was demonstrated.
Conclusion: The tapes utilized removed a similar amount of SC. The tapes have a different propensity to cause barrier disruption. Some individuals do not demonstrate increased TEWL despite an equivalent mass of SC being removed compared to those who do show a response.     

Effect of the vehicle on the amount of stratum corneum removed by tape stripping

Background: The penetration of topically applied substances into the stratum corneum can non‐invasively be studied using the tape stripping procedure. This method was applied to investigate in vivo the penetration of a fragrance, vanillin, applied in ethanol and a w/o emulsion. Methods: Twenty tape strips were removed from each skin area treated with vanillin in ethanol or w/o emulsion, respectively. The concentration of vanillin was determined for each tape strip. In addition, the pseudo‐absorption of the corneocytes was determined to calculate the SC profile. Results: The vanillin concentration was correlated both with the tape number and with the stratum corneum profile. Depending on whether the tape number or the profile of the stratum corneum were correlated with the vanillin concentration, different distributions within the stratum corneum were obtained. Different amounts of stratum corneum were removed with 20 tape strips dependent on the vehicle applied previously. The application of the w/o emulsion led to the removal of nearly the half the amount of corneocytes stripped from the ethanol‐treated area. Conclusions: The results obtained underline the general necessity to correlate the amount of stratum corneum with the amount of substance in penetration studies.     

Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography

Abstract Intercellular lipids in the stratum corneum (SC) are responsible for the barrier function of mammalian skin. The main components of the SC lipids are ceramides, cholesterol, and free fatty acids, as established by thin-layer chromatographic analysis of lipids extracted from the human and mammalian SC. Up to now, for lipid analysis the extracts of the entire SC has been used and information on whether the lipid composition changes with the depth in the SC is scarce. Tape stripping is a technique which removes corneocyte layers step by step with an adhesive film. The use of this technique for lipid analysis was hampered by the contamination of lipid extracts with compounds co-extracted from the tape with organic solvents used for the extraction of SC lipids. The aim of the present study was to establish a suitable analytical method for the determination of the local SC lipid composition. For this purpose, the SC samples were collected by sequential stripping with Leukoplex tape in five healthy volunteers. The lipids were extracted with ethyl acetate:methanol mixture (20:80) and separated by means of HPTLC. The results of this study revealed that the free fatty acid level is highest and the cholesterol and ceramide levels lowest in the uppermost SC layers (about 4 strippings). The levels remained unchanged in the underlying SC layers. In these layers, the ceramide level was about 60 wt% and the free fatty acid and cholesterol levels were about 20 wt% each. Ceramides could be separated into seven different fractions and the relative amounts of individual ceramide fractions did not significantly change with the SC depth. Cholesterol sulfate levels were about 5% of total cholesterol and did not change with the SC depth, except for the for the first strip where the level was about 1%. The method developed makes it possible to study the differences in the SC lipid profile in healthy and diseased human skin with relation to the SC lipid organization and to the skin barrier function in vivo. Received: 2 August 2000 / Revised: 20 December 2000 / Accepted: 26 January 2001     

Aberrant lipid organization in stratum corneum of patients with atopic dermatitis and lamellar ichthyosis

There are several skin diseases in which the lipid composition in the intercellular matrix of the stratum corneum is different from that of healthy human skin. It has been shown that patients suffering from atopic dermatitis have a reduced ceramide content in the stratum corneum, whereas in the stratum corneum of lamellar ichthyosis patients, the amount of free fatty acids is decreased and the ceramide profile is altered. Both patient groups also show elevated levels of transepidermal water loss indicative of an impaired barrier function. As ceramides and free fatty acids are essential for a proper barrier function, we hypothesized that changes in the composition of these lipids would be reflected in the lipid organization in stratum corneum of atopic dermatitis and lamellar ichthyosis patients. We investigated the lateral lipid packing using electron diffraction and the lamellar organization using freeze fracture electron microscopy. In atopic dermatitis stratum corneum, we found that, in comparison with healthy stratum corneum, the presence of the hexagonal lattice (gel phase) is increased with respect to the orthorhombic packing (crystalline phase). In lamellar ichthyosis stratum corneum, the hexagonal packing was predominantly present, whereas the orthorhombic packing was observed only occasionally. This is in good agreement with studies on stratum corneum lipid models that show that the presence of long-chain free fatty acids is involved in the formation of the orthorhombic packing. The results of this study also suggest that the ceramide composition is important for the lateral lipid packing. Finally, using freeze fracture electron microscopy, changes in the lamellar organization in stratum corneum of both patient groups could be observed.     

Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients

Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1-KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation-related proteases. We report quantification by enzyme-linked immunosorbent assay (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of atopic dermatitis (AD) patients by ELISA, and examine their variation with clinical phenotype, correlation with blood levels of eosinophils, lactate dehydrogenase (LDH) and immunoglobulin E. The overall SC serine protease activities were also measured. In the SC of AD, all KLKs, except KLK11, were significantly elevated. The elevation of chymotrypsin-like KLK7 was predominant, compared with trypsin-like KLKs. The SC overall plasmin- and furin-like activities were significantly elevated, while trypsin- and chymotrypsin-like activities did not differ significantly. In the serum of AD patients, KLK8 was significantly elevated and KLK5 and KLK11 were significantly decreased. However, their serum levels were not modified by corticosteroid topical agents. The alterations of KLK levels in the SC of AD were more pronounced than those in the serum. KLK7 in the serum was significantly correlated with eosinophil counts in the blood of AD patients, while KLK5, KLK8 and KLK11 were significantly correlated with LDH in the serum. In conclusion, we report abnormal kallikrein levels in the SC and the serum of AD patients. KLKs might be involved in skin manifestation and/or focal/systemic inflammatory reactions in AD. Our data may contribute to a better understanding of the pathogenesis of AD.     

Increased stratum corneum turnover induced by subclinical irritant dermatitis

The chronic effects of the irritant sodium lauryl sulphate (SLS) on stratum corneum (SC) barrier function, determined by transepidermal water loss (TEWL) measurements and on epidermal cell kinetics, estimated by stratum corneum turnover time (SCTT) determination (dansyl chloride staining method), were investigated in 18 healthy female volunteers. SLS (7.5%) was applied without occlusion for 20 min once daily, over a period of 3 weeks (5 days a week) on dansyl chloride-stained skin and on untreated skin. SCTT of untreated skin (19.3 +/- 0.8 days; mean +/- SEM) was not changed by daily treatment with water (control) (19.3 +/- 2.0) but was significantly reduced by SLS (10.9 +/- 0.6; P less than or equal to 0.0001; compared to controls). However, TEWL was increased in SLS-treated sites 1.5-fold after 4 days of treatment (5.3 +/- 0.6 vs. 3.5 +/- 0.3; P less than 0.001). At the end of the second week, TEWL was increased 2.6-fold and after 3 weeks TEWL was 3.3 times higher than in controls 13.0 +/- 1.6 vs. 3.9, P less than or equal to 0.0001). The intensity of SLS-induced irritation as measured by TEWL was significantly correlated with baseline TEWL (r = 0.50; P less than or equal to 0.02) and significantly negatively correlated with SCTT of SLS treated sites (r = -50; P less than or equal to 0.02) but not with SCTT of untreated skin (r = 0.19).     

Analysis of free and protein-bound ceramides by tape stripping of stratum corneum from dogs

The free and protein-bound ceramides of dog stratum corneum (SC) were analyzed by thin-layer chromatography after tape stripping of the abdomen of five dogs. The sphingoid bases were identified by gas–liquid chromatography as sphingosine, phytosphingosine, and 6-hydroxysphingosine. Electrospray ionization-ion trap mass spectrometry was used to characterize the protein-bound ceramides containing sphingosine and omega-hydroxy long-chain fatty acids. Although the molecular species were the same ones in all dogs, wide quantitative variations in the patterns of SC ceramides were observed in different breeds of dogs. The free ceramide concentration changed with the depth of SC, with a higher concentration in the deep layers, whereas the concentration of protein-bound ceramides remained constant. These results show that canine SC is close to that of humans with respect to ceramides.     

Skin barrier function in patients with completely healed atopic dermatitis

Although it has been well established that the dry skin often seen in patients with atopic dermatitis shows a deranged barrier function, there is no unanimity of opinion as to whether the barrier in normal-appearing skin of patients with the disease is deranged or not. Hence, it remains unclear whether individuals with atopic dermatitis constitution have an intrinsic derangement of skin barrier function or not. To settle this problem, in the present study we examined transepidermal water loss and stratum corneum water content in normal appearing skin of the upper back of 16 patients with completely healed atopic dermatitis who had been free from skin symptoms for 5 years or more, 30 patients with active atopic dermatitis, and 39 healthy subjects. The transepidermal water loss values and the stratum corneum water content values in normal-appearing skin of the completely healed patients were not different from the values in normal controls. These findings indicate that skin barrier function is not disturbed in patients with completely healed atopic dermatitis.     

Examination of stratum corneum barrier function in vivo by infrared spectroscopy

It is generally accepted that the stratum corneum (SC) is the least permeable layer of the epidermis. Histologically, though, the SC is a non-uniform, inhomogeneous membrane, and the question "Is barrier function distributed uniformly across the SC thickness?" has been posed. To address this issue, human ventral forearm SC has been studied in vivo by attenuated-total-reflectance Fourier-transform infrared spectroscopy during the course of sequential tape-stripping. Because the intercellular lipids of the SC and the degree of hydration of the membrane have been shown to be crucial determinants of barrier function, attention has been focused on the spectral features, which report specifically on these parameters. The degree of disorder of the SC intercellular lipids has been found to decrease over the outer cell layers (up to three tape-strips) and then to remain essentially constant. The amount of lipids decreases similarly such that a 60% reduction (relative to the "no-strip" baseline) is observed after about four tape-strips. A plausible explanation for these measurements is that the lipids near the surface are a mixture of (a) "true" intercellular lipid (which is expected to be highly ordered), and (b) sebaceous lipid (which contains much greater amounts of low-melting components, such as fatty acids). The sequential infrared (IR) spectra provide at least circumstantial evidence to support this hypothesis. As expected, the IR spectra show that SC hydration increases from the surface towards the SC-stratum granulosum interface. Taken together, the results imply that the SC is indeed non-uniform. The properties of the outer layers (those removed by the first 3-4 tape-strips) change significantly with increasing depth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioavailability of clobetasol propionate-quantification of drug concentrations in the stratum corneum by dermatopharmacokinetics using tape stripping.

The concentration of clobetasol propionate in the stratum corneum after application of three different formulations was determined, quantifying the influence of the formulations on the bioavailability of the drug. The stratum corneum was sampled by tape stripping. The concentrations of clobetasol propionate were determined quantitatively by HPLC. After application of Clobetasol Propionate Cream USP, 0.05%, and Temovate Cream, 0.05%, identical amounts of the drug were found in the stratum corneum, whereas after application of Temovate epsilon Emollient, 0.05%, the quantity was clearly decreased. From results obtained measuring the drug concentration in the adjacent sites of the skin where the creams had not been applied, it became clear that clobetasol propionate in Temovate epsilon Emollient, migrated to a large extent in the lateral direction. This explains the lower concentration measured for this formulation in the skin areas where the cream had been applied. In general, a lateral distribution of the applied drug must be taken into account when positioning the application areas on the forearm.     

Quantification of stratum corneum removal by adhesive tape stripping by total protein assay in 96-well microplates

BACKGROUND/AIMS: Examination of stratum corneum (SC) content with tape stripping and a colorimetric method is increasingly used. We examined the possible use of microplates in tandem with a colorimetric method to examine SC removed with tape stripping. As a corollary to this examination, the homogeneity of tape strips was examined. METHOD: The commonly used Lowry assay was adapted for 96-well plates. Tapes were divided into four regions and sample disks of 5 mm diameter were taken from each and analyzed for SC mass using the adapted Lowry assay. RESULTS: Homogeneity of SC removal over different areas across a tape strip is limited. CONCLUSION: Quantification of SC by means of a 96-well microplate-based colorimetric method is feasible and shortens the time of analysis. However, when using D-Squame tape disks, SC removal on a limited area of the tape is not predictive for SC removal on the entire tape as removal is inhomogenous. Therefore, SC protein extraction should be performed on a large enough area, eventually on the entire tape when quantifying SC mass removed by tape stripping.     

The water content of the stratum corneum in patients with atopic dermatitis. Measurement with the Corneometer CM 420

The dry looking skin seen in many patients with atopic dermatitis reflects a defect in the epidermal barrier, the stratum corneum, as demonstrated by an increased transepidermal water loss (TEWL) and a decreased ability of the stratum corneum to bind water. The absolute amount of water within the stratum corneum is of importance both for barrier properties and for the clinical appearance of the skin. This water content was measured with a new instrument, the Corneometer CM 420, which takes advantage of the high dielectric constant of water. Forty patients with atopic dermatitis were studied--20 with dry skin and 20 with clinically normal skin on non-eczematous areas. The stratum corneum in dry skin was found to have a lower content of water than that in the clinically normal skin (p less than 0.01). Clinically normal skin in patients with atopic dermatitis did not differ significantly from normal control skin. An experiment was performed in vitro in an attempt to correlate the values obtained with the Corneometer to the absolute amount of water within the corneum.     

Distinct barrier integrity phenotypes in filaggrin-related atopic eczema following sequential tape stripping and lipid profiling

Background Filaggrin gene (FLG) loss‐of‐function mutations have been shown to represent the strongest so far known genetic risk factor for atopic dermatitis (AD). Whereas the barrier characteristics in FLG mutation carriers under baseline conditions have been investigated, there are only limited data on the permeability barrier function in filaggrin‐AD under compromised conditions. Aim We investigated: (i) stratum corneum (SC) integrity/cohesion; (ii) barrier recovery after controlled mechanical and irritant‐induced barrier abrogation; and (iii) the lipid composition of the non‐lesional and lesional skin of AD patients harbouring the European R501X, 2282del4, 3702delG, R2447X or S3247X FLG variants. Methods Thirty‐seven AD patients (14 FLG mutation carriers and 23 non‐carriers) and 20 healthy controls participated in the study. Stratum corneum integrity/cohesion was assessed by measurement of transepidermal water loss (TEWL) and amount of removed protein following sequential tape stripping. Barrier recovery was monitored by repeated measurements of TEWL and erythema up to 96 h after barrier abrogation. Samples for lipid analysis were obtained from non‐lesional and lesional skin using the cyanoacrylate method. Results Tape stripping revealed distinct genotype‐related impairment of the SC integrity/cohesion. No differences in the rate of barrier recovery among the groups were found. The SC lipid analysis revealed significant differences regarding the percentage amount of cholesterol, ceramide/cholesterol ratio and triglycerides in the uninvolved skin as well as the amounts of free fatty acids, CER[EOH] and triglycerides in the skin lesions of the AD FLG mutation carriers. Conclusions Our results provide evidence for discernible FLG‐related barrier integrity phenotypes in atopic eczema.     

Microanalysis of an antimicrobial peptide, beta-defensin-2, in the stratum corneum from patients with atopic dermatitis

Background  Antimicrobial peptides, such as defensin and cathelicidin, have recently been reported to play important roles in host defence and in cutaneous innate immunity. Although β-defensin-2 has been reported to be downregulated in the skin of patients with atopic dermatitis (AD), little is known about its role in the colonization of Staphylococcus aureus in the stratum corneum of patients with AD. A precise evaluation of these peptides in the stratum corneum as an antimicrobial barrier against S. aureus colonization has not yet been performed.
Objectives  To compare β-defensin-2 levels in the skin of patients with AD and healthy controls.
Methods  We developed a microanalytical technique to measure β-defensin-2 in the stratum corneum using a combination of immunoprecipitation and Western blotting.
Results  β-Defensin-2 in the stratum corneum was significantly higher in AD lesional skin compared with healthy control skin. The β-defensin-2 content in AD lesional skin also increased in proportion to the severity of the disease. Counting bacterial colonies revealed higher populations of S. aureus on lesional and nonlesional skin surfaces of patients with AD compared with healthy controls. Comparison of S. aureus colony numbers and β-defensin-2 levels demonstrated a positive correlation ( r  =   0·342, P  =   0·004, n  =   67) between both factors.
Conclusions  Collectively, these findings suggest that β-defensin-2 is induced in response to bacteria, injury or inflammatory stimuli and is not associated with vulnerability to S. aureus colonization in the skin of patients with AD.