Knock‐down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents

Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT‐based 3D‐skin equivalent (NSE) after knock‐down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock‐down (FLG‐KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG‐KD alone does not necessarily affect the functionality of a proper barrier function.     

特应性皮炎患儿与健康儿童皮肤屏障功能的对比

目的探讨特应性皮炎(AD)患儿与健康儿童皮肤屏障功能的差异。方法 0～7岁的AD患儿和健康儿童各60名,根据不同年龄段分成2组,0～2岁组和2～7岁组各30例。依次进行角质层含水量、pH值、经表皮水分丢失量(TEWL)的测量,使用SPSS13.0统计软件分析。结果 0～2岁、2～7岁AD患儿与健康儿童比较,角质层含水量除前臂无差异外,前额和颊前均明显低于健康对照组;皮肤表面pH值均明显高于健康对照组;0～2岁的AD患儿TEWL值除前臂无差异外,前额和颊前均明显高于健康对照组,而2～7岁AD患儿TEWL值均明显高于健康对照组。结论 AD患儿与健康儿童比较,皮肤屏障功能存在障碍。表现为角质层含水量、皮肤表面pH值、TEWL值有不同程度的差异。     

Susceptibility to irritants: role of barrier function, skin dryness and history of atopic dermatitis

The susceptibility of the skin to various irritants was investigated with the aim of determining the role of the barrier function of the stratum corneum, skin dryness and whether a history of atopic dermatitis (AD) was a factor. The transepidermal water loss (TEWL) was measured using an evaporimeter and skin hydration using a Corneometer and by visual scoring. The group with a history of AD (n = 20) had a lower pre-exposure barrier function and a higher TEWL value following irritant exposure than the group with a history of allergic contact dermatitis (n = 18) and a control group (n = 18). Clinically dry skin was more susceptible than normal skin, though no difference was noted in the pre-exposure barrier function. The increased susceptibility to irritants in those with a past history of AD was probably due to impaired barrier function and/or the presence of a dry skin.     

Functional analyses of the skin surface of the areola mammae: comparison between healthy adult male and female subjects and between healthy individuals and patients with atopic dermatitis

Background Although the nipple and areola of the breast constitute a unique and prominent area on the chest, so far no study has been done on the functional properties of their skin surfaces. Objective To study the stratum corneum (SC) covering the areola using noninvasive methods. Methods Eighteen adult healthy subjects comprising nine men and nine women and 18 age‐ and sex‐matched patients with atopic dermatitis (AD), none of whom had visible skin lesions, participated in the study. Transepidermal water loss (TEWL), skin surface hydration and skin surface lipid levels were measured on the areola and adjacent breast skin. The size of the skin surface corneocytes of these skin regions was assessed. Results All the healthy subjects showed significantly higher TEWL accompanied by smaller sized corneocytes on the areola than on the adjacent breast skin. Only female subjects revealed a significantly higher skin surface hydration state together with significantly increased skin surface lipid levels on the areola than on the adjacent breast skin. These sex differences were observed even in patients with AD. Comparison between healthy individuals and the patients with AD demonstrated higher TEWL, decreased skin surface hydration state and lower skin surface lipid levels associated with smaller sized corneocytes in the areola in the patients with AD, especially in male patients. Conclusions In adults, the SC barrier function and SC water‐binding capacity of the areola were functionally poorer than in the adjacent skin, being covered by smaller sized corneocytes and lower amounts of skin surface lipids, especially in men and in patients with AD.     

Normal recovery of the stratum corneum barrier function following damage induced by tape stripping in patients with atopic dermatitis

Patients with atopic dermatitis (AD) constantly inflict mechanical damage to their skin by scratching induced by pruritus. On excoriated lesions of the cheek we found exceedingly high levels of transepidermal water loss (TEWL) as compared to those in the normal skin of healthy subjects. However, it is not clear whether the skin of patients with AD also shows an abnormally slow recovery after mechanical damage. We compared the recovery of the barrier function of the stratum corneum (SC). after its complete removal by tape stripping, in patients with AD and age-matched healthy control subjects. On the normal-looking skin of the flexor forearm, we found no difference in the recovery process of the water barrier function of the SC between the two groups. This suggests that ability to reconstruct SC barrier function after mechanical damage is not impaired in AD patients.     

Percutaneous penetration of sodium lauryl sulphate is increased in uninvolved skin of patients with atopic dermatitis compared with control subjects

BACKGROUND: Involved regions of the skin in patients with atopic dermatitis (AD) have been shown to have higher transepidermal water loss (TEWL), indicating a compromised skin barrier. Whether uninvolved skin also has diminished barrier characteristics is controversial. OBJECTIVES: To study the penetration of sodium lauryl sulphate (SLS) into uninvolved skin of patients with AD compared with the skin of control subjects. METHODS: Percutaneous penetration was assessed using the tape stripping technique on the stratum corneum (SC). Twenty patients with AD and 20 healthy subjects were exposed to 1% SLS for 4 h on the mid-volar forearm. After the end of exposure the SC was removed by adhesive tape. The amount of SLS was determined in each consecutive strip. Fick's second law of diffusion was used to deduce the diffusivity and the partition coefficient of SLS between water and the SC. RESULTS: The SC thickness was similar in both groups; however, the TEWL was higher in patients with AD compared with that of the control group (mean+/-SD 8.4+/-4.3 and 6.3+/-2.0 g m-2 h-1, respectively). There was a correlation between SC thickness and TEWL in control subjects but no correlation was found in patients with AD. The diffusivity of SLS through uninvolved AD skin was higher compared with normal skin (mean+/-SD 12.7+/-5.8x10(-9) and 6.2+/-3.0x10(-9) cm-2 h-1, respectively), while the partition coefficient between SC and water was lower (mean+/-SD 137+/-64 and 196+/-107, respectively). CONCLUSIONS: The results show a different penetration profile of SLS into the SC of patients with AD compared with control subjects. This indicates that even noninvolved skin in patients with AD has altered barrier characteristics, emphasizing the importance of skin protection and prevention of skin contact with chemicals.     

特应性皮炎与丝聚蛋白研究进展

丝聚蛋白(FLG)基因的失功能突变可使表皮FLG含量减少或缺失,进而引起表皮屏障功能发生改变,增加特应性皮炎(AD)的患病风险。本文对FLG在表皮屏障形成中的作用、FLG基因结构、FLG基因在AD患者中的突变情况及FLG基因突变与AD临床表型的相关性进行综述。     

Characteristics of skin surface morphology and transepidermal water loss in clinically normal-appearing skin of patients with atopic dermatitis: a video-microscopy study

In patients with atopic dermatitis (AD), it is debatable whether clinically normal-appearing skin is equal to non-atopic normal skin. The aim of this study was to quantitatively evaluate the characteristics of normal-appearing skin of AD. We examined the value of skin surface morphological changes using a new, simple, computer-assisted method with a video microscope. We also investigated the physiological function as represented by transepidermal water loss (TEWL) levels in 44 patients with AD and 15 normal controls. The morphological changes were represented by a variation coefficient score that reflected the irregularity of skin ridges, named the surface irregularity index (SII). There were significant differences between the normal-appearing skin of AD and non-atopic normal skin in both SII (P<0.001) and in TEWL (P<0.01). Especially for the SII, there were significant differences between AD subgroups subdivided by peripheral blood eosinophil count (Eo), serum lactate dehydrogenase level, and clinical score. TEWL values were significantly higher in the high-Eo AD group (n=15) than in the low-Eo AD group (n=29) (P<0.05). These findings indicate that clinically normal-appearing skin of AD patients with high disease activity differs from non-atopic normal skin in both surface morphology and physiology and that these changes reflect the current disease activity.     

Improvement in skin barrier function in patients with atopic dermatitis after treatment with a moisturizing cream (Canoderm)

Patients with atopic skin show a defective barrier function both in rough and in clinically normal skin, with an increasing risk of developing contact dermatitis. Moisturizing creams are often used in the treatment of dry skin. The purpose of this study was to investigate the influence of treatment with a urea-containing moisturizer on the barrier properties of atopic skin. Fifteen patients with atopic dermatitis treated one of their forearms twice daily for 20 days with a moisturizing cream. Skin capacitance and transepidermal water loss (TEWL) were measured at the start of the study and after 10 and 20 days. On day 21 the skin was exposed to sodium lauryl sulphate (SLS) and on day 22 the irritant reaction was measured non-invasively. Skin capacitance was significantly increased by the treatment, indicating increased skin hydration. The water barrier function, as reflected by TEWL values, tended to improve (P = 0.07), and the skin susceptibility to SLS was significantly reduced, as measured by TEWL and superficial skin blood flow (P < 0.05). Thus, it seems that certain moisturizers could improve skin barrier function in atopics and reduce skin susceptibility to irritants. The mechanism and the clinical relevance need further investigation.     

Comparison of skin barrier function and sensory nerve electric current perception threshold between IgE-high extrinsic and IgE-normal intrinsic types of atopic dermatitis

Background  Two types of atopic dermatitis (AD) have been proposed, with different pathophysiological mechanisms underlying this seemingly heterogeneous disorder. The extrinsic type shows high IgE levels presumably as a consequence of skin barrier damage and feasible allergen permeation, whereas the intrinsic type exhibits normal IgE levels and is not mediated by allergen-specific IgE.
Objectives  To investigate the relationship between pruritus perception threshold and skin barrier function of patients with AD in a comparison between the extrinsic and intrinsic types.
Methods  Enrolled in this study were 32 patients with extrinsic AD, 17 with intrinsic AD and 24 healthy individuals. The barrier function of the stratum corneum was assessed by skin surface hydration and transepidermal water loss (TEWL), and pruritus perception was evaluated by the electric current perception threshold (CPT) of sensory nerves upon neuroselective transcutaneous electric stimulation.
Results  Skin surface hydration was significantly lower and TEWL was significantly higher in extrinsic AD than intrinsic AD or normal controls. Although there was no statistically significant difference in CPT among extrinsic AD, intrinsic AD and normal controls, CPT was significantly correlated with skin surface hydration and inversely with TEWL in intrinsic AD and normal controls, but not extrinsic AD. Finally, CPT was correlated with the visual analogue scale of itch in the nonlesional skin of patients with extrinsic but not intrinsic AD.
Conclusions  Patients with extrinsic AD have an impaired barrier, which increases the pre-existing pruritus but rather decreases sensitivity to external stimuli. In contrast, patients with intrinsic AD retain a normal barrier function and sensory reactivity to external pruritic stimuli.     

Use of Raman spectroscopy for the early detection of filaggrin‐related atopic dermatitis

Background: Filaggrin (FLG) gene mutations, which result in complete or incomplete loss of proFLG/FLG peptides, have been reported as an important predisposing factor for atopic dermatitis (AD) and secondary atopic phenotypes such as atopic asthma. Method: The presence of the protein FLG in the skin was evaluated at birth on 12 infants using Raman spectroscopy; these 12 infants were monitored for 1 year to see whether they developed AD. Three different statistical analysis procedures, two of which involved principal component analysis (PCA), were performed on the Raman spectra in order to determine the FLG content. Results: The infants who had a lower FLG content, determined using any of the three statistical analysis procedures proposed, were also the ones that clinically developed AD. Conclusion: The results suggest that Raman spectroscopy and statistical analysis such as PCA could be used as an early detection procedure for FLG ‐related AD and as a possible quantitative marker for FLG gene mutations.     

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.     

上海两社区特应性皮炎患儿的皮肤屏障功能研究

【摘要】 目的 探讨上海两个社区特应性皮炎（AD）患儿及健康对照儿童皮肤屏障功能及AD皮损严重程度与皮肤屏障功能的相关性。方法 3 ~ 12岁AD患儿169例和健康对照儿童142例来自上海长宁新泾社区和嘉定菊园社区,检测前臂伸侧、屈侧及脸颊、胫前4个部位非皮损区的角质层含水量和经皮失水量（TEWL）。并用欧洲AD评分标准（SCORAD）对AD患儿临床严重程度进行评分。结果 AD患儿前臂伸侧、屈侧及脸颊、胫前四个部位的TEWL值均高于健康对照儿童（P ＜ 0.05）,角质层含水量在前臂伸侧和胫前均显著低于健康对照儿童（P ＜ 0.05）。AD患儿SCORAD与TEWL均值呈正相关,与角质层含水量均值呈负相关。结论 皮肤屏障功能可以作为评价AD临床严重程度的指标之一。     

Kontaktallergie und Atopie

Retrospective studies demonstrate that the prevalence of skin sensitization does not significantly differ between atopic and non-atopic patients. In children and adolescents the risk for sensitization seems to occur independently from AD. According to the results of a recent study, AD patients are overrepresented in the group of polysensitized patients. IgE-mediated sensitization as well as an early onset of AD and duration of the disease have been identified as possible risk factors for skin sensitization to contact allergens. A defective permeability barrier with increased epidermal water loss is a hallmark of AD and contributes to sensitization against common allergens. A highly significant association between FLG mutations and the risk of early onset, severe, persistent AD and an increased risk for asthma has been shown in several studies. A more recent study revealed an association between FLG mutations and increased nickel sensitization, but not other contact allergens. However, further large prospective studies with well-characterized patients are necessary to clarify the correlation between impaired skin barrier, atopic dermatitis and allergic contact dermatitis.     

Evaluation of out–in skin transparency using a colorimeter and food dye in patients with atopic dermatitis

Background  Atopic dermatitis is a disease of skin barrier dysfunction and outside stimuli can cross the skin barrier.
Objectives  To examine a new method for evaluating the outside to inside skin transparency with a colorimeter and yellow dyes.
Methods  In study 1, a total of 28 volunteer subjects (24 normal and four with atopic dermatitis) participated. After provocation with yellow dye, the skin colour of all the subjects was measured using a colorimeter. The skin transparency index was calculated by the changes of the skin colour to yellow. Other variables of skin function, including transepidermal water loss (TEWL) and stratum corneum hydration, were also measured. In study 2, the skin transparency index was evaluated for a cohort of 38 patients with atopic dermatitis, 27 subjects with dry skin and 29 healthy controls.
Results  In study 1, the measurement of skin colour (b*) using tartrazine showed good results. There was a significant relationship between the skin transparency index with tartrazine and the atopic dermatitis score ( P  =   0·014). No other measurements of skin function, including the TEWL, were correlated. In study 2, the skin transparency index score obtained with tartrazine in the patients with atopic dermatitis was significantly higher than that of the controls and those with dry skin ( P  <   0·001 and P  =   0·022, respectively). However, the TEWL in patients with atopic dermatitis was not significantly higher than that of patients with dry skin and the TEWL in subjects with dry skin was not higher than that of the controls.
Conclusions  This method, which used a colorimeter and food dye, is noninvasive, safe and reliable for the evaluation of out–in skin transparency and can demonstrate the characteristic dysfunction in the skin barrier in patients with atopic dermatitis.     

Correlation of clinical features and skin barrier function in adolescent and adult patients with atopic dermatitis

BACKGROUND: Xerotic changes in atopic skin are considered to be related to a decrease in the water permeability barrier. Whether abnormal skin barrier function is the main cause of atopic dermatitis (AD) or a secondary change of the disease is still controversial. Noninvasive bioengineering methods, including the measurement of the transepidermal water loss (TEWL) and water capacitance, have been commonly used to evaluate skin barrier function. AIM: To evaluate the correlation between the clinical features of each evaluation site (severity of AD) and skin barrier function. METHODS: TEWL, capacitance, and pH were checked on five evaluation sites: postauricle, forearm, abdomen, thigh, and popliteal fossa. The subjects included 25 patients, both adolescents and adults, with AD and 25 age-matched normal controls. The clinical severity, from 0 (no clinical manifestation) to 3 (severe), was also scored for erythema, induration/papulation, lichenification, and xerosis on each evaluation site of the AD patients. RESULTS: Based on the data, we found that the clinical severity score was correlated with TEWL and capacitance in more than one-half of the evaluation sites. Erythema and induration/papulation showed a statistically significant correlation with TEWL in most cases (P < 0.05, four sites). Lichenification and xerosis showed a significant correlation with capacitance in most cases (P < 0.05, four sites). In most cases, severity scoring of the clinical features did not show a significant correlation with skin pH. The patients showed higher TEWL and lower capacitance than normal controls (P < 0.05, all five sites). CONCLUSIONS: The results of our study suggest that skin barrier function, measured by TEWL and capacitance, and clinical severity show a statistically significant correlation in patients with AD.     

Acute irritant threshold correlates with barrier function,skin hydration and contact hypersensitivity in atopic dermatitis and rosacea

The aim of the study was to disclose interactions between epidermal barrier, skin irritation and sensitization in healthy and diseased skin. Transepidermal water loss (TEWL) and stratum corneum hydration (SCH) were assessed in adult patients with atopic dermatitis (AD), rosacea and healthy controls. A 4‐h patch test with seven concentrations of sodium lauryl sulphate was performed to determine the irritant threshold (IT). Contact sensitization pattern was revealed by patch testing with European baseline series. Subjects with a lower IT had higher TEWL values and lower SCH. Subjects with positive allergic reactions had significantly lower IT. In AD, epidermal barrier deterioration was detected on both volar forearm and nasolabial fold, while in rosacea, impeded skin physiology parameters were observed on the facial skin only, suggesting that barrier impediment is restricted to the face in rosacea, in contrast with AD where the abnormal skin physiology is generalized.     

Ceramide-dominant barrier repair lipids alleviate childhood atopic dermatitis: changes in barrier function provide a sensitive indicator of disease activity

BACKGROUND: It is currently fashionable to consider atopic dermatitis (AD), like other inflammatory dermatoses, as immunologic in pathogenesis ("inside-outside" hypothesis). Accordingly, topical glucocorticoids and other immunosuppressive agents are mainstays of therapy, but the risk of toxicity from these agents is not insignificant, particularly in children. Alternatively, because stratum corneum (SC) permeability barrier function is also abnormal in AD, it has been hypothesized that the barrier abnormality could drive disease activity. Yet commonly used emollients and moisturizers do not correct the SC ceramide deficiency, the putative cause of the barrier abnormality. OBJECTIVES: We assessed the efficacy of a newly developed, ceramide-dominant, physiologic lipid-based emollient, when substituted for currently used moisturizers, in 24 children who were also receiving standard therapy for stubborn-to-recalcitrant AD. METHODS: All subjects continued prior therapy (eg, topical tacrolimus or corticosteroids), only substituting the barrier repair emollient for their prior moisturizer. Follow-up evaluations, which included severity scoring of atopic dermatitis (SCORAD) values and several biophysical measures of SC function, were performed every 3 weeks for 20 to 21 weeks. RESULTS: SCORAD values improved significantly in 22 of 24 patients by 3 weeks, with further progressive improvement in all patients between 6 and 20 or 21 weeks. Transepidermal water loss levels (TEWL), which were elevated over involved and uninvolved areas at entry, decreased in parallel with SCORAD scores and continued to decline even after SCORAD scores plateaued. Both SC integrity (cohesion) and hydration also improved slowly but significantly during therapy. Finally, the ultrastructure of the SC, treated with ceramide-dominant emollient, revealed extracellular lamellar membranes, which were largely absent in baseline SC samples. CONCLUSION: These studies suggest that (1) a ceramide-dominant, barrier repair emollient represents a safe, useful adjunct to the treatment of childhood AD and (2) TEWL is at least as sensitive an indicator of fluctuations in AD disease activity as are SCORAD values. These studies support the outside-inside hypothesis as a component of pathogenesis in AD and other inflammatory dermatoses that are accompanied by a barrier abnormality.     

The effect of aqueous cream BP on the skin barrier in volunteers with a previous history of atopic dermatitis

Background The emollient aqueous cream BP is frequently used for the treatment of atopic dermatitis (AD), yet it is associated with a high rate of adverse cutaneous reactions. It contains the harsh anionic surfactant sodium lauryl sulphate, a known negative environmental factor associated with the exacerbation of AD. Objectives To investigate the effect of aqueous cream BP on stratum corneum (SC) integrity and skin barrier function in volunteers with a predisposition to a defective skin barrier. Methods Thirteen volunteers with a previous history of AD (no symptoms for 6 months) applied aqueous cream BP twice daily to the volar side of one forearm for 4 weeks. The other forearm was left untreated as a control. Permeability barrier function and SC integrity were determined before and after treatment by measuring transepidermal water loss (TEWL) in conjunction with tape‐stripping. For comparison, 13 volunteers with current AD were recruited for assessment, without treatment, of SC integrity and skin barrier function at unaffected sites. Results Topical application of aqueous cream BP resulted in significant elevation of baseline TEWL and a concomitant decrease in SC integrity. Measurements made after no treatment in volunteers with current AD, at unaffected sites, suggest that application of aqueous cream BP negatively affects the skin barrier towards the damaged state associated with onset of flares of the disease. Conclusions Aqueous cream BP used as a leave‐on emollient caused severe damage to the skin barrier in volunteers with a previous history of AD. Aqueous cream BP should not be used as a leave‐on emollient in patients with AD.