New non‐invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape‐stripped samples

The corneodesmosomes in the stratum corneum are critical for the maintenance of stratum corneum integrity. To evaluate the normal and diseased keratinization states in the epidermis, we studied the distribution of desmoglein 1 (DSG1), a major component of corneodesmosomes, in samples of the stratum corneum obtained by tape stripping, a non‐invasive method. Samples were collected from lesional skin of four patients with psoriasis and three with lichen planus, and from non‐lesional skin of three volunteers. Upper stratum corneum cells were obtained by tape stripping and skin biopsies were obtained from adjacent sites. Tape‐stripped samples were examined by immunofluorescence microscopy using anti‐DSG1 monoclonal antibody, in combination with histopathology of skin biopsies. In normal human stratum corneum, which shows basket‐woven orthokeratosis, DSG1‐containing fluorescent dots were distributed on the lateral cell–cell contact areas of plasma membrane, but not on the dorsal/ventral plasma membrane, and formed a well‐ordered hexagonal network structure. In psoriatic stratum corneum, fluorescent dots were distributed throughout the cell membrane at ventral aspects of corneocytes as well as at the lateral cell–cell contacts. In lichen planus, fluorescent dots were distributed homogeneously and/or heterogeneously on the ventral surface in some cells. Adjacent cells lacked DSG1 at the lateral cell–cell contacts, but were instead separated by distinctive black‐gap lines. These results suggest that the intercellular adhesion by DSG1 may depend on the lateral plasma membrane in normal human stratum corneum, on the dorsal/ventral plasma membrane in lichen planus, and on both lateral and dorsal/ventral plasma membranes in psoriatic stratum corneum. Tape stripping and DSG1 immunofluorescence visualizes adhesion features of corneocytes and has considerable potential for evaluation of abnormal keratinization and the process of healing in response to treatment.     

Physiological and functional changes in the stratum corneum restored by oestrogen in an ovariectomized mice model of climacterium

Significant decreases in hormonal levels at menopause induce physiological and functional discomfort in the skin. Representative changes at menopause are based on so‐called dry skin. However, there is no evidence to explain the mechanism, even though hydration of the stratum corneum (SC) in women at menopause is comparable with that at premenopause but is enhanced by hormone replacement therapy. This study objective was to evaluate structural and functional changes in the SC in ovariectomized mice model of menopause. Hydration of the SC, recovery of the permeability barrier function, integrity and cohesion of the SC, and irritant dermatitis were analysed in mice that underwent ovariectomy with or without replacement of 17ß‐estradiol. In ovariectomized mice, hydration of the SC was reduced, recovery of permeability barrier function after acute disruption was impaired, and integrity of the SC was weakened and was associated with increased cohesion and increased levels of irritant dermatitis. Oestrogen replacement treatment restored all changes. Immunohistochemistry revealed reduced levels of expression of desmoglein‐1 and differentiation markers of epidermis in ovariectomized mice compared with control mice and mice with oestrogen replacement treatment. These changes might be directly associated with weakened integrity and impaired permeability barrier function of the SC in ovariectomized mice. This study results reveal that so‐called dry skin at menopause is caused by not only lower hydration of the SC but also complicated structural and functional changes in the SC and skin.     

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.     

Cell surface glycans in the human stratum corneum: distribution and depth‐related changes

During the formation of the stratum corneum (SC) barrier, the extracellular spaces of viable epidermis, rich in glycans, are filled with a highly organized lipid matrix and the plasma membranes of keratinocytes are replaced by cornified lipid envelopes. These structures comprise cross‐linked proteins, including transmembrane glycoproteins and proteoglycans, covalently bound to a monolayer of cell surface ceramides. Little is known about the presence and distribution of glycans on the SC corneocytes despite their possible involvement in SC hydration, cohesion and desquamation. In this work, we visualized ultrastructurally and quantified the distribution of glycans on the surface of native and delipidated corneocytes. The cells were harvested at different depths of the SC, allowing us to define the relationship between the distribution of various glycans, proteoglycans and glycoproteins, and other changes occurring in SC. At the cell periphery, we found a correlation between the depth‐related alterations of corneodesmosome glycoproteins and α‐d ‐mannosyl and N‐acetyl‐d ‐glucosamine‐labelling patterns. Elimination of the terminal sugars, α‐linked fucose and α‐(2,3) linked sialic acid, was less abrupt, but also the initial extent of their peripheral distribution was overall lower than that of concanavalin A and wheat germ agglutinin lectin‐detected glycans. Diffuse labelling of heparan sulphate glycosaminoglycans disappeared completely from the outermost corneocytes, whereas that of several simple carbohydrates could be detected at all SC levels. Our results suggest that specific glycan distribution may participate in the progressive changes of SC, as it evolves from the SC compactum to the SC disjunctum, towards desquamation.     

Sun‐induced changes of stratum corneum hydration vary with age and gender in a normal Chinese population

Background/objectives: Previous studies have demonstrated that sun‐induced alteration of epidermal permeability barrier function varies with gender and age. In the present study, we assess the stratum corneum (SC) hydration in sun‐exposed males and females. Methods: A total of 168 subjects (84 males and 84 females) aged 19–75 years were enrolled. A multifunctional skin physiology monitor was used to measure SC hydration. Results: In comparison with non‐sun exposure, sun exposure does not cause a significant change in SC hydration in either young males or young females, whereas in aged females, a significant reduction of SC hydration is seen on the forehead and the dorsal hand of sun‐exposed subjects. SC hydration on the canthus of both aged males and aged females is significantly lower than that of young subjects. Additionally, SC hydration on the dorsal hand of aged females is also significantly lower as compared with young females. Sun‐induced reduction of SC hydration is more evident on the dorsal hand of aged females than that of males (P<0.001). Moreover, the SC rehydration capacity is significantly lower in sun‐exposed aged females than in age‐matched males. Conclusion: These results demonstrated that sun‐induced changes of the SC hydration property vary with age and gender.     

Macrophage migration inhibitory factor (MIF) in the stratum corneum: a marker of the local severity of atopic dermatitis

Different biomarkers are used to evaluate the severity of atopic dermatitis (AD); however, it remains difficult to determine the severity of localized skin lesions. MIF plays an essential role in the pathophysiology of skin inflammation. To establish whether the MIF level in the stratum corneum (SC) serves as a marker of the severity of AD lesions, we examined the SC MIF (scMIF) levels in AD patients. The SC of the cheek, neck and upper arm skin was collected using tape stripping, and the scMIF levels were measured. Consequently, the scMIF levels were found to be significantly higher in the involved skin lesions than the uninvolved areas within the same patient. Moreover, the scMIF levels were significantly correlated with the severity of local skin lesions. These findings suggest that the scMIF level can be used as an effective marker for evaluating the local severity of AD.     

Polymorphisms in the interleukin-1 gene influence the stratum corneum interleukin-1 alpha concentration in uninvolved skin of patients with chronic irritant contact dermatitis

Background:  Interleukin (IL)-1α and its receptor antagonist IL-1ra play a role in skin inflammation. Several polymorphisms in the IL1 gene cluster, coding for IL-1α, IL-1ra, and IL-1β, influence their protein expression. Within this cluster, strong linkage disequilibrium has been shown.
Objective:  We studied the association between the polymorphisms IL1A -889 (C→T) and IL1B -31 (T→C) and the concentration of IL-1α and IL-1ra in the stratum corneum (SC).
Method:  In 124 patients with chronic irritant contact dermatitis, we genotyped the IL1A -889 and IL1B -31 polymorphisms and determined the amount of IL-1α and IL-1ra on tape strips obtained from uninvolved skin of the volar forearm.
Results:  The SC IL-1α concentration was 23% and 47% lower in subjects with IL1A -889 C/T genotype and T/T genotype, respectively, compared with wild-type genotype. In subjects with IL1B -31 C/C genotype, the IL-1α concentration was 51% lower compared with C/T and T/T genotypes. The ratio IL-1ra/IL-1α increased twofold in IL1A -889 C/T genotype and threefold in T/T genotype compared with wild type.
Conclusions:  We have shown a clear effect of IL1 genotype on protein expression in the SC. This altered expression may be responsible for the interindividual differences in the inflammatory response of the skin.     

Increased carbonyl protein level in the stratum corneum of inflammatory skin disorders: A non‐invasive approach

The stratum corneum (SC) is the interface of body and environment, and is continuously exposed to oxidative stress, resulting in carbonyl modification of proteins. We have developed a simple and non‐invasive method to assess carbonyl protein (CP) level in the SC, applied it to various kinds of skin, and revealed a link between the stratum corneum carbonylated protein (SCCP) level and water content in the SC. The purpose of the present study is to examine the SCCP level in inflammatory skin disorders associated with xerosis. Psoriasis vulgaris (PV) and atopic dermatitis (AD) are typical inflammatory skin disorders, of which the stratum corneum shows markedly low water content. SC samples were non‐invasively collected from the lesional and non‐lesional areas of PV and AD by adhesive tape stripping, and their carbonyl groups were determined by reaction with fluorescein‐5‐thiosemicarbazide. The average fluorescence intensity of the SC was calculated as SCCP level. Higher SCCP level was observed in the lesional area of PV as compared with non‐lesional area or healthy control. Lesional area of AD also exhibited higher SCCP level than corresponding non‐lesional area, of which SCCP level was slightly higher than the healthy control. These data suggest the involvement of oxidative modification of the SC protein, at least in part, in generation of xerotic skin in inflammatory skin disorders as well as dry skin in healthy subjects.     

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.     

Th 1/Th2细胞因子检测在鉴别脓毒症不同病原菌感染中的临床价值

目的探讨Th1/Th2细胞因子对于脓毒症患者早期病原学诊断的临床价值。方法回顾分析96例脓毒症患者的临床资料。依据患者病原学结果进行分组:革兰氏阴性(G-)菌感染组(n=35)和革兰氏阳性(G+)菌感染组(n=61),比较两组患者一般资料、白细胞计数(WBC)、外周血Th1/Th2细胞因子的差异,分析各指标对脓毒症早期病原菌感染的诊断价值。选取同期正常体检者81例作为对照组。结果使用IL-6、IL-10或联合二者预测G-菌阳性的ROC曲线下面积分别为0.862、0.833、0.891,P值均小于0.05。用于预测G-菌阳性的IL-6阈值为53.08 pg/mL,灵敏度为82.0%,特异度85.7%;IL-10相应阈值为10.08 pg/mL,灵敏度为80.3%,特异度80.0%;联合IL-6与IL-10预测G-菌阳性的灵敏度为91.8%,特异度为77.1%,小于阈值提示G+菌感染。结论Th1/Th2细胞分泌的细胞因子IL-6、IL-10可以用于鉴别脓毒症患者早期G-或者G+菌感染,联合IL-6和IL-10判断灵敏度更好,有助于临床抗细菌感染治疗的药物选择。     

Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis: dependence on phenotype, severity and therapy

BACKGROUND: Human tissue kallikreins (KLKs) are a family of 15 trypsin-like or chymotrypsin-like secreted serine proteases (KLK1-KLK15). Multiple KLKs have been quantitatively identified in normal stratum corneum (SC) and sweat as candidate desquamation-related proteases. OBJECTIVES: To quantify KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of patients with psoriasis, and their variation between lesional and nonlesional areas and with phenotype, therapy and severity. The overall SC serine protease activities were also measured. METHODS: Enzyme-linked immunosorbent assays and enzymatic assays were used. RESULTS: The lesional SC of psoriasis generally contained significantly higher levels of all KLKs. KLK6, KLK10 and KLK13 levels were significantly elevated even in the nonlesional SC. The overall trypsin-like, plasmin-like and furin-like activities were significantly elevated in the lesional SC. Plasmin-like activity was significantly elevated also in the nonlesional SC. The SC chymotrypsin-like activity was only slightly elevated in psoriasis. KLK7 serum levels did not differ between normal volunteers and patients with psoriasis. Serum KLK6, KLK8, KLK10 and KLK13 levels in patients with untreated psoriasis significantly correlated with Psoriasis Area and Severity Index score. Serum KLK5 and KLK11 levels decreased in patients with psoriasis after therapy, especially with etretinate. Patients with erythrodermic psoriasis exhibited significantly higher serum KLK levels than normal subjects or patients with psoriasis vulgaris or arthropathic psoriasis. CONCLUSIONS: We found aberrant KLK levels in the SC and serum of patients with psoriasis and suggest that KLKs might be involved in the pathogenesis of this disease.     

Topical suplatast tosilate (IPD) ameliorates Th2 cytokine-mediated dermatitis in caspase-1 transgenic mice by downregulating interleukin-4 and interleukin-5

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by elevated serum levels of IgE. AD is associated with Th2 cytokines including interleukin (IL)-4, IL-5, IL-13 and IL-10. Systemic administration of suplatast tosilate (IPD) is currently used to treat Th2 cytokine-mediated AD. OBJECTIVES: To evaluate the effect of topical IPD on skin lesions of AD using a genetically engineered AD mouse model (K14/caspase-1 transgenic mouse: KCASP1Tg). METHODS: IPD ointment (3%) and white petrolatum (WP) were applied to KCASP1Tg mice every other day from 6 to 14 weeks after birth. Histopathological analysis of skin lesions and measurement of mRNA expression of cytokines in skin lesions and spleen cells were carried out. We also compared changes in serum parameters between IPD-treated and WP-treated KCASP1Tg mice. RESULTS: WP-treated mice developed dermatitis at 8 weeks after birth. However, skin lesions in IPD-treated mice were limited. Histopathologically, skin lesions in WP-treated KCASP1Tg mice showed marked inflammatory changes with increased mast cell infiltration. However, mice treated with IPD showed minimum skin lesions with scarce mast cell infiltration. WP-treated KCASP1Tg mice had significant elevation in the serum levels of histamine, IgE and IL-18 as compared with IPD-treated KCASP1Tg mice. mRNA expression of IL-4 and IL-5 in the skin lesions from WP-treated KCASP1Tg mice was significantly higher than in those from IPD-treated mice. In the spleen, the expression of IL-4, IL-5 and interferon-gamma was significantly increased in WP-treated KCASP1Tg mice as compared with their IPD-treated counterparts. CONCLUSIONS: This study shows that topical therapy with IPD inhibits the expression of IL-4 and IL-5 and ameliorates skin manifestations in an AD mouse model, suggesting the potential usefulness of topical IPD for the treatment of AD.