A non-invasive tape absorption method for recovery of inflammatory mediators to differentiate normal from compromised scalp conditions

Background/aims: A simple non-invasive tape (Sebutape^TM) adsorption method was used to recover inflammatory proteins from normal and compromised human scalp (i.e. dandruff and seborrheic dermatitis) in order to assess the inflammatory and immunologic changes relevant to these clinical conditions. Methods: The scalps of subjects identified by a dermatologist as having either dandruff (n = 18), seborrheic dermatitis (n = 19) or normal scalp (n = 16) were visually graded to obtain total adherent scalp flaking scores (TASFS). Sebutape samples were then collected from both the high and low TASFS scalp sites using a one-minute tape application. To recover inflammatory molecules, tapes were extracted in buffered saline with sonication and the tape extracts analysed using commercial immunoassay methods for pro-inflammatory cytokines [i.e. interleukin-1α (IL-1α), IL-1β, IL-1 receptor antagonist (IL-1ra), IL-8 and tumor necrosis factor-α (TNF-α)] and the immunologic cytokines [i.e. IL-2, IL-4, IL-10, IL-12, IL-15 and interferon gamma (IFN-γ)]. Nitric oxide (NO) was also assayed on tape extracts using the Greiss reaction. To account for differences in protein loading on the tapes all cytokine and NO results were normalized using the total protein (TP) amounts recovered in tape extracts. Results: The IL-1α/TP levels recovered from dandruff and seborrheic scalps were significantly decreased (P = 0.03) compared to normal appearing scalp levels. The scalp levels of IL-1ra/TP and the ratio of IL-ra to IL-1α were significantly (P = 0.002) or directionally (P = 0.07) higher in seborrheic dermatitis scalps and dandruff scalps, respectively, compared to normal scalps. The IL-1ra and the IL-1ra/IL-1α ratio values correlated well with the TASFS. The TNF-α/TP levels recovered from dandruff scalps were significantly higher (P = 0.02) than levels recovered from seborrheic dermatitis and normal scalp subjects. IL-2/TP was significantly increased (P = 0.01) and IFN-γ and NO significantly decreased (P = 0.05) in the seborrheic dermatitis scalp samples compared to normal controls. Conclusion: The Sebutape method has proven useful for distinguishing normal from diseased scalp conditions. The cytokines recovered from the scalp tape samples showed distinct patterns that differentiated dandruff, seborrheic dermatitis and normal scalp populations. These methods may also prove useful for monitoring the clinical efficacy of therapeutic actives for treating dandruff and seborrhea.     

Effects of disinfectants and detergents on skin irritation

We investigated the biological response of regular human skin to alcohol-based disinfectants and detergents in a repetitive test design. Using non-invasive diagnostic tools such as transepidermal water loss, laser-Doppler flowmetry and corneometry, we quantified the irritative effects of a propanol-based hand disinfectant (Sterillium), its propanol mixture (2-propanol 45% w/w and 1-propanol 30% w/w), sodium lauryl sulfate (SLS) 0.5% and distilled water. The substances were applied in a 2-D patch test in a repetitive occlusive test design to the back. Additionally, we performed a wash test on the forearms that was supposed to mimic the skin affection in the normal daily routine of health care workers. In this controlled half-side test design, we included the single application of the hand rub, SLS 0.5% and water as well as a tandem application of the same substances. Patch test and wash test showed similar results. The alcohol-based test preparations showed minimal irritation rather comparable to the application of water. However, the detergent SLS produced stronger barrier disruption, erythema and dryness than the alcohol-based preparations. There was no additional irritation at the combined use of SLS and disinfectants. By contrary, there was even a decrease in barrier disruption and erythema induced by the tandem application of SLS followed by alcohol-based disinfection compared with the use of SLS alone. These findings show a less irritant effect of alcohol-based disinfectants on the skin than detergents. Our study shows that there is no summation of irritating effects of a common detergent and propanol and that the combination of washing and disinfection has a rather protective aspect compared with washing alone.     

Stratum corneum cytokines and skin irritation response to sodium lauryl sulfate

Little is known about cytokines involved in chronic irritant contact dermatitis. Individual cytokine profiles might explain at least part of the differences in the individual response to irritation. Our objective was to investigate the relation between baseline stratum corneum (SC) cytokine levels and the skin response to a single and a repeated irritation test. This study also aimed to determine changes in SC cytokine levels after repeated irritation. Transepidermal water loss (TEWL) and erythema were measured in 20 volunteers after single 24-hr exposure to 1% sodium lauryl sulfate (SLS), and during and after repeated exposure to 0.1% SLS over a 3-week period. SC cytokine levels were measured from an unexposed skin site and from the repeatedly exposed site. Interleukin (IL)-1alpha decreased by 30% after repeated exposure, while IL-1RA increased 10-fold and IL-8 increased fourfold. Baseline IL-1RA and IL-8 values were predictors of TEWL and erythema after single exposure (r = 0.55-0.61). 6 subjects showed barrier recovery during repeated exposure. Baseline IL-1RA and IL-8 levels are likely to be indicators of higher skin irritability after single exposure to SLS. Barrier repair in some of the subjects might explain the lack of agreement between the TEWL response after single and repeated irritation.     

Validity and ethics of the human 4-h patch test as an alternative method to assess acute skin irritation potential

For more than 50 years, the Draize rabbit skin irritation test has reigned supreme as the regulatory method of choice for the identification of skin irritant chemicals. To date no in vitro alternative test has been validated as an adequate replacement. However, one potential option, to test the endpoint of concern (skin irritation) in the species of concern (man) has been overlooked. The advent of predictive in vitro tools for the identification of substances corrosive to the skin has opened up the practical possibility of carrying out safe and ethical studies on small panels of humans. The human 4-h patch test has been developed to meet the needs of identifying chemical skin irritation potential, providing data which is inherently superior to that given by a surrogate model, such as the rabbit. This paper reviews in detail the present state of the human 4-h patch test, highlighting its advantages and noting its utility as the 'gold standard' on which to build future in vitro models.     

Historical perspective on the use of visual grading scales in evaluating skin irritation and sensitization

Visual assessment of skin reactions has long been used to evaluate the safety of chemicals and preparations that contact the skin, and to meet regulatory requirements. This article reviews the history of visual grading scales, and the results of investigations into the reliability of the method. Some examples are provided to illustrate the diverse array of protocols that use visual scoring to evaluate skin irritation. Furthermore, as bioengineering methods are developed that can quantitate certain aspects of skin irritant and sensitization reactions, it is important to consider whether such measures should supplement or replace visual assessment. Examples of investigations comparing the outcomes of studies that use visual scoring and those that use bioengineering methods are discussed. These examples provide little evidence that bioengineering measures provide an improvement in overall quality in comparison with current testing methods that rely on visual assessment. In addition, such measuring techniques can add considerably to the complexity of testing protocols. When benefits and cost are weighed in the balance, the visual assessment scales popularized by Draize and others remain an effective, practical method of evaluation.     

Characterization of skin‐resident T cells using a crawl‐out method and flow cytometry

A large fraction of the skin‐homing T‐cell population resides in the skin even under resting, non‐inflammatory conditions. Here, we used a crawl‐out culture method to retrieve T cells from human skin and characterized them using flow cytometric analysis. On average, 48000 viable, non‐proliferating cells were retrieved per biopsy. We found that human skin contains a larger fraction of IL‐17‐, IL‐4‐, IL‐10‐ and IL‐22‐positive T cells as compared with paired blood samples. Our research indicates that it is feasible to use the crawl‐out method in combination with flow cytometry to characterize T‐cell subpopulations in patient‐derived skin biopsies. This method enables further study of the skin immune system and could function as a valuable tool for evaluation of the effects of immunotherapy in skin diseases.     

The role of mitogen- and stress-activated protein kinase 1 and 2 in chronic skin inflammation in mice

Mitogen- and stress-activated protein kinase 1 and 2 (MSK1/2) are two kinases phosphorylated by both ERK1/2 and p38 MAPK. Recently, MSK1 and 2 have been reported to act as negative regulators of acute inflammation. In this study, we investigated the role of MSK1/2 in chronic skin inflammation using an oxazolone-induced allergic contact dermatitis model in MSK1/2 knockout mice and wild-type mice. MSK1/2 knockout mice were demonstrated to have significantly increased inflammation compared with wild-type mice. This was measured by an increased ear thickness, elevated infiltration of neutrophils in the skin and increased inflammatory histological changes. Furthermore, we found significantly elevated levels of the proinflammatory cytokines Tumor necrosis factor-α (TNF-α), IL-1β and IL-6 at both mRNA and protein levels in MSK1/2 knockout mice compared with wild-type mice after oxazolone treatment. In addition, the mRNA expression of the chemokine Thymus and activation regulated chemokine (TARC) was demonstrated to be significantly elevated in oxazolone-treated MSK1/2 knockout mice compared with wild-type mice. The increased expression of TARC was paralleled by increased infiltration of cells positive for the TARC receptor, CCR4, in the dermis of MSK1/2 knockout mice. Our results indicate that MSK1/2 are involved in the activation of feedback mechanisms that dampen oxazolone-induced skin inflammation.     

The effect of repeated open exposure to low levels of nickel on compromised hand skin of nickel-allergic subjects

Nickel is a very common contact allergen, with some reports showing that more than 10% of women are affected. It is therefore important to be able to assess the risk of elicitation of nickel dermatitis in such individuals, particularly in relation to repeated exposure to low levels of this metal. It has been shown previously that under the conditions of a 48-h occluded patch test, some subjects will react to as little as 0.5 ppm nickel on compromised skin. In the present study, the effect of repeated open nickel application combined with repeated treatment with the anionic surfactant sodium dodecyl sulphate has been evaluated on the hand skin of 4 nickel-allergic subjects. Despite 2 of these being known to be particularly sensitive, there was no evidence of a nickel-allergic reaction following 2 × daily immersion of hand skin sites in up to 1 ppm nickel over a period of 23 days.     

Application of a 4-h human patch test method for comparative and investigative assessment of skin irritation

A human 4-h patch test has recently been developed for testing the irritation hazard potential of chemicals. The original method was developed for comparative irritation assessments relative to benchmark irritants using simple statistical tests. In this context, the method has been shown to be robust in intra laboratory testing over time. Recent inter laboratory testing has also established the consistency of the method in assessment of the relative irritation potential of selected chemicals. These data help to position the method as a suitable replacement for animal test methods in assessment of skin irritation hazard. In addition, the method has great utility for investigating different parameters of clinical skin irritation. Using kinetic response patterns and curve fitting analysis, we have compared the relative irritation potential of chemicals in greater detail, using as a basis the time required for test subjects to respond as well as the incidence of positive responses. Also, using the response to 20% sodium dodecyl sulfate (SDS) as a benchmark, we've been able to examine the intersubject variation in clinical skin irritation responses. In general, subjects most reactive to 20% SDS, in terms of the exposure time required to produce a positive response, were relatively more sensitive to a 2nd irritant chemical as well. However, this was not an absolute correlation in that some test subjects showed divergent patterns of response. The method was also used to compare directly the relative skin reactivity of different populations, based on race (Caucasian versus Asian) or on neurosensory skin sensitivity. Our results using this acute exposure test method indicate little difference in visually assessed skin irritation among these diverse human subpopulations.     

A study of a method for distribution analysis of skin color

Background/aims: The objective and quantitative assessment of the skin is important in medical and cosmeceutical research. Assessment of color is an important element for analyzing the surface of the skin, which is usually determined subjectively by a doctor or using color analysis devices. These devices, however, cannot provide correct color information because color is construed from the mean value of the observation region, and analysis of color distribution is impossible. The purpose of this paper is to develop an objective analysis method to permit skin color measurement of each pixel unit of an image and analyze the distribution of skin surface color. Methods: The Skin Color Distribution Analyzer (SCDA) is an analysis method newly developed at the Research Institute for Skin Image at Korea University. The SCDA system presented in this paper performed a novel form of quantitative and objective analysis of skin color distribution using each pixel color model parameter found in image wavelength information. In this paper, distribution analysis was conducted on normal skin and skin lesions and skin affected by artificially induced irritant contact dermatitis and pigmented nevous. The method selected a grade using a color model parameter. Twenty healthy Korean males participated in this study. A comparative study of the eight anatomical areas was performed, including the exposure and non‐exposure parts and the medial aspect and the lateral aspect of the forearm. A reliability test for the SCDA system was also conducted with a spectrometer (SPEC) using the color analysis method. Results: Each skin lesion was precisely segmented by grade and each parameter hada different statistical significance for results of analysis of distribution in pigmented nevous and the artificially induced irritant contact dermatitis. Parameters L^*, b^*, a^*, and EI showed salient traits. Showed resemble measured result in the SCDA system and the SPEC of normal skin. The exposed site, in comparison with the non‐exposed site, showed a notable difference in the L^* parameter and a significant statistical difference in the x and z parameters, except b^*. The comparison of the medial and lateral aspects of the forearm showed a notable difference in the L^* parameter and a significant statistical difference in the parameters except y and b^*. In the reliability test result using the SCDA system and the SPEC, the SCDA system was highly reliabile in terms of the CV value in all color model parameters. Conclusions: The color distribution analysis method using the SCDA system has revealed an aspect that the existent method of medical research has not shown, and is considered to be more reliable than other methods. This method can provide better study findings because it can be applied to other fields in addition to the medical science field and the ripple effect is thought to be bigger in other science field too.     

A study of matrix metalloproteinase expression and activity in atopic dermatitis using a novel skin wash sampling assay for functional biomarker analysis

Background Atopic dermatitis (AD) is a chronic inflammatory skin condition that is characterized by a defective skin barrier. Despite the well‐recognized role of proteases in skin barrier maintenance, relatively little is known of the contribution made by matrix metalloproteinases (MMPs) to the inflammatory process in AD. Objectives To test a simple, novel ex vivo bioassay technique in an analysis of the MMPs present in wash samples taken from the skin surface of patients with AD. Methods Saline wash samples were collected from eczematous and unaffected areas of the skin of patients with AD and from the skin of normal controls. Wash samples were analysed for their MMP content using a functional peptide cleavage assay, gelatin zymography and an antibody array. Results Using a functional substrate cleavage assay, skin wash samples from AD lesions were shown to contain 10‐ to 24‐fold more MMP activity than those from normal control skin (P < 0·02) and fivefold more than those from unaffected AD skin (P < 0·05); this activity was inhibited by a broad‐spectrum MMP inhibitor Ro 31‐9790. Gelatin zymography and antibody array analysis revealed substantial levels of MMP‐8 (neutrophil collagenase) and MMP‐9 (92‐kDa gelatinase) in AD skin wash samples as well as lower levels of MMP‐10 (stromelysin 2) and tissue inhibitor of metalloproteinases (TIMP)‐1 and TIMP‐2; low levels of MMP‐1 (fibroblast collagenase), MMP‐3 (stromelysin 1) and TIMP‐4 were also detected. Conclusions A simple skin wash technique suitable for the quantitative and functional analysis of biomolecules in AD is described. Using this method we show that MMPs, and in particular MMP‐8 and MMP‐9, represent an important potential component of the pathology of AD. The method is expected to prove useful in advancing our understanding of AD and in identifying biomarkers for the evaluation of new therapies.     

A simple method to assess severity of polymorphic light eruption

BACKGROUND: The severity of polymorphic light eruption (PLE) is highly variable. The results of studies of the prevalence, pathogenesis, provocation and treatment of PLE may be highly dependent on the severity of disease in the patients studied. OBJECTIVES: To produce a simple, valid and reproducible method to assess the severity of PLE. PATIENTS AND METHODS: Eighty patients were asked about the PLE they had experienced during the preceding 12 months, using a standardized interview comprising 16 questions. The answer to each question received a score. A PLE Severity Index (PLESI) was formulated, consisting of 10 questions, with a possible total score of 2-100. The internal consistency of the PLESI (the extent to which the responses to different questions correlated with each other) was assessed by reliability analysis, using Cronbach's method. Twenty patients were re-interviewed 7-27 days later to assess the repeatability of the PLESI. The ease of provocation of PLE by exposure at 24-h intervals to solar-simulated radiation was assessed on a five-point scale in nine of the 80 subjects (the EOPSSR score). RESULTS: The value of Cronbach's alpha for the PLESI was 0.77. The distribution of the PLESI was consistent with a normal distribution, with a mean value of 52.7 and standard deviation of 19.4. It had a coefficient of repeatability of 20.1. The PLESI was positively correlated with EOPSSR (rs =0.69, P = 0.039) and the number of years since onset of PLE (rs = 0.25, P = 0.03). There was no association between the PLESI and the duration of persistence of the eruption after ceasing sun exposure (rs = 0.12, P = 0.30), the development of tolerance as summer progressed (rs = -0.14, P = 0.39), gender (P = 0.50) or skin type (P = 0.87). CONCLUSIONS: This study has (i) validated the concept that a single score can reflect disease severity in PLE by showing that the principal characteristics of the condition, including, for example, the extent of anatomical distribution and the ease of provocation of the eruption, correlate with each other; (ii) formulated the PLESI, which is a simple, valid and reproducible way of assessing disease severity; we suggest it could be used worldwide to determine the severity of PLE among patients enrolled in future PLE research; (iii) shown that the ease with which the eruption is provoked by solar-simulated radiation correlates with the severity of the condition; and (iv) shown that the duration of persistence of the eruption after sun exposure does not correlate with the severity of the condition.     

Scale-sensitive fractal analysis using the patchwork method for the assessment of skin roughness

Background/aims: As skin roughness and wrinkles are easily perceived by the consumer, quantifying skin surface structures is a vital parameter for cosmetic product development. As more tools are available for measuring three-dimensional (3-D) surface data, instead of two-dimensional (2-D) profile lines, new algorithms are desirable, to take advantage of the information gathered.
Methods: The patchwork method tiles topographic data sets virtually and analyzes the change in apparent area as a function of tile scale. The patchwork method and conventional 2-D profilometric analysis were applied to 24 topographic skin data sets. The data sets were derived before and after application of 15% glycerol solution on the skin of eight volunteers.
Results: One hour after application, skin roughness decreased by 20.8%, as measured by conventional analysis, and by 23.3%, as measured by the patchwork method. For both methods, the differences were not significant.
Conclusions: The patchwork method can be applied to skin data and renders results similar in intensity and direction to conventional 2-D analysis. It is advantageous over conventional 2-D analysis in three ways: it makes use of the full topologic information, it requires no high-pass or low-pass filtering, and it is independent of the anisotropy of the skin.     

Implementation of fatty acid carriers to skin irritation and the epidermal barrier

Acute perturbations are followed by barrier repair and enhanced lipid synthesis, as well as cellular fatty acid trafficking, yet irritation of the skin may be induced by repeat disturbance of barrier function. Recently, new insights in cellular fatty acid transport and metabolism have evolved with respect to skin irritation and barrier disturbances: (1) Employing sodium dodecyl sulfate, skin irritation is accompanied by the induction of an epidermal (E) cytosolic fatty acid binding protein (FABP) associated with enhanced barrier repair. Whether E-FABP contributes to the water barrier function in normal skin remains to be elucidated; (2) Cutaneous inflammation, as it occurs in irritant contact dermatitis, can be reduced by peroxisome proliferating activated receptor (PPAR) agonists, such as linoleic acid, with clinical effects comparable to that of glucocorticoids; (3) PPARalpha agonists accelerate barrier recovery and enhance lamellar body synthesis, neutral lipid synthesis, in particular that of ceramides and cholesterol; (4) PPARalpha agonists increase the minimal erythema dose in UVB-irradiated human skin. This review provides a brief overview of the current understanding of mammalian fatty acid (FA) metabolism with respect to epidermal barrier abrogation and repair, including new insights into cellular FA transport and metabolism.     

A simple and efficient method for DNA extraction from skin and paraffin-embedded tissues applicable to T-cell clonality assays

PCR-based clonality assay of rearranged T-cell receptor genes gamma and beta (TCRG and TCRB) in a number of cases could be essential to discriminate between cutaneous T-cell lymphomas and reactive lymphoproliferative lesions in the skin. However, extraction of good-quality DNA from skin specimens (especially formalin-fixed paraffin-embedded) remains a challenge. Common procedures, being labour-intensive and time-consuming and requiring toxic solvents such as phenol and chloroform, still may end up with DNA sample of insufficient quality. We herewith present a simple and efficient method for DNA isolation based on ammonia extraction of tissue, followed by neutralization and simultaneous salting out of proteins with acetic acid. We have analysed 30 samples - 24 fresh (16 skin, two spleen and six lymph node) and six paraffin-embedded. Standard procedure (proteinase K digestion, followed by phenol/chloroform extraction) has been carried out simultaneously. We observed good PCR signal for TCRG rearrangements in 30 samples processed with the new protocol and only in 20 extracted with proteinase K/phenol/chloroform. For TCRB, the success rate was 29 of 30 with the new protocol, compared to 11 of 30 with conventional protocol. The proposed method of DNA extraction should improve the value of T-cell clonality assay, because insufficient DNA quality and quantity may bias analysis towards monoclonality and therefore cause false-positive results.     

A screening of skin changes, with special emphasis on neurochemical marker antibody evaluation, in patients claiming to suffer from "screen dermatitis" as compared to normal healthy controls

Abstract In the present study, facial skin from so-called “screen dermatitis” patients were compared with corresponding material from normal healthy volunteers. The aim of the study was to evaluate possible markers to be used for future double blind or blind provocation investigations. Differences were found for the biological markers calcitonin generelated peptide (CGRP), somatostatin (SOM), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI), neuropeptide tyrosine (NPY). protein S-100 (S-100). neuron-specific enolase (NSR), protein gene product (PGP) 9.5 and phenyl-ethanolamine N-methyltransferase (PNMT). The overall impression in the blind-coded material was such that it turned out easy to blindly separate the two groups from each other. However, no single marker was 100% able to pin-point the difference, although some were quite powerful in doing so (CGRP, SOM. S-100). However, it has to be pointed out that we cannot, based upon the present results, draw any definitive conclusions about the cause of the changes observed. Whether this is due to electric or magnetic fields, a surrounding airborne chemical, humidity, healing, stress factors, or something else, still remains an open question. Blind or double-blind provocations in a controlled environment are necessary to elucidate possible underlying causes for the changes reported in this investigation.