Consecutive Tape Strips Research Articles

080 The proteome of hand eczema assessed by tape stripping

Hand eczema (HE) is a highly prevalent, often multifactorial and potentially debilitating skin disease. Few studies have investigated subtype-specific biomarkers, and no studies have so far investigated the proteome of HE using tape strips. Here, we assessed the protein expression in subtypes of HE using two consecutive tape strips. Tape strips were collected from lesional and non-lesional skin of patients with HE (n=34) and healthy skin of age-matched controls (n=13). Patients with HE included both patients with (n=16) and without atopic dermatitis (AD) (n=18).

The stratum corneum transcriptome in atopic dermatitis can be assessed by tape stripping

BackgroundSkin biopsies represent a gold standard in skin immunology and pathology but can cause pain and induce scarring. Non-invasive techniques will facilitate study recruitment of e.g. patients with paediatric atopic dermatitis (AD), hand eczema or facial dermatitis. ObjectiveBy RNA sequencing, we examined whether the stratum corneum transcriptome in AD skin can be assessed by tape stripping, as compared to the epidermal transcriptome of AD in skin biopsies. To make the procedure clinically relevant tape strips were stored and shipped at room temperature for up to 3 days. MethodsNine adult Caucasian AD patients and three healthy volunteers were included. Tape samples were collected from non-lesional and lesional skin. Biopsies were collected from lesional skin and were split into epidermis and dermis. Total RNA was extracted, and shotgun sequencing was performed. ResultsShotgun sequencing could be performed on skin cells obtained from two consecutive tape strips which had been stored and shipped at room temperature for up to three days. The most prominent differences between the tape strip and biopsy derived transcriptome were due to structural genes, while established molecular markers of AD, including CCL17, CCL22, IL17A and S100A7-S100A9, were also identified in tape strip samples. Furthermore, the tape strip derived transcriptome showed promise in also analysing the skin microbiome. ConclusionOur study shows that the stratum corneum (SC) transcriptome of AD can be assessed by tape stripping the skin, supporting that this method may be central in future skin biomarker research.NCBI GEO data accession: GSE160501

Advancement through epidermis using tape stripping technique and Reflectance Confocal Microscopy

The tape stripping technique is increasingly used in research regarding skin barrier function. However, number of tape strips varies between studies, and literature considering advancement into stratum corneum/epidermis in relation to number of tape strips is scarce. The aim of this pilot study was to assess the advancement through epidermis using tape stripping technique in healthy volunteers. A total of ten healthy volunteers were included. From all volunteers 0, 5, 15 and 35 consecutive tape strips (D-squame) were taken from four adjacent skin areas on the middle volar forearm, followed by Reflectance Confocal Microscopy (RCM) of the four areas to assess epidermal thickness. Squame Scan was used to determine amount of protein removed. Stratum corneum was completely removed in all volunteers after 35 tape strips. Advancement into epidermis was predominantly achieved by the first 15 tape strips, removing 25% of the total epidermis, whereas 35 tape strips removed 33% of epidermis. Protein removal per tape decreased with increasing depth. Information on advancement into the epidermis according to number of tape strips taken, is a significant step forward. The possibility to obtain samples from different layers of epidermis may lead to an improved understanding of skin barrier properties.

Noninvasive analysis of skin proteins in healthy Chinese subjects using an Orbitrap Fusion Tribrid mass spectrometer.

The objective of this study was to perform noninvasive analysis of skin proteins in a healthy Chinese population using label-free nanoflow liquid chromatography-mass spectrometry (nLC-MS). Five consecutive tape strippings were obtained from the volar forearm skin of healthy Chinese subjects. Proteins were extracted, and trypsin-digested peptides were analyzed by a nanochromatography instrument coupled to an Orbitrap Fusion Tribrid mass spectrometer. Data-dependent acquisition allowed protein identification, which was performed by using Proteome Discoverer software (v2.2). In this study, we identified 80 common proteins that were expressed in the skin of healthy Chinese volunteers and divided these proteins into 16 categories, including keratins, cornified envelope proteins, and enzymes associated with substance metabolism. These proteins were closely associated with multiple functions of the skin barrier. This study provides a noninvasive method to analyze healthy human epidermal proteins, which are closely associated with the skin barrier. In addition, this study provides a reference for further studies on the application of proteomic technologies to investigate the role of human epidermal proteins in health and disease.

Measurements of AMPs in stratum corneum of atopic dermatitis and healthy skin\u2013tape stripping technique

Decreased levels of antimicrobial peptides (AMPs) in atopic dermatitis (AD) have previously been reported and have been linked to the increased susceptibility to skin infections found in AD patients. This study intents to identify AMPs: hBD-2, hBD-3, RNase7, psoriasin and LL-37 in AD patients and healthy controls, and determine concentrations in consecutive depths of the outer most skin layers. Tape stripping was used on lesional and non-lesional skin. From each skin site, 35 consecutive tape strips were collected and pooled in groups of 5. Commercially available ELISA kits were used to determine AMP concentration in stratum corneum samples. hBD-2, hBD-3, RNase7 and psoriasin were identified in stratum corneum samples. hBD-3-level was markedly higher in AD non-lesional skin compared to healthy controls, and a similar trend was observed for RNase7. Most AMPs were distributed evenly through 35 tape strips, implying a homogeneous distribution of antimicrobial defense in the outer most skin layers. The findings indicate that AD patients may not suffer from a general baseline deficiency in AMPs, and that the innate immune defense is present throughout the stratum corneum, both insights of importance for understanding the role of AMPs in AD.

The effect of photodamage on the female Caucasian facial stratum corneum corneome using mass spectrometry-based proteomics.

The effect of photodamage on facial stratum corneum (SC) is still poorly understood. To describe the SC proteome from tape strippings of Caucasian SC from photoexposed cheek and photoprotected post-auricular (PA) site, a global analysis of photodamage on the skin will be developed leading to a better understanding of keratinocyte signalling pathways and identification of new molecular targets for the treatment of photoaged skin. Female Caucasian subjects had nine consecutive tape strippings taken from their cheeks and PA site. Proteins were extracted and the trypsin-digested peptides were analysed by nanochromatography coupled to a high-resolution mass spectrometer. Data-dependent acquisition allowed protein identification that was processed by Paragon algorithm of Protein Pilot software. Changes in the levels of epidermal differentiation proteins were apparent indicating poor epidermal differentiation and SC maturation (keratins, cornified envelope (CE) proteins) on photoexposed cheeks. Differences in protease-anti-protease balance were observed for corneodesmolysis (favouring desquamation) and filaggrinolysis (favouring reduced filaggrin processing). 12R-LOX, a CE maturation enzyme, was reduced in photodamaged skin but not transglutaminases. Changes in signal keratinocyte transduction pathway markers were demonstrated especially by reduced levels of downstream signalling markers such as calreticulin (unfolded protein response; UPR) and increased level of stratifin (target of rapamycin; mTOR). Evidence for impaired proteostasis was apparent by reduced levels of a key proteasomal subunit (subunit beta type-6). Finally, key antioxidant proteins were upregulated except catalase. Clear examples of poor keratinocyte differentiation and associated metabolic and signalling pathways together with reduced SC maturation were identified in photodamaged facial SC. Corneocyte immaturity was evident with changes in CE proteins. Particularly, the reduction in 12R-LOX is a novel finding in photodamaged skin and supports the lack of SC maturation. Moreover, filaggrinolysis was reduced, whereas corneodesmolysis was enhanced. From our results, we propose that there is a poor cross-talk between the keratinocyte endoplasmic reticulum UPR, proteasome network and autophagy machinery that possibly leads to impaired keratinocyte proteostasis. Superimposed on these aberrations is an apparently enhanced mTOR pathway that also contributes to reduced SC formation and maturation. Our results clearly indicate a corneocyte scaffold disorder in photodamaged cheek SC.

Facial skin pigmentation is not related to stratum corneum cohesion, basal transepidermal water loss, barrier integrity and barrier repair.

Hypotheses have been developed for the evolutionary selection of skin pigmentation one of which relates to improved skin barrier function. The aim of this study was to compare facial skin condition on photoexposed (cheek) and photoprotected (post-auricular) sites of naturally pigmented subjects of different ethnicities (Fitzpatrick skin phototypes II/III and V/VI) and Albino African subjects to understand better the relationship between facial stratum corneum (SC) barrier function, skin surface pH and skin pigmentation. Expert grading of skin conditions, capacitance, skin surface pH and skin barrier function measurements were performed. For the latter, transepidermal water loss (TEWL) measurements before (basal TEWL), after 3, 6 and 9 consecutive tape strippings (SC integrity) and 3.5 and 24 h post tape stripping (barrier recovery) were taken. Amounts of SC protein removed during stripping were estimated using infrared densitometry (SC cohesion). Firstly, correlation analysis of the biometric data of the Black African and Caucasian subjects showed there to be no relationship between skin surface pH and ITA° values nor pH and ITA° with basal TEWL. Neither skin surface pH nor ITA° correlated with SC integrity and barrier recovery measurements, but skin surface pH correlated with SC cohesion. ITA° values were correlated with skin hydration. Secondly, on comparing the three ethnic groups, severe skin photodamage was observed in the Albino African subjects and their SC was thicker. Whereas their basal TEWL was elevated, superior values for SC integrity and barrier recovery were measured. No differences in basal TEWL, SC integrity and barrier recovery were found between the other two subject groups. Equally, SC cohesion and skin surface pH values were similar among the three groups. There was no relationship between ITA° values and basal TEWL, SC integrity, SC cohesion and barrier recovery, but ITA° was correlated with skin hydration. Skin surface pH, irrespective of ITA° values, correlated with SC cohesion, indicating a greater intracorneal cohesion at lower pH values. Thus, pigmentation has no effect on SC barrier properties but was related to skin hydration. On comparing the three ethnic groups, Albino African SC was found to be superior to the Caucasian and Black African subjects in terms of SC integrity and barrier recovery but not basal TEWL. The Albino African subjects also have a thicker SC which contributes to their better SC integrity. No differences in skin barrier functionality or skin surface pH were observed for the other two groups. Skin hydration was, however, greatest in the Black African subjects. Our data support the evolutionary hypothesis that pigmentation protects the skin from UV irradiation and thereby the skin barrier but not the skin pigmentation-/pH-driven adaptive skin barrier hypothesis.

Surface distribution and depths profiling of particulate organic UV absorbers by Raman imaging and tape stripping

Tape stripping in conjunction with scanning Raman microscopy was used for assessing the lateral and vertical distribution of an organic particulate UV filter, methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT), in a sunscreen formulation. On the volar forearms of three volunteers, 1 mg cm(-2) formulation containing 10% MBBT was applied, and the average amount of MBBT was measured by Raman scanning microscopy in 15 consecutive tape strippings. The recovery of MBBT was 91.1% with 30.2% localizing on the skin surface (first strip), 42.5% in the upper stratum corneum (strips 2-5) and from 3.6 down to 0.8% in each of the 10 consecutive layers. The concentration of surface deposits of MBBT differed by a factor of 300 between folds, furrows and pores on the one hand and the interjacent ridges on the other hand. Seventy-five per cent of the applied particles occupied a fifth of the evaluated area - where concentrating in folds and furrows - as was confirmed by 3-D reconstruction. On interjacent ridges, 8.6% of MBBT distributed as very thin films preferentially. MBBT localized at sites not connected with the surface, such as in truncated pores or as potentially penetrated material amounted to 0.06% or to a twentieth of the 1.4% found in the lowest skin strippings. Scanning Raman microscopy in combination with tape stripping documented the lateral and vertical distribution quantitatively and at cellular (12.5 μm) lateral resolution. Our results confirmed an earlier report on the vertical distribution of organic particles applied to skin and was in line with similar reports on TiO(2) distribution.

Profiling of serine protease activities in human stratum corneum and detection of a stratum corneum tryptase‐like enzyme

Seasonal variation in stratum corneum (SC) biophysical and biological characteristics has been described previously. In particular, the winter season has been shown to affect more severely the properties of facial skin compared with forearm skin. Moreover, when compromised, such as in dry skin conditions, facial SC has been shown to contain increased inflammatory cytokines and proteases. Nevertheless, there have been no comparative studies of the activities and depth activity of several proteases in the SC on different body sites and at different times of the year. In this study, we examined the distribution of key serine protease activities (kallikrein 5, kallikrein 7, urokinase, plasmin and a tryptase-like enzyme) in different layers of the SC on the cheek and the forearm by analysis of consecutive tape strippings of healthy Caucasian subjects during winter and summer. The protein content of the tape strippings was quantified by absorption measurements with a recently developed and novel infrared densitometer SquameScan 850A while the SC enzyme activities were determined using fluorogenic peptide substrates. Transepidermal water loss (TEWL), skin pH and skin hydration were higher on the cheek than on the forearm. In the same way, the activity of the inflammatory-related proteases plasmin, urokinase and tryptase was approximately five to eight times and the activity of the desquamatory-related proteases kallikrein 5 and kallikrein 7 approximately two to four times higher on the cheek than on the forearm. There were no gender-related differences in these enzyme activities except for the increased kallikrein 7 in the forearm skin of the female subjects in winter. Reduced kallikrein 5 was associated with increased SC cohesion, as judged by increased protein removal, in forearm skin in the winter months of the year although the skin was clinically normal. It can be concluded that (i) protected skin areas show lower TEWL, skin pH and skin hydration and less protease activities than skin areas that are exposed to the environment, possibly indicating subclinical inflammation on these body sites, (ii) in normal healthy forearm skin, the outer SC exhibits greater serine protease activity than its deeper layers, (iii) compared with the forearm, urokinase- and plasmin-like activities are elevated on SC strippings from the cheek, confirming activation of the plasminogen cascade, and (iv) tryptase-like activity in the SC is also elevated in samples from the cheek, possibly indicating involvement of mast cells in these barrier-compromised body sites or the synthesis of a novel tryptase-like enzyme by keratinocytes. Although elevation of the activities of urokinase, plasmin, kallikrein 5, kallikrein 7 and now a tryptase-like enzyme was observed on SC derived from skin of clinically normal cheeks, we anticipate even higher activities in skin conditions where the epidermal barrier is further impaired.

Development, Evaluation and Data Acquired with a Tape-Stripping Technique for Measuring Dermal Exposure to Budesonide at a Pharmaceutical Manufacturing Site

Although corticosteroids have been used for over 50 years as anti-inflammatory and anti-proliferative agents, few studies have examined their exposure levels and health effects on workers employed in the corticosteroid manufacturing industry. The aims of the study reported here were to develop a tape-stripping technique for monitoring budesonide (a corticosteroid used in inhalators for treating respiratory diseases) and to apply the method in a pilot study to estimate the potential dermal exposure to budesonide among workers at a pharmaceutical formulation site. The tape-stripping method was evaluated by applying 0.5 and 2.07 microg of budesonide dissolved in ethanol on tape strips. The same amounts were also applied on a cleaned glass plate and human skin of volunteers, which were then stripped by series of tapes immediately, and 30 min later, the amounts collected by the tapes were measured. Finally, the technique was used to study the exposure of budesonide among eight employees at a pharmaceutical industry site. Three exposure sites were tested: the tip of the forefinger, palm of the hand and ventral part of the lower arm. Five consecutive tape strips per sampling site were used in both the recovery studies and the field study. The mean overall recoveries from spiked tapes and the glass plate were 96 and 81%, respectively, while for human skin the corresponding figure was 38%, (for applications of 2.07 microg; no detectable amounts were recovered from human skin after 0.5 microg applications). The recovered amount was found on two consecutive tapes after 0 min, but only on the first tape strip after 30 min. The inter-individual variability was 4-fold. In the field, quantifiable amounts were found for four of eight employees and a concentration gradient was detected along the two or three consecutive tape strips. The tip of the forefinger and the palm of the hand were the most highly exposed sites to budesonide. A tape-stripping method can be used to determine potential dermal exposure to budesonide. The results also indicate that budesonide is taken up by the skin of operators who are exposed to the substance at their workplace.

Novel Method for Studying Photolability of Topical Formulations: A Case Study of Titanium Dioxide Stabilization of Ketoprofen

Sunlight may decompose active substances and excipients in pharmaceuticals. This may cause formulation problems as well as induce adverse skin reactions. The photodecomposition of topical preparations may occur on the skin surface, but also deeper in the skin after penetration of light into the viable tissues. The aim of the present study was to investigate whether microparticles of titanium dioxide could protect against photodecomposition using ketoprofen as a photolabile model substance. The results showed quality differences between titanium dioxide, where surface-coated particles were superior to pharmaceutical grades in reducing the degradation in vitro. The protective effect was also studied in humans. The skin was treated for 3 h with the gels and then exposed to ultraviolet (UV) light (11.7 J/cm2 UVA and 5.4 mJ/cm2 UVB). Layers of the stratum corneum were then removed by consecutive tape strippings and assayed for content of ketoprofen. The remaining amount was higher in the different stratum corneum compartments after treatment with a gel containing 4% coated titanium dioxide compared with a transparent gel. Thus, surface-coated microparticles of titanium dioxide may well be of clinical benefit in protecting photolabile drug substances against sunlight.

Determination of keratin protein in a tape-stripped skin sample from jet fuel exposed skin.

Chemical contaminants or their metabolites may bind to and react with keratin proteins in the stratum corneum of the skin. Here, we present a tape-stripping method for the removal and quantification of keratin from the stratum corneum for normalization of extracted concentrations of naphthalene (as a marker for jet fuel exposure) from 12 human volunteers before and after exposure to jet fuel (JP-8). Due to the potential for removal of variable amounts of squamous tissue from each tape-strip sample, keratin was extracted and quantified using a modified Bradford method. Confirmation of the extraction of keratin was verified by western blotting using a monoclonal mouse anti-human cytokeratin antibody. Naphthalene was quantified in the sequential tape strips collected from the skin between 10 and 25 min after a single dose of JP-8 was initially applied. The penetration of jet fuel into the stratum corneum was demonstrated by the fact that the average mass of naphthalene recovered by a tape strip decreased with increased exposure time and subsequent tape strips and that the evaporation of naphthalene was observed to be negligible. There were no significant differences in the amount of keratin or naphthalene removed by tape strips between males and females, between age groups, races or degrees of skin pigmentation. We conclude that (i) the amount of keratin removed with tape strips was not affected by up to a 25 min exposure to JP-8 and (ii) there was a substantial decrease in the amount of keratin removed with consecutive tape strips from the same site, thus, adjusting the amount of naphthalene by the amount of keratin measured in a tape-strip sample should improve the interpretation of the amount of this analyte using this sampling approach. Although we found that normalization of the naphthalene to the amount of keratin in the tape-strip samples did not affect the ability of this method to quantify the dermal exposure to JP-8 under these laboratory conditions, the actual concentration of naphthalene (as a marker for JP-8 exposure) per unit of keratin in a tape-strip sample can be determined using this method and may prove to be required when measuring occupational exposures under field conditions.

A tape-stripping method for measuring dermal exposure to multifunctional acrylates

Current methods for measuring dermal exposure to skin irritants and allergens, such as acrylates, have significant drawbacks for exposure assessment. A noninvasive sampling method has been developed and tested for measuring dermal exposure to a multifunctional acrylate employing a tape stripping of the nonviable epidermis (stratum corneum). Samples were subsequently extracted and a gas chromatographic method was employed for quantitative analysis of tripropylene glycol diacrylate (TPGDA). This method was tested in 10 human volunteers exposed to an a priori determined amount of TPGDA or a UV-radiation curable acrylate coating containing TPGDA (UV-resin) at different sites on hands and arms. On the average, the first tape stripping removed 94% (coefficient of variation 16%) of the theoretical quantity of deposited TPGDA and 89% (coefficient of variation 15%) of the theoretical quantity of deposited TPGDA in UV-resin 30min after exposure. Quantities of TPGDA recovered from two consecutive tape strippings accounted for all of the test agent, demonstrating both the efficiency of the method to measure dermal exposure and the potential to determine the rate of absorption with successive samples over time. In general, the amount removed by the first stripping was greater for TPGDA than for UV-resin while the second stripping removed approximately 6 and 21% of TPGDA and UV-resin, respectively. However, when the amounts removed with the first tape stripping for TPGDA or UV-resin from the five different individual sites were compared, no significant differences were observed (P=0.111 and 0.893, respectively). No significant difference was observed in recovery between TPGDA and UV-resin for the first tape stripping when calculated as a percentage of the theoretical amount (P=0.262). The results indicate that this tape-stripping technique can be used to quantify dermal exposure to multifunctional acrylates.

A tape-stripping method for measuring dermal exposure to multifunctional acrylates

Current methods for measuring dermal exposure to skin irritants and allergens, such as acrylates, have significant drawbacks for exposure assessment. A noninvasive sampling method has been developed and tested for measuring dermal exposure to a multifunctional acrylate employing a tape stripping of the nonviable epidermis (stratum corneum). Samples were subsequently extracted and a gas chromatographic method was employed for quantitative analysis of tripropylene glycol diacrylate (TPGDA). This method was tested in 10 human volunteers exposed to an a priori determined amount of TPGDA or a UV-radiation curable acrylate coating containing TPGDA (UV-resin) at different sites on hands and arms. On the average, the first tape stripping removed 94% (coefficient of variation 16%) of the theoretical quantity of deposited TPGDA and 89% (coefficient of variation 15%) of the theoretical quantity of deposited TPGDA in UV-resin 30min after exposure. Quantities of TPGDA recovered from two consecutive tape strippings accounted for all of the test agent, demonstrating both the efficiency of the method to measure dermal exposure and the potential to determine the rate of absorption with successive samples over time. In general, the amount removed by the first stripping was greater for TPGDA than for UV-resin while the second stripping removed approximately 6 and 21% of TPGDA and UV-resin, respectively. However, when the amounts removed with the first tape stripping for TPGDA or UV-resin from the five different individual sites were compared, no significant differences were observed (P=0.111 and 0.893, respectively). No significant difference was observed in recovery between TPGDA and UV-resin for the first tape stripping when calculated as a percentage of the theoretical amount (P=0.262). The results indicate that this tape-stripping technique can be used to quantify dermal exposure to multifunctional acrylates.