Visualizing tropoelastin in a long-term human elastic fibre cell culture model.

Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin-fibrillin-1 fibre network. Future investigations will allow the tracking of TE produced under various conditions. In tissue engineering applications the fluorescent fibre network can be visualized under various conditions or it serves as a tool for investigating fibre degradation processes in disease-in-a-dish-models.

Effective treatment for sensitive skin: 4-t-butylcyclohexanol and licochalcone A.

BACKGROUND: More than 50% of adults report to suffer from sensitive skin. This common condition is characterized by subjective sensations such as prickling, burning, skin tightness or pruritus, and is often accompanied by objective symptoms like inflammation and erythema. OBJECTIVE: The objective of this study was to develop an active ingredient concept for the treatment of sensitive skin. We tested compounds regarding their potential to (i) decrease the release of proinflammatory mediators, which among others induce erythema and (ii) counteract the hyperresponsiveness of nerve fibres and, thus, exert effects on cutaneous neurosensory dysfunction. METHODS: 4-t-butylcyclohexanol, licochalcone A and acetyl dipeptide-1 cetyl ester were analysed in vitro regarding their potential to (i) decrease the release of PGE2 and activation of NFκB and to (ii) inhibit TRPV1 activation or the release of neuronal CGRP. To assess subjective and objective symptoms of skin sensitivity in vivo, two controlled, single-blind, randomized studies were conducted with 4-t-butylcyclohexanol and the combination with licochalcone A. RESULTS: In vitro, 4-t-butylcyclohexanol significantly reduced TRPV1 activation, while acetyl dipeptide-1 cetyl ester had no effect on receptor activation. Licochalcone A significantly decreased NFκB signalling and PGE2 secretion, at lower concentrations than acetyl dipeptide-1 cetyl ester. A formulation containing 4-t-butylcyclohexanol showed a significant immediate anti-stinging/anti-burning effect in vivo, and a cream base containing a combination of 4-t-butylcyclohexanol and a licochalcone A-rich licorice extract reduced shaving-induced erythema. CONCLUSION: In vitro and in vivo data indicate that the combination of the TRPV1 antagonist 4-t-butylcyclohexanol and the potent anti-inflammatory licochalcone A provide an effective active ingredient concept for the treatment of sensitive skin, as the topical application resulted in an immediate relief from symptoms such as erythema and stinging.

The matricellular protein periostin contributes to proper collagen function and is downregulated during skin aging.

BACKGROUND: Periostin is a secreted 90kDa matricellular protein, which is predominantly expressed in collagen-rich tissues. Collagen is the most abundant protein in mammals and has great tensile strength. Recent investigations have shown that periostin influences collagen fibrillogenesis and biomechanical properties of murine connective tissues. OBJECTIVE: We investigated the function of periostin concerning collagen homeostasis during intrinsic and extrinsic skin aging. For this purpose, human skin samples of young and old donors as well as samples of photoaged and sun-protected skin areas were analyzed for periostin expression. Using in vitro models, we determined the cell types responsible for periostin expression and performed functional analyses with periostin knockdown cells. METHODS: TaqMan Real-Time PCR, UV irradiation, knockdown experiments, immunostaining, electron microscopy, collagen degradation assay, collagen crosslink analysis. RESULTS: Periostin expression is highest in the papillary dermis and downregulated during skin aging. Fibroblasts and non-follicular skin derived precursors were identified as main source for periostin expression in human skin. Periostin knockdown in fibroblasts has no effect on collagen expression, but results in an increased fibril diameter and aberrant collagen structure. This leads to an increased susceptibility of collagen toward proteases, whereas recombinant periostin protects collagen fibrils from degradation. CONCLUSION: Our data show that periostin plays an important role for proper collagen assembly and homeostasis. During skin aging periostin expression decreases and contributes to the phenotype of aged skin.

Quantification of cyclobutane pyrimidine dimers in human epidermal stem cells.

The common procedures that are used to quantify cyclobutane pyrimidine dimers (CPD) comprise the extraction of cellular DNA followed by the detection of this nucleic acid modification by immunoblotting or electrophoretic methods. Consequently, these approaches provide an averaged damage intensity value of a whole population of cells and are not applicable to studies where a small subgroup such as somatic stem cells are intended to be investigated and the individual cellular damage is of interest. Here, we describe a strategy to isolate epidermal stem cells from minimum human epidermis samples and a subsequent immunocytochemical quantification of cellular CPDs. Besides the determination of the DNA damage status, this technique allows for the examination of cellular CPD intensity distributions.

4-n-butylresorcinol, a highly effective tyrosinase inhibitor for the topical treatment of hyperpigmentation.

BACKGROUND: Hyperpigmentary disorders like melasma, actinic and senile lentigines are a major cosmetic concern. Therefore, many topical products are available, containing various active ingredients aiming to reduce melanin production and distribution. The most prominent target for inhibitors of hyperpigmentation is tyrosinase, the key regulator of melanin production. Many inhibitors of tyrosinase are described in the literature; however, most of them lack clinical efficacy. METHODS: We were interested in evaluating the inhibition of skin pigmentation by well-known compounds with skin-whitening activity like hydroquinone, arbutin, kojic acid and 4-n-butylresorcinol. We compared the inhibition of human tyrosinase activity in a biochemical assay as well as inhibition of melanin production in MelanoDerm skin model culture. For some compounds, the in vivo efficacy was tested in clinical studies. RESULTS: Arbutin and hydroquinone only weakly inhibit human tyrosinase with a half maximal inhibitory concentration (IC(50)) in the millimolar range. Kojic acid is 10 times more potent with an IC(50) of approximately 500 µmol/L. However, by far the most potent inhibitor of human tyrosinase is 4-n-butylresorcinol with an IC(50) of 21 µmol/L. In artificial skin models, arbutin was least active with an IC(50) for inhibition of melanin production > 5000 µmol/L. Kojic acid inhibited with an IC(50) > 400 µmol/L. Interestingly, hydroquinone inhibited melanin production in MelanoDerms with an IC(50) below 40 µmol/L, probably due to a mechanism different from tyrosinase inhibition. Again, 4-n-butylresorcinol was the most potent inhibitor with an IC(50) of 13.5 µmol/L. In vivo efficacy of 4-n-butyl-resorcinol was confirmed in clinical studies. Subjects with age spots on the forearm treated twice daily two age spots with a formula containing 4-n-butylresorcinol and two control age spots with the corresponding vehicle. Within 8 weeks, 4-n-butylresorcinol reduced visibly the appearance of age spots, while the control spots showed no improvement. A second study showed that 4-butylresorcinol was more effective than 4-hexylresorcinol and 4-phenylethylresorcinol. CONCLUSION: The present in vitro and in vivo data prove the high inhibitory capacity of 4-n-butylresorcinol on human tyrosinase activity, exceeding by far the potency of hydroquinone, arbutin and kojic acid. The resulting clinical improvement of skin hyperpigmentations reveals 4-n-butylresorcinol as a very valuable active compound for the management of pigmentation disorders.

Effects of glyceryl glucoside on AQP3 expression, barrier function and hydration of human skin.

BACKGROUND/AIM: Aquaporins (AQPs) present in the epidermis are essential hydration-regulating elements controlling cellular water and glycerol transport. In this study, the potential of glyceryl glucoside [GG; alpha-D-glucopyranosyl-alpha-(1->2)-glycerol], an enhanced glycerol derivative, to increase the expression of AQP3 in vitro and ex vivo was evaluated. METHODS: In vitro studies with real-time RT-PCR and FACS measurements were performed to test the induction by GG (3% w/v) of AQP3 mRNA and protein in cultured human keratinocytes. GG-containing formulations were applied topically to volunteer subjects and suction blister biopsies were analyzed to assess whether GG (5%) could penetrate the epidermis of intact skin, and subsequently upregulate AQP3 mRNA expression and improve barrier function. RESULTS: AQP3 mRNA and protein levels were significantly increased in cultured human keratinocytes. In the studies on volunteer subjects, GG significantly increased AQP3 mRNA levels in the skin and reduced transepidermal water loss compared with vehicle-controlled areas. CONCLUSION: GG promotes AQP3 mRNA and protein upregulation and improves skin barrier function, and may thus offer an effective treatment option for dehydrated skin.

Poly(A) tail shortening correlates with mRNA repression in tropoelastin regulation.

BACKGROUND: It has been shown for various organisms that expression of tropoelastin (TE) is high during fetal and neonatal growth and that it is reduced in adulthood by an unknown mechanism. OBJECTIVE: To highlight the process of TE mRNA repression in vivo, total RNA from human skin biopsies was analyzed and TE mRNA expression was compared in fetal and adult donors. METHODS: TaqMan Real-Time PCR, Poly(A) tail length assay, immunoblot. RESULTS: In this study a more than 30-fold reduction of mature TE mRNA was detected whereas the decline on pre-mRNA level was not pronounced. This finding supports the hypothesis that the repression of mature TE mRNA is for the most part due to posttranscriptional mechanisms. Since deadenylation-dependent mRNA destabilization is the major decay pathway for most mRNAs, poly(A) tail length of mature TE mRNA was analyzed in fetal and adult human skin, lung and uterus, showing a profound reduction of poly(A) tail length in the adult samples. While TE mRNA is repressed in adult tissues in vivo, TGF-ß(1) has been shown to induce expression of TE mRNA in vitro on the posttranscriptional level. To analyze the underlying mechanism, TE mRNA poly(A) tail length was analyzed in human dermal fibroblasts after treatment with TGF-ß(1)in vitro. Besides the expected increase in TE expression, TGF-ß(1) treatment resulted in a significant stabilization of TE mRNA poly(A) tail length. CONCLUSION: Our findings correlate for the first time TE expression level with poly(A) tail length and suggest that maintenance of poly(A) tail and deadenylation of TE mRNA might be general mechanisms involved in the regulation of TE expression.

Characterization of fibrillar collagen types using multi-dimensional multiphoton laser scanning microscopy.

In dermal photodamage the ratio of the collagen types III to I changes. This makes the investigation of the fibrillar collagen type characteristics interesting for skin research. In this study collagen types were characterized using 5-dimensional multiphoton laser scanning microscopy (5D-IVT) that can be applied in vivo. Second harmonic generation (SHG) signals and fluorescence lifetimes of the collagen autofluorescence were analysed. Collagen type I generates a higher SHG intensity and a longer fluorescence lifetime compared to collagen type III. Thus, the SHG intensity decrease found in photodamaged skin might be explained by the increase in collagen type III. Calculating the in vivo relevant increase of collagen type III gives a negligible difference in fluorescence lifetime not qualifying this method for the determination of collagen type changes in dermal photodamage in vivo in human skin. However, for pathologies that exhibit higher differences in collagen types 5D-IVT analysis might be a suitable method.

Qualification of a precise and easy-to-handle sweat casting imprint method for the prediction and quantification of anti-perspirant efficacy.

A time- and cost-effective sweat casting method using the forearm as test site to assess the efficacy of several anti-perspirant formulations with a low number of test subjects has been evaluated and qualified. The imprint sweat casting method is based on a 2-component silcone-imprint technique to measure the efficacy of more than eight products in parallel with the same test subject. In studies using aluminum chlorohydrate (ACH) formulations as test anti-perspirants, a clear-cut correlation could be demonstrated between sweat gland activities measured by the imprint method and gravimetric measurement of sweat gland activities. Concentration-dependent inhibition of sweat gland activity could be observed with the imprint technique up to an ACH concentration of 15%, and all formulations containing 2% ACH or above resulted in statistically significant reduction of sweat gland activity (P < 0.001) when compared with untreated control areas. Furthermore, the SDs of individual studies using the imprint technique were in a range of +/-20% of sweat gland activity, which can be regarded rather low for in vivo measurements of a complex process like sweat secretion. A group-wise comparison between the measurements of anti-perspirant activity as determined by the imprint protocol and the Food and Drug Administration (FDA) Guideline compliant gravimetric hot-room protocol revealed that the test results for anti-perspirant activity obtained with the imprint protocol are similar to those obtained with the hot-room protocol. Moreover, the data generated with the imprint protocol have a high predictive value for the outcome of a later guideline-compliant hot-room test. As the imprint casting method tends to be a little more sensitive for formulations with low anti-perspirant activity, and seems to be associated with less interassay variability than the standard gravimetric hot-room test, the imprint casting method may select products which later fail to pass the standard gravimetric hot-room test. Meanwhile the imprint sweat casting has proven to be a robust method useful to support efficacy-oriented product development. Therefore, in later stages of utilization it might even evolve into an efficient claim substantiation tool.

Aging skin is functionally anaerobic: importance of coenzyme Q10 for anti aging skin care.

The functional loss of mitochondria represents an inherent part in modern theories trying to explain the cutaneous aging process. The present study shows significant age-dependent differences in mitochondrial function of keratinocytes isolated from skin biopsies of young and old donors. Our data let us postulate that energy metabolism shifts to a predominantly non-mitochondrial pathway and is therefore functionally anaerobic with advancing age. CoQ10 positively influences the age-affected cellular metabolism and enables to combat signs of aging starting at the cellular level. As a consequence topical application of CoQ10 is beneficial for human skin as it rapidly improves mitochondrial function in skin in vivo.

Pros and cons: cryo-electron microscopic evaluation of block faces versus cryo-sections from frozen-hydrated skin specimens prepared by different techniques.

Over the last two decades, several different preparative techniques have been developed to investigate frozen-hydrated biological samples by electron microscopy. In this article, we describe an alternative approach that allows either ultrastructural investigations of frozen human skin at a resolution better than 15 nm or sample throughput that is sufficiently high enough for quantitative morphological analysis. The specimen preparation method we describe is fast, reproducible, does not require much user experience or elaborate equipment. We compare high-pressure freezing with plunge freezing, and block faces with frozen-hydrated slices (sections), to study variations in cell thickness upon hydration changes. Plunge freezing is optimal for morphological and stereological investigations of structures with low water content. By contrast, high-pressure freezing proved optimal for high-resolution studies and provided the best ultrastructural preservation. A combination of these fast-freezing techniques with cryo-ultramicrotomy yielded well-preserved block faces of the original biological material. Here we show that these block faces did not exhibit any of the artefacts normally associated with cryo-sections, and--after evaporating a heavy metal and carbon onto the surface--are stable enough in the electron beam to provide high-resolution images of large surface areas for statistical analysis in a cryo-SEM (scanning electron microscope). Because the individual preparation steps use only standard equipment and do not require much experience from the experimenter, they are generally more usable, making this approach an interesting alternative to other methods for the ultrastructural investigation of frozen-hydrated material.

Contact angle measurement - a reliable supportive method for screening water-resistance of ultraviolet-protecting products in vivo.

Substantivity of sunscreen formulations is affected by the wash-out rate of ultraviolet-absorber and -reflector compounds in water. Water-resistance of sunscreen formulations is currently determined according to a standardized European Cosmetic Toiletry and Perfumery Association (COLIPA) protocol, encompassing the determination of a minimal erythemal dose before and after a defined immersion step in water. It can be supposed that the higher the wettability of a treated skin area, the higher is the wash-out rate of sunscreen compounds. This present report addresses the validity of determining the wettability of treated skin alone as a measure for the water-resistance of sunscreen products. The report addresses the robustness, accuracy and congruence of a recently developed wettability test, based on the measurement of the contact angle (CA) of a sessile water drop on treated skin areas. Contact angle data of 66 sunscreen formulations are compared with the corresponding results of 81 water-resistance tests, using the sun protection factor (SPF)/immersion/SPF method. Sunscreen products tested by the CA method were applied to the skin of the volar forearm of test subjects at a defined dose and drying-time, using a standardized application and recording device. Contact angles between a sessile water drop and skin were recorded by a Charge-Coupled Device (CCD) camera and subjected to automatic contour analysis. Taking the SPF/immersion/SPF method as gold standard, accuracy parameters of the CA method were determined. By using an appropriate cut-off level of CAs, the CA method has a specificity and positive-predictive value of 100%, and turns out to be a reliable screening method to identify water-resistant formulations. Based on our findings, those formulations that give CAs above 30 degrees may be categorized water-proof without further testing by the COLIPA water-resistance method.

Alterations in the epidermal-dermal melanin axis and factor XIIIa melanophages in senile lentigo and ageing skin.

BACKGROUND: Senile lentigo (SL) is a pigmentation disorder that occurs predominantly on the dorsa of the hands, the forearms and the face; its incidence increases with age. Histological hallmarks of SL lesions are hyperpigmentation of the epidermis and elongation of the epidermal rete ridges. Various factors such as alpha-melanocyte-stimulating hormone, endothelin-1 or stem cell factor are involved in the onset and maintenance of the increased pigmentation. Alterations of the dermal compartment have not yet been analysed in detail in SL. OBJECTIVES: To study the occurrence and distribution of melanin in the dermis from SL and aged skin, biopsies from 12 subjects were morphologically analysed by light and electron microscopy in comparison with unaffected skin. METHODS: Punch biopsies of SL and adjacent skin from 12 male or female volunteers aged 52-81 years were prepared for light and electron microscopy and samples were analysed by morphological, morphometric, histochemical and immunohistochemical methods. RESULTS: The epidermis from SL revealed morphological features such as hyperpigmentation of basal keratinocytes and the formation of elongated rete ridges. S100+ melanocytes in the stratum basale were not markedly increased, indicating that the hyperpigmentation is predominantly due to changes in melanin synthesis, distribution or turnover. Quantification of epidermal cells expressing the proliferation marker Ki67 did not show an increase of this parameter in SL, indicating that at least in the established lesion cell proliferation is not enhanced. We further focused on the dermal compartment and observed granulated cells which were more abundant in SL. Electron microscopic and histochemical analysis revealed that the granulation of these cells is based on melanosomes, mostly present in large melanosomal complexes. Immunohistochemistry using antibodies to CD68 and factor XIIIa (FXIIIa) showed these melanophages to be predominantly FXIIIa+ dermal dendrocytes, which were about six times more abundant than CD68+ macrophages. CONCLUSIONS: In SL an increased number of melanophages was found compared with unaffected skin from the same subject. These melanophages were identified as FXIIIa+ dermal dendrocytes. Possible functional consequences of the massive melanin uptake by dermal dendrocytes are discussed.

Mid-infrared spectroscopy on skin using a silver halide fibre probe in vivo.

BACKGROUND/AIM: Mid-infrared spectroscopy is a versatile method for in vivo investigation of skin after topical treatment with skin care products. METHODS: FTIR-spectrometer (Bruker Optics) with a flexible silver halide fibre probe (Infrared Fiber Sensors). RESULTS: Absorbance spectra from 700 to 3000 cm(-1) have been recorded to gain information about proteins (amide-I and amide-II vibrations at 1650 and 1550 cm(-1)), esters (1740 cm(-1)), carboxylic acid (1710 cm(-1)), polyalcohols (1050 cm(-1)) and hydrocarbons (CH(n) vibrations at 2800-3000 cm(-1)). CONCLUSIONS: Using the particular light guide, we were able to measure for the first time the effects of lip care products on lips directly. Furthermore, water binding and glycerol content of the skin could be determined simultaneously, as well as the replenishment of lipids by lipid-enriched bath oil.

Are sweat glands an alternate penetration pathway? Understanding the morphological complexity of the axillary sweat gland apparatus.

To build an effective barrier against the penetration of extrinsic agents is one of the skin's main functions. The barrier properties of the stratum corneum and the epidermis have been subject to extensive studies in the past while the role of skin appendages as possible pathways of penetration are only rarely described. In order to study the possible penetration barriers in these complex appendages, a careful investigation of their morphology and ultrastructure has to be done. Studying the morphology of axillary skin appendages requires clear-cut criteria for the differentiation between eccrine, apocrine and apoeccrine glands. Therefore we studied the distribution of proteins described to be specific for either eccrine or apocrine glands (CD15, CD44, S-100 and milk fat globulin) on axillary skin samples from healthy young adults by immunofluorescence. Additionally, we examined the distribution of cytoskeletal proteins such as cytokeratins (1/10/11, 14, 18) and F-actin. For a more detailed understanding of the possible versatile barrier elements of the axillary sweat glands, we studied the distribution of tight-junction-associated proteins (occludin, claudin 1, claudin 4). The coils and the dermal duct may provide an active barrier built of tight junctions as occludin and claudin 4 are co-localized. However, the intra-epidermal duct did not show any co-localization of the investigated proteins. By combining morphological features as revealed by F-actin staining and the distribution of the above-mentioned proteins, immunocytochemical typing of eccrine and apocrine glands becomes possible. With this tool, we could also confirm the existence of apoeccrine glands and locate them in their 'natural environment'.

Stimulation of skin's energy metabolism provides multiple benefits for mature human skin.

As an organism ages, there is a decline in mitochondrial function and cellular energy balance. This decline is both accelerated by and can cause the formation of reactive oxygen species (ROS) that damage nuclear and mitochondrial DNA, lipid membranes as well as structural and catalytic proteins, especially those involved in energetic pathways of cells. Further, ROS have also been linked to some of the detrimental skin changes that occur as a result of photoaging. We have previously shown that levels of Coenzyme Q10 (CoQ10), a component of the respiratory chain in mitochondria, are reduced in skin cells from aging donors, and that topical supplementation can ameliorate processes involved in skin aging. Creatine is another important component of the cellular energy system and phosphocreatine, its phosphorylated form, functions as a reservoir for high energy phosphates. Unfortunately the creatine system and thus the energy storage mechanism in skin are negatively affected by aging and conditions of oxidative stress. This article reviews some of our in vivo data about the synergistic effects of combining a stabilized form of Creatine with CoQ10 and clearly depicts their beneficial effects as active ingredients in topical formulations.

Topical activity of ascorbic acid: from in vitro optimization to in vivo efficacy.

We present here a new cosmetic formula system containing 3% ascorbic acid based on an optimized oil-in-water (O/W) emulsion. This formulation demonstrated a good long-term stability of the active ingredient and also of the emulsion itself. It could be deduced from in vitro release studies that this O/W emulsion enabled a better release of the hydrophilic active agent than an alternative W/O emulsion. By measuring the ultraweak photon emission, which is a well-established parameter for the oxidative stress in the skin, the high in vivo antioxidant capacity of 3% ascorbic acid was demonstrated after 1 week of product application. This placebo-controlled study also proved that ascorbic acid in an O/W cream reduced oxidative stress in human skin significantly better than the derivative sodium ascorbyl-2-phosphate, a more stable vitamin C replacement commonly used in cosmetic formulations. With increasing age, the number of papillae in the epidermal-dermal junction zone in human skin are reduced. This implies a possible consequence of reduced mechanical resistance of the skin and impaired supply of the epidermis with nutrients. In a 1-month placebo-controlled study on 25 human volunteers, a significant increase in the number of dermal papillae after application of the 3% ascorbic acid cream was demonstrated, using a confocal laser scanning microscope. Fine lines and wrinkles are a characteristic sign of aged and especially photo-aged skin. Application of 3% ascorbic acid in a 12-week placebo-controlled usage study indicated a significant reduction of facial wrinkles. Altogether, 3% ascorbic acid in a cosmetic O/W emulsion has been shown to be appropriately stable and to enable a good release of the active agent in vitro as a precondition for a high efficacy in vivo. Application in vivo resulted in a significant reduction of oxidative stress in the skin, an improvement of the epidermal-dermal microstructure and a reduction of fine lines and wrinkles in aged skin. These results were received within a relatively short period of time of product application.

Influence of cleansing on stratum corneum tryptic enzyme in human skin.

Desquamation in human skin is a well-balanced process of de novo production of corneocytes and their shedding from the skin surface. The proteolysis of corneodesmosomes is an important step in the final desquamation process. In the degradation of these adhesion molecules, the stratum corneum tryptic enzyme (SCTE) plays a key role. In initial studies with extracts of porcine epidermis, SCTE was shown to be inactivated by low concentrations of sodium lauryl ether sulphate (SLES). These in vitro findings were supported by in situ results obtained by measuring the release of fluorescent dyes coupled to trypsin-specific substrates incubated on human skin cross-sections. Moreover, in further studies, it could be demonstrated that the SCTE activity in the human horny layer decreases after in vivo application of cleansing products containing SLES. After repeated washing of human volunteers with tap water, a standard market cleansing product (SLES/betaine system) or a new improved cleansing product (SLES/betaine/disodium cocoyl glutamate system), the specific SCTE activity was determined in extracts from the uppermost layers of the stratum corneum. It could be shown that after application of the new formula the remaining SCTE activity was significantly higher than after use of the standard market formula. This ex vivo approach has proven to be very helpful for measuring surfactant effects on human skin enzymes. Using this assay, we developed an improved shower gel formula, which leads to a significantly higher skin enzyme activity after application, compared to a standard market formula.