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.

Design and application of a screening and training protocol for odour testers in the field of personal care products.

The assessment of odours and in particular of human axillary odour is an integral part of the research and development of deodorant and anti-perspirant products. One method to perform odour assessment is the odour evaluation that is carried out by experts, designated as odour testers or sniffers. Product development decisions are therefore based on human assessment. As for every scientific measurement, the influencing factors need to be standardized or regularly calibrated as effectively as possible for reasons of quality assurance. We therefore developed a screening and training concept aiming to examine the general suitability of odour testers by determining the individual odour sensitivity for relevant odours. This newly developed method is based on the national and international standards and guidelines EN 13725:2003, VDI 3882 sheet 1 and ASTM-1207. Suitable odour testers are subsequently trained to correlate their individual odour intensity perception with an intensity calibration scale in order to achieve reproducible results. Training sessions held on a regular basis help to achieve a greater homology in the response of an existing panel. Our established screening and training protocol has already been successfully put into practice and is also subject to permanent improvement with regard to practical requirements.

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.

Field evaluation of the efficacy of proprietary repellent formulations with IR3535 and picaridin against Aedes aegypti.

Seven proprietary repellent formulations (3 hydro-alcoholic spray solutions and 4 skin lotions) with active ingredient IR3,535 (ethyl butylacetylaminopropionate, EBAAP) or Picaridin (hydroxyethyl isobutyl piperidine carboxylate, KBR 3,023, Bayrepel) were tested in a field study on 10 test persons over a period of 10 h for their efficacy at preventing bites. The tests were conducted in Belo Horizonte, Brazil on field populations of the yellow fever mosquito Aedes aegypti. The concentration of the active substances ranged from 10% to 20%. All the tested samples provided lasting protection (time to first bite) over several hours: ranging from 5 h 20 min to 6 h 50 min with a mean of approximately 6 h. The longest protection until the second bite (=first confirmation bite) was approximately 7 h 40 min, whereas the shortest protection was 6 h 50 min. The longest protection until the third bite (=second confirmation bite) was 8 h 35 min, whereas the shortest protection was 7 h 40 min. In the control tests in which none of the samples were applied, the mean times until the first, second and third bites were 26, 46 and 59 min, respectively. The basis for this field study was provided by two American guidelines, which have the greatest international acceptance. The first is a draft guideline from the Environmental Protection Agency (EPA (United States Environmental Protection Agency), Product performance test guidelines. OPPTS 810.3700. Insect repellents for human skin and outdoor premises. Public Draft, 1999) and the second is a standard from the American Society for Testing and Materials (ASTM (American Society for Testing and Materials International), E 939-94 (reapproved 2,000): standard test method of field testing topical applications of compounds as repellents for medically important and pest arthropods (including insects, ticks, and mites): I. Mosquitoes, 2,000). Both guidelines recommend measuring the duration of protection until the first and second bites and also determining the relative protection efficacy in terms of a 95% protection level. The ASTM standard permits different repellents to be applied, whereas the EPA guidelines only permit the use of a single repellent (in different concentrations) on the extremities (forearms or lower leg). In the study presented here, to exclude any possibility of different repellents or concentrations of a single repellent having a reciprocal effect on each other, each test person had repellent samples applied to only one of their forearms. The other forearm was used as a control for making comparative checks every hour and for determining the biting pressure. There was no significant difference in protection times between the two active substances.

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.

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.

Dead but highly dynamic--the stratum corneum is divided into three hydration zones.

Topically applied water exerts mechanical stress on individual corneocytes as well as on the whole stratum corneum (SC), resulting in an alteration of barrier function. In this study we used complete skin biopsies and showed that the SC reacts to water stress as a highly optimized and well-regulated structure against osmotic changes. Following a relatively new cryo-processing protocol for cryo-SEM, it is possible to reliably maintain and investigate the hydrated state of the SC and individual corneocytes after treatment with solutions of different ionic strength. Treatment with distilled water results in swelling of SC cells together with formation of massive water inclusions between adjacent cell layers. Treatment with 5-20% NaCl reveals three different hydration zones within the SC: Corneocytes near the live-dead transition zone can swell to nearly double their thickness. The second zone is the most compact, as the corneocytes here show the smallest thickness variation with all treatments. Within the outermost zone, again a massive swelling and loosening of intracellular filament packing can be observed. We therefore conclude that the SC itself is subdivided into three functional zones with individual water penetration and binding potentials. Since the second zone remains nearly unaffected by water stress, we propose that this zone hosts the functional SC barrier.

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.

Dielectric spectroscopy of concentrated cosmetic W/O-emulsions: possibilities to distinguish product changes caused by coalescence, sedimentation and variation of ingredients.

A quick result whether a newly developed cosmetic water-in-oil (W/O)-emulsion shows constant sensory behavior (stable) or whether it changes its behavior over time (instable) is an important aspect in cosmetic research. In order to observe changes as quickly as possible, analytic methods are used. An established method is rheology, a sensitive method that gives direct information on sensory aspects. Additional information concerning the kind of instability allows a more focused improvement of formulation in the case of instabilities. In order to gain this information, an additional analytic method with a more ingredient specific focus is needed. In this article, the possibilities of using dielectric spectroscopy in order to get additional information are discussed. For concentrated W/O-emulsions a dependence of emulsion behavior from volume median droplet diameter d(v50) is visible by both methods: rheology and dielectric spectroscopy. A variation of droplet size distribution at constant volume median droplet diameter d(v50) does not change the results for the examined emulsions. Quantitative information about mean droplet size is possible with calibration. Because of their different physical forci, rheology and dielectric spectroscopy complement each other in high-sensitive detection of coalescence. In contrast to the mechanical properties of W/O-emulsion, dielectric spectroscopy gives additional information concerning some reasons of change in emulsion structure. Mechanism like sedimentation and coalescence can be distinguished and the phase where changes take place (oil phase or water phase) can be located. The latter is possible by a new parameter - the correlation between maximum of dielectric loss epsilon''(f(R)) and the relaxation frequency f(R). Their correlation can be described by a simple power law. By coupling rheology and dielectric spectroscopy, an improvement in fast emulsion development and production control may be achieved without losing the advantage of a quick and easy measurement procedure.

Adsorption kinetics of surfactant mixtures from micellar solutions as studied by maximum bubble pressure technique.

The adsorption kinetics of micellar solutions of anionic/cationic SDS/DATB mixtures with mixing ratios of 10/1 and 10/2, respectively, are studied experimentally by means of the maximum bubble pressure method. For long adsorption times the adsorption of the highly surface-active anionic/cationic complex leads to a decrease of dynamic surface tension in comparison to the single SDS system. However, the situation is the reverse for short adsorption times where the dynamic surface tension is increased by addition of the cationic surfactant, although the overall concentration is increased. This unexpected behavior is explained by partial solubilization of free SDS molecules into micelles formed by SDS/DTAB complexes. With increasing overall concentration, when eventually the CMC of SDS is reached, the anionic/cationic complex itself is solubilized by SDS micelles. Finally, no complex micelles, which for their part can solubilize an excess of SDS molecules, are present. Hence, the dynamic properties of the solution are no longer influenced by the depletion of SDS molecules and the mixture tends to behave like a pure SDS solution.

From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging.

Structural investigation of tissue biopsies requires the coupling of optimal structural sample preservation with detailed information collected at the light and electron microscopic level. Unfortunately, although cryo-immobilization by high-pressure freezing provides the best structural preservation, it is used routinely only for electron microscopic (EM) investigations, whereas for light microscopy chemical fixation protocols have been established. These chemically invasive fixation protocols have the drawback of introducing unpredictable fixation artefacts. Therefore, comparative histopathological (i.e. light microscopic) and ultrastructural (i.e. EM) results are usually obtained from parallel samples that have not been prepared identically and never by examining exactly the same features in exactly the same, optimally preserved sample. Finally, finding an area of interest for EM investigation within a complex tissue is like searching for a needle in a haystack. To overcome these handicaps, we modified the well-established freeze-substitution technique (FS) to allow us to investigate resin-embedded cryo-immobilized tissue by confocal laser scanning microscopy (CLSM) prior to EM examination. Thus (1) selected cells throughout the whole tissue block can be depicted by CLSM and subsequently (2) selectively prepared by targeted sectioning for follow-up investigation of the identical structure by transmission electron microscopy. This is facilitated by the addition of specific fluorescent dyes during the first FS exchange step. Selective binding properties of various dyes to different cellular structures allow a direct histological description of the tissue at the light microscope level. After embedding and preparation of a blockface, the specimen can first be examined by CLSM. For areas of interest, the depth in the resin block is determined followed by removal of the tissue lying above. Then, the cell layer can be cut into a series of ultrathin sections and examined by EM for determination of the subcellular and nanostructural organization.

The influence of pre-irradiation on the predictability of in vivo UVA protection with a new in vitro method.

BACKGROUND/PURPOSE: UVA protection of sunscreen formulations is becoming increasingly important especially because of recent investigations on the long-term skin damage associated with UVA light. The development of a new in vitro method to measure UVA protection performance made it possible to predict reliably the in vivo UVA protection performance of representative sunscreen formulations found presently in the European and US market (1). This study was performed in order to determine the applicability of the method developed by Wendel et al. (1) to photostable and photolabile filter combinations and in order to measure the influence of sample pre-irradiation on predicting the in vivo performance. This was done by subjecting six photostable and six photolabile filter combinations to a standard irradiation. Then the in vitro UVA protection afforded by each combination was measured and compared with the persistent pigment darkening (PPD) values determined in vivo. RESULTS: The results clearly showed that pre-irradiation does not affect the in vitro PPD factor of the photostable and photolabile samples in the same way. Almost identical values were determined for the stable filter combinations with and without pre-irradiation, whereas distinct reductions in the in vitro factors by as much as 93% were observed after irradiation in the group of less stable filter combinations. Comparison of the in vivo and in vitro PPD factors showed that all 12 samples comprise a homogeneous distribution with identical factors before irradiation. After pre-irradiation only the factors for the six less stable products were selectively reduced. The correlation with the data determined on the skin was clearly poorer for these products after irradiation. CONCLUSION: Overall, the results showed that pre-irradiation should not to be used for the assessment of UVA protection using this method. Furthermore, it can be assumed that normalizing the in vitro absorbance curves to the labelled SPF of the sunscreen will adequately take into account the photochemical behaviour of UV filters on the skin during sun exposure.

Distribution of sunscreens on skin.

The effectiveness of sunscreens was originally achieved by incorporation of soluble organic UV absorbers such as cinnamates and others into cosmetic formulations. Determinations of the sun protection factor (SPF) of emulsions containing different organic UV absorbers clearly indicate that the efficacy depends on the absorption characteristics of each single UV filter substance. Nowadays, micronised pigments such as titanium dioxide or zinc oxide have also been found to be protective against harmful UV rays. Our investigations using optical and electron microscopy proved that neither surface characteristics, particle size nor shape of the micronised pigments result in any dermal absorption of this substance. Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis.

Hydration dynamics of human fingernails: an ellipsometric study.

We use spectroscopic ellipsometry to obtain the complex refractive index, ñ=n+ik, of human fingernails. By studying the change of ñ upon hydration and dehydration, we reveal three different time domains with typical time constants of 4, 150, and 3200 min. A simple model that takes into account the presence of one fast and one slow process is fully consistent with the observed hydration and dehydration dynamics. We attribute these processes to "free" water incorporated between the keratin filaments and water more tightly "bound" in keratin complexes, respectively. From our model we determine the hydration profiles of "free" and "bound" water during, both, hydration and dehydration.

A quick, practical test procedure to evaluate the performance of instruments used for in vitro UV protection measurements.

The in vitro determination of the UV protection of sunscreens is usually performed by means of transmission measurements with special photometers. Many different instruments are used. Besides numerous commercially available instruments, which are equipped by the manufacturer for the specific measurement, other modular instruments are used. We present here a quick and practical method to evaluate the performance of these instruments with respect to their measuring ranges and to compare the uniformity and reliability of the results obtained with these instruments.

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.