Effects of petrolatum, a petrolatum depositing body wash and a regular body wash on biomarkers and biophysical properties of the stratum corneum.

BACKGROUND: An important trend in the personal care industry involves the development of body wash products that not only clean the skin without damage but deposit conditioning ingredients to improve skin barrier function. OBJECTIVE: The objective of this study was to develop skin biomarker measures to quantify the treatment effects of body wash products. METHODS: We employed analysis of structural proteins (keratin 1,10,11 and involucrin), a natural moisturizing factor (pyrrolidone carboxylic acid) and an inflammatory mediator (IL-1ra/IL-1α) from adhesive discs with dry skin grading, TEWL and capacitance measurements to compare the effects of direct application of petrolatum, a high petrolatum depositing body wash, and a regular body wash on dry leg skin in a standard leg-wash treatment protocol. RESULTS: High depositing body wash and petrolatum had positive effects on stratum corneum barrier function as judged by biomarker analysis, biophysical measurements and skin grading compared to the regular body wash product. CONCLUSIONS: The results clearly indicate that a combination of biomarker and biophysical property measurements is effective for determining the skin benefits of moisturizing body wash products.

CONTEXTE: Une tendance importante dans l'industrie des soins personnels inclut le développement de produits de lavage corporel qui non seulement nettoient la peau sans l'endommager, mais déposent des ingrédients de traitement pour améliorer la fonction de la barrière cutanée. OBJECTIF: L'objectif de cette étude était de développer des mesures de biomarqueurs cutanés permettant de quantifier les effets du traitement des produits de lavage corporel. MÉTHODES: Nous avons utilisé l'analyse de protéines structurelles (kératine 1,10,11 et involucrine), un facteur hydratant naturel (acide carboxylique de pyrrolidone) et un médiateur inflammatoire (IL-1ra/IL-1a) provenant de disques adhésifs avec cotation de la sécheresse cutanée, mesures de perte d'eau transépidermique (transepidermal water loss, TEWL) et de capacitance pour comparer les effets de l'application directe de vaseline, d'un produit de lavage corporel avec dépôt élevé de vaseline et d'un produit de lavage corporel ordinaire sur la peau sèche des jambes, dans un protocole de traitement de lavage des jambes standard. RÉSULTATS: Le produit de lavage corporel à dépôt élevé et la vaseline avaient des effets positifs sur la fonction de barrière de la couche cornée, comme évalué par l'analyse des biomarqueurs, les mesures biophysiques et la cotation de la peau, comparé au produit de lavage corporel ordinaire. CONCLUSIONS: Les résultats indiquent clairement qu'une combinaison de mesures des biomarqueurs et des propriétés biophysiques est efficace pour déterminer les bienfaits pour la peau des produits de lavage corporel hydratants.

Quantitation of 24-Hour Moisturization by Electrical Measurements of Skin Hydration.

PURPOSE: The purpose of this study was to quantify the effects of several moisturizers on hydration of the stratum corneum by measuring their effect on electrical conductance over a 24-hour period. DESIGN: Double-blind, randomized controlled trial. SUBJECTS AND SETTING: Twenty-five healthy female volunteers aged 18 to 65 years with dry skin on the lower legs and no other known dermatologic pathology participated in the study. Additional exclusion criteria were pregnant or taking anti-inflammatory steroids. The study was carried out in a clinical research facility in Winnipeg, Manitoba, Canada. METHODS: Subjects underwent a 3-day conditioning period using a natural soap bar on the lower legs and no application of moisturizer to the skin. Participants then came to the test site and equilibrated for at least 30 minutes under controlled conditions of temperature and humidity. After baseline hydration measurements on test sites on the lower legs of each subject, a single application of each of 5 test products at a dose of 2 mg/cm was made. Skin hydration was assessed by electrical conductance measurements with a specialized probe. The probe was briefly placed on the skin surface with light pressure, and the measurement recorded in units of microsiemens (µS). Conductance was measured at 2, 4, 6, 8, and 24 hours after product applications. RESULTS: Although all but 1 of the test products increased conductance at 2 hours, only 2 moisturizers containing high levels of glycerin (products C and E) maintained increased conductance relative to baseline at 24 hours, +37.8 (P < .001) and +103.5 (P < .001), respectively. CONCLUSIONS: Moisturizers containing high levels of glycerin can provide a measurable moisturization benefit as determined by skin conductance for at least 24 hours after a single application.

Targeted deactivation of cancer-associated fibroblasts by ß-catenin ablation suppresses melanoma growth.

Cancer-associated fibroblasts (CAFs) are the crucial components of the dynamic tumor microenvironment, which not only supports the growth and metastasis of melanoma but also contributes to drug resistance in melanoma treatment. We recently discovered that loss of ß-catenin signaling deactivated stromal fibroblasts and reduced the production of paracrine factors and extracellular matrix proteins. Based on this finding, we aimed to determine whether melanoma growth could be suppressed by targeted deactivation of CAFs via ß-catenin ablation using a combination of in vitro and in vivo approaches. Using an in vitro three-dimensional (3D) tumor co-culture model, we showed that ß-catenin-deficient fibroblasts lost the ability to respond to melanoma cell stimulation and to support the growth of B16F10 melanoma cells. To determine the in vivo effects of CAF deactivation on melanoma growth, we designed a novel genetic approach to ablate ß-catenin expression in melanoma-associated fibroblasts only after melanoma tumor was formed. As expected, our observation showed that development of B16F10 melanoma was significantly delayed when ß-catenin expression was ablated in CAFs. We determined that inhibition of tumor growth was due to decreased melanoma cell proliferation and increased cell death. Further analysis revealed that CAF deactivation caused the downregulation of the MAPK/ERK signaling cascade and S and G2/M phase cell cycle arrest in B16F10 melanoma cells. Overall, our data emphasize the significance of targeting CAFs as a potential novel therapeutic approach to improve melanoma treatment by creating a tumor-suppressive microenvironment through tumor-stroma interactions.

Effects of season stratum corneum barrier function and skin biomarkers.

The skin on the lower legs of 25 female subjects was evaluated first in the winter, and then again in the summer of the same subjects. Barrier function was determined by measuring transepidermal water loss (TEWL), and skin hydration and dryness were evaluated by electrical measurements (Corneometer ® CM825) and visual grading. Stratum corneum (SC) was sampled using 10 sequential D-Squame sampling discs and analyzed for 2-pyrrolidone-5-carboxylic acid (PCA), keratin-1,10,11, interleukin 1α (IL-1α), interleukin 1 receptor antagonist (IL-1ra), selected ceramides, cholesterol, cholesterol sulfate, and selected free fatty acids. TEWL as well as the visual dryness grades were significantly lower in the summer while hydration was higher. PCA was significantly higher in the summer as were the keratins. The ratio IL-1ra:IL-1α, an indicator of skin inflammation, was significantly lower in the summer. The amount of protein removed by the tape strips was also significantly lower in summer indicating better SC cohesion. Among the SC lipids measured, total ceramides, individual ceramides, total fatty acids, and cholesterol were higher in summer compared to winter. Stearic acid and cholesterol sulfate were not significantly different between winter and summer.

Potential of native Thai aromatic plant extracts in antiwrinkle body creams.

Antioxidant activities of 10 essential oils and 10 absolutes extracted from Thai aromatic plants were evaluated and compared to thyme oil, trolox, quercetin, and kaempferol by two independent assays: the 2, 2-diphenyl-1-1-picrylhydrazyl (DPPH*) radical scavenging assay and the thiobarbituric acid reactive species (TBARS) assay for lipid peroxidation. We found that four essential oils including ginger oil (Zingiber officinale Roscoe), Wan-sao-long leaf oil (Amomum uliginosum Koen), lemongrass oil (Cymbopogon citratus), holy basil oil (Ocimum sanctum L.), and the absolute of dwarf ylang-ylang [Cananga odorata Hook. f. & Thomson var. fruticosa (Craib) J. Sinclair] exhibited high antioxidant activity in both DPPH and TBARS assays and possessed satisfactory fragrance properties. These were then combined into an essential oil blend (EOB) and retested for antioxidant activity. The EOB also exhibited high antioxidant activity in the above assays. It was then incorporated into a stable cream base as EOB body cream. The EOB body cream was found to be best able under storage in stress conditions and presented significantly higher antioxidant activity than its' cream base both before and after stability testing. The effect of EOB body cream on skin surface topography was evaluated in 29 healthy volunteers using the Skin Visiometer (SV 600 FW, CK Electronic GmbH, Germany). Three parameters, Ra, Rz (roughness), and surface, were analyzed. After 4 weeks of application, the EOB body cream showed significant reductions in surface and Rz compared with before treatment (p < 0.05, paired t-test), and with untreated and placebo treatment (p < 0.05, Duncan test). These results indicate that the essential oils and absolutes from Thai plants may serve as potential sources of natural antioxidants for spa and cosmetic products designed to prevent or treat signs of skin aging.

Influence of tumour necrosis factor-α polymorphism-308 and atopy on irritant contact dermatitis in healthcare workers.

BACKGROUND: Chronic irritant hand dermatitis is an issue for healthcare workers and may negatively impact infection control. OBJECTIVES: We examined the effects of a G to A transition at position -308 on the tumour necrosis factor-α (TNF-α) gene on chronically damaged skin of healthcare workers during exposure and recovery from repetitive hand hygiene, after intensive treatment, and on the irritant response in normal skin. PATIENTS/MATERIALS/METHODS: In 68 healthcare workers with irritant hand dermatitis, we genotyped TNF-α-308 and measured the epidermal response via quantitative digital imaging, erythema, dryness, and barrier integrity. RESULTS: Excess hand erythema decreased with hand hygiene exposure and increased during time off for AA/GA genotypes, but had opposite effects for GG. AA/GA had smaller reductions in dryness with lotion treatment and larger reductions in excess erythema than GG. The atopic diathesis and heightened neurosensory irritation resulting from water and lactic acid significantly influenced the responses. Repeated exposure to water and sodium lauryl sulfate (0.05, 0.1%) produced higher erythema in normal skin for AA/GA than for GG. CONCLUSIONS: This study provides evidence that the TNF-α polymorphism at -308 and an atopic history impact the severity of irritation and recovery from exposure and response to treatment for common hand skin products in both chronic irritant hand dermatitis and normal skin.

Regional variation in the free amino acids in the stratum corneum.

Regional differences in water-binding free amino acids (FAAs) in the stratum corneum (SC) may be expected, since differences in skin biophysical properties are well known. The objective was to determine whether differences in skin hydration as a function of body site may arise from differences in the chemical makeup of the skin, specifically the FAAs. Levels were quantified from serial SC samples collected from the forearm, calf, back, torso, and jaw in two studies using HPLC methods. FAA levels were higher from the calf versus the forearm and lower from the jaw compared to torso and back skin. Body site variations in skin hydration could not be attributed to differences in FAA levels.

Comparative thermodynamic and spectroscopic properties of water interaction with human stratum corneum.

BACKGROUND/PURPOSE: The water content of skin has a significant impact on skin properties; sufficient hydration is necessary to keep the skin supple, flexible, and smooth. To understand more completely the water retention properties of the human skin barrier, physical macroscopic properties must be related to the structural organization of the stratum corneum (SC). Water, lipids, and natural moisturizing factor (NMF) influence the molecular structures that affect the properties of SC, including water sorption and binding enthalpy. In the research reported here, isothermal microcalorimetry was used to study the interaction of water vapor with isolated human SC in intact, delipidized, and water-washed delipidized forms to identify the influences of the principal components of SC on water sorption. The calorimetric data are interpreted in conjunction with spectroscopic results to identify the conformational changes in keratins induced by lipid and NMF removal and to assess the influence of these changes on water binding in SC. METHODS: Isothermal calorimetry was used to measure the integral heat of water vapor sorption on intact, delipidized, and water-washed delipidized human SC at 32 degrees C as a function of relative humidity using back and thigh skin from three donors. Calorimetric measurements were combined with water vapor sorption measurements to determine the differential thermodynamic properties of these systems. Attenuated total reflection-Fourier transform infrared spectroscopy was used to investigate effects of extraction on protein secondary structure. RESULTS: The magnitudes of the differential enthalpy, entropy, and free energy were greatest for intact SC and least for water-washed delipidized SC. Water sorption followed a similar trend. Delipidization led to a significantly reduced binding enthalpy at low water content; water washing the delipidized SC had only a small additional effect on binding enthalpy. Delipidization converts a fraction of keratin alpha-helixes to turns and random coils, while water sorption converts a fraction of keratin alpha-helixes to beta-sheets, turns, and random coils. CONCLUSIONS: The results of this study are consistent with a water sorption model in which keratin-keratin hydrogen bonds are replaced by keratin-water hydrogen bonds. Delipidization reduces the fraction of dry keratin that is in the alpha-helix conformation, suggesting that lipids hold the keratins in a conformation conducive to optimal hydration.

Mechanisms regulating skin pigmentation: the rise and fall of complexion coloration.

Skin pigmentary abnormalities are seen as aesthetically unfavorable and have led to the development of cosmetic and therapeutic treatment modalities of varying efficacy. Hence, several putative depigmenting agents aimed at modulating skin pigmentation are currently being researched or sold in commercially available products. In this review we will discuss the regulation of processes that control skin complexion coloration. This includes direct inhibition of tyrosinase and related melanogenic enzymes, regulation of melanocyte homeostasis, alteration of constitutive and facultative pigmentation and down-regulation of melanosome transfer to the keratinocytes. These various processes, in the complex mechanism of skin pigmentation, can be regulated individually or concomitantly to alter complexion coloration and thus ameliorate skin complexion diseases.

Equilibrium water content in native vernix and its cellular component.

Vernix caseosa is a naturally occurring substance coating the skin of newborn humans. Structurally, vernix contains fetal corneocytes embedded in a hydrophobic lipid matrix. Despite a relatively high water content approximating 80.7%, vernix exhibits slow water release. In this study, we quantified and contrasted the water release and uptake properties of native vernix and its isolated cellular component over the full range of water activity. Theoretical water sorption models (D'Arcy-Watt, and Frenkel-Halsey-Hill (FHH), and Guggenheim-Anderson-de Boer (GAB)) were fit to the vernix water sorption data. Each of the theoretical models provided a satisfactory description of the equilibrium water content of vernix over the water activity range 0.15-1.0. Vernix corneocytes without the surrounding lipid matrix exhibited markedly increased equilibrium water binding at water activities greater than 0.62 compared to native vernix. Resorption experiments showed full recovery of water content in both native vernix and isolated corneocytes supporting a structured internal domain. These results provide the first quantitative characterization of the water handling properties of native vernix and its cellular component. Such information may prove useful in the design of alternative skin care moisturizing formulations.

Water-handling properties of vernix caseosa and a synthetic analogue.

A naturally occurring barrier cream, vernix caseosa, is the viscous material synthesized by the sebaceous glands in the late gestational human fetus. Vernix functions as a moisturizer by increasing the skin hydration and water-holding capacity of treated skin. Vernix films are semi-permeable, i.e., in the range that facilitates barrier repair. Antioxidant, disinfectant, and skin cleansing functions are also present. Premature infants have a markedly immature epidermal barrier and the excessive water loss can lead to fluid and electrolyte imbalances, along with high evaporative heat loss. Application of petrolatum-based, low-water creams on these infants has decreased TEWL and improved the skin condition. However, in infants of 500-750 g, this treatment was associated with an increased incidence of late-onset nosocomial infection, and questions regarding efficacy and safety have been raised. The water-handling properties, semi-permeability and multi-functionality, suggest that application of vernix may promote the development and restoration of premature or other compromised skin. The present study focuses on the development of barrier creams to simulate the water-handling properties of native vernix. Barrier creams were prepared as high-water-phase emulsions containing various lipid mixtures. Several stable creams with high water content exhibited slow water release and water vapor transport rates in the range to facilitate barrier repair. The results showed the importance of emulsion type in preventing water release. Preparations with vernix-like lipids demonstrated water release profiles closer to the native vernix benchmark than those with conventional lipids. The work resulted in a synthetic vernix barrier cream prototype for evaluation on skin and to which additional functionality, e.g., anti-infective and antioxidant activity, could be added.

Comparative efficacy and safety of deoxyarbutin, a new tyrosinase-inhibiting agent.

Several tyrosinase inhibitors have been developed and utilized to ameliorate various cutaneous hyperpigmentary disorders and complexion discolorations. Deoxyarbutin (dA) (i.e., 4-[(tetrahydro-2H-pyran-2-yl)oxy]phenol), designed using quantitative structure-activity relationships (QSAR), demonstrates effective inhibition of mushroom tyrosinase and skin-lightening capability (1). However, its comparative safety, effectiveness, and reversibility to other known tyrosinase inhibitors in human melanocytes had not been determined. The effect of dA was assessed in cultured human skin cells, on xenographs, and with a clinical trial. Using cultured human melanocytes, the maximum concentration of dA that allowed 95% viability was fourfold greater than for hydroquinone (HQ), indicating that dA is less cytotoxic/cytostatic than HQ. The viability of cultured human keratinocytes and fibroblasts was also less compromised by increasing concentrations of dA as opposed to HQ. At the maximum concentration allowing normal cellular viability, dA effectively inhibited tyrosinase activity and melanin content in human melanocytes, whereas HQ was marginally inhibitory. Upon removal of dA, tyrosinase activity and melanin content was normalized within five days. Topical application of dA on human xenografts resulted in a gradual and visually apparent skin lightening effect during an eight-week period. In a clinical trial, dA facilitated fading of pre-tanned skin to a statistically significant greater extent than either HQ or no treatment. These results demonstrate that dA is a potentially safe, effective, and reversible tyrosinase inhibitor.

Quantitative model of cellulite: three-dimensional skin surface topography, biophysical characterization, and relationship to human perception.

Gynoid lipodystrophy (cellulite) is the irregular, dimpled skin surface of the thighs, abdomen, and buttocks in 85% of post-adolescent women. The distinctive surface morphology is believed to result when subcutaneous adipose tissue protrudes into the lower reticular dermis, thereby creating irregularities at the surface. The biomechanical properties of epidermal and dermal tissue may also influence severity. Cellulite-affected thigh sites were measured in 51 females with varying degrees of cellulite, in 11 non-cellulite controls, and in 10 male controls. A non-contact high-resolution three-dimensional laser surface scanner was used to quantify the skin surface morphology and determine specific roughness values. The scans were evaluated by experts and naive judges (n=62). Body composition was evaluated via dual-energy x-ray absorptiometry; dermal thickness and the dermal-subcutaneous junction were evaluated via high-resolution 3D ultrasound and surface photography under compression. Biomechanical properties were also measured. The roughness parameters Svm (mean depth of the lowest valleys) and Sdr (ratio between the roughness surface area and the area of the xy plane) were highly correlated to the expert image grades and, therefore, designated as the quantitative measures of cellulite severity. The strength of the correlations among naive grades, expert grades, and roughness values confirmed that the data quantitatively evaluate the human perception of cellulite. Cellulite severity was correlated to BMI, thigh circumference, percent thigh fat, architecture of the dermal-subcutaneous border (ultrasound surface area, red-band SD from compressed images), compliance, and stiffness (negative correlation). Cellulite severity was predicted by the percent fat and the area of the dermal-subcutaneous border. The biomechanical properties did not significantly contribute to the prediction. Comparison of the parameters for females and males further suggest that percent thigh fat and surface area roughness deviation are the distinguishing features of cellulite.

Perspective of Targeting Cancer-Associated Fibroblasts in Melanoma.

Melanoma is known as an exceptionally aggressive and treatment-resistant human cancer. Although a great deal of progress has been made in the past decade, including the development of immunotherapy using immune checkpoint inhibitors and targeted therapy using BRAF, MEK or KIT inhibitors, treatment for unresectable stage III, stage IV, and recurrent melanoma is still challenging with limited response rate, severe side effects and poor prognosis, highlighting an urgent need for discovering and designing more effective approaches to conquer melanoma. Melanoma is not only driven by malignant melanocytes, but also by the altered communication between neoplastic cells and non-malignant cell populations, including fibroblasts, endothelial and inflammatory cells, in the tumor stroma. Infiltrated and surrounding fibroblasts, also known as cancer-associated fibroblasts (CAFs), exhibit both phenotypical and physiological differences compared to normal dermal fibroblasts. They acquire properties of myofibroblasts, remodel the extracellular matrix (ECM) and architecture of the diseased tissue and secrete chemical factors, which all together promote the transformation process by encouraging tumor growth, angiogenesis, inflammation and metastasis and contribute to drug resistance. A number of in vitro and in vivo experiments have shown that stromal fibroblasts promote melanoma cell proliferation and they have been targeted to suppress tumor growth effectively. Evidently, a combination therapy co-targeting tumor cells and stromal fibroblasts may provide promising strategies to improve therapeutic outcomes and overcome treatment resistance. A significant benefit of targeting CAFs is that the approach aims to create a tumor-resistant environment that inhibits growth of melanomas carrying different genetic mutations. However, the origin of CAFs and precise mechanisms by which CAFs contribute to melanoma progression and drug resistance remain poorly understood. In this review, we discuss the origin, activation and heterogeneity of CAFs in the melanoma tumor microenvironment and examine the contributions of stromal fibroblasts at different stages of melanoma development. We also highlight the recent progression in dissecting and characterizing how local fibroblasts become reprogrammed and build a dynamic yet optimal microenvironment for tumors to develop and metastasize. In addition, we review key developments in ongoing preclinical studies and clinical applications targeting CAFs and tumor-stroma interactions for melanoma treatment.

Effect of soaking and natural moisturizing factor on stratum corneum water-handling properties.

Stratum corneum (SC) hydration is partially regulated by water-soluble molecules, natural moisturizing factor (NMF) that is associated with the corneocytes. Routine water exposure, e.g., bathing, may deplete NMF and alter the SC water-handling properties. We determined the effects of bathing and solvent extraction on the volar forearm skin of eleven healthy volunteers. Acetone/ether (A/E) was used to remove surface and upper SC lipids. Adjacent sites were soaked for ten minutes or treated with the A/E-plus-soak combination. Subsequently, an NMF formulation was applied to the treated sites, and transepidermal water loss (TEWL), hydration, and moisture accumulation rate (MAT) were measured. A/E extraction increased TEWL, but did not effect MAT. Soaking produced a short-term increase in TEWL, followed by a decrease, and substantially reduced MAT, an effect that was maintained for five hours. NMF application significantly decreased TEWL and significantly increased MAT for all sites. The replacement experiment suggests that the MAT reduction occurred as a result of extraction of hygroscopic NMF components. The effects of soaking and NMF application are more readily detected by the MAT technique, whereas TEWL is more sensitive to A/E extraction. The results support the use of multiple assessments of barrier function and raise questions about the effects of cumulative repeated water exposure on SC function.

Noninvasive skin measurements after CO(2) and erbium laser resurfacing.

BACKGROUND: Improvement in skin after laser resurfacing depends on treatment to a precise level. The target treatment level will depend on skin type, anatomic location, and severity of the presenting problem. The development of noninvasive skin measurements that correlate with histologic changes would provide a useful intraoperative guide for treatment. OBJECTIVE: The relationship among different types of lasers and levels of applied energy; objective real-time noninvasive measures of the skin; and histologic condition were studied in an attempt to gain information that could aid in achieving safer and more predictable laser skin resurfacing. METHODS: The paraspinal skin of Yorkshire hybrid pigs was used for the study. In pig 1, study sites were treated with the Ultrapulse CO(2) laser (Coherent, Inc, Santa Clara, CA) with the CP-G at 300 ml for 0 to 5 passes. The Sciton Contour erbium: YAG llaser (Contour, Sciton, Palo Alto, CA) set at 200 mum of pure ablation was used to treat a separate but adjacent area for 0 to 5 passes. Treated sites and control sites were evaluated by measurements of biomechanical properties, digital imaging, and barrier integrity. In pig 2, the study was repeated with the Sciton Contour erbium:YAG laser at 10 different energy levels. Full-thickness punch biopsy specimens were obtained immediately after treatment and correlated with the biomechanical measures. RESULTS: After the third pass with the CO(2) laser, there was no additional tissue ablation or change in surface biophysical measurements. There was an apparent increase in inflammation in the lower dermis with passes 4 and 5 seen on day 2 biopsies that suggested deeper thermal effects. The erbium laser continued to ablate with increasing applied energy. The change in viscoelastic creep correlated with the laser energy applied and inversely correlated with the remaining dermal depth of the skin after laser treatment. CONCLUSIONS: The measurement of immediate changes in viscoelastic creep should be further studied as a means of guiding intraoperative erbium laser resurfacing. Histologic evaluation suggests that the erbium:YAG laser may be a more effective tool for dermal ablation than the CO(2) laser.

Hydrolytic enzymes of the interfollicular epidermis differ in expression and correlate with the phenotypic difference observed between light and dark skin.

Degradation of melanosomes in light skin (LS, i.e. phototype I/II) appears to occur more rapidly than dark skin (DS, i.e. phototype IV/V). Hydrolytic enzymes known to reside and be expressed in a differential pattern within the interfollicular epidermis are implicated in playing a role in epidermal differentiation and potentially melanosome degradation. The aim of this present study was to evaluate the differential expression of hydrolytic enzymes that may correlate with physiological and phenotypic differences seen between DS and LS. Expression of six hydrolytic enzymes was confirmed by microarray analysis of the suprabasal epidermis from LS and DS. Specific lysosomal hydrolases identified by microarray analysis were analyzed by indirect immunofluorescence (IIF) and immunoblot analysis. Immunogold electron microscopy (IEM) was completed to visualize cellular expression of the hydrolytic enzyme cathepsin L2 (Cath L2) and biochemical assay was performed to ascertain Cath L2 activity. Immunoblotting of light and dark epidermal lysates demonstrated that of the six enzymes initially analyzed, both prostatic acid phosphatase (ACPP) and Cath L2 were reproducibly upregulated in DS and LS, respectively. IIF and IEM analyses of Cath L2 in tissue confirmed this differential expression. Biochemical analysis of Cath L2 in light and dark epidermal lysates displays increased activity of Cath L2 in LS. The results of this study confirm differential expression of ACPP and Cath L2 in DS and LS at gene and protein level. Additionally, Cath L2 displays increased activity in LS-derived epidermal lysates. This study indentified two acid hydrolases that may play a role in melanosome degradation and pigment processing.

Epidermal keratinocytes from light vs. dark skin exhibit differential degradation of melanosomes.

Modification of skin complexion coloration has traditionally been accomplished by interruption or attenuation of melanogenesis and/or melanosome transfer. Post-transfer modification of pigmented melanosomes provides an attractive and distinct avenue of modulating skin pigmentation. The processing of melanosomes during keratinocyte (KC) terminal differentiation and the degradative variability observed between light and dark skin (LS and DS) remains enigmatic. To evaluate this, we developed a model system to investigate the loss of fluorescently labeled and isolated melanosomes by cultured human KCs. The extent of melanosome loss has been qualitatively assessed using transmission electron microscopy and indirect immunofluorescence with confocal microscopy, and quantitatively assessed using flow cytometry analysis. Results show that melanosomes are incorporated into the cytoplasm of both light and dark keratinocytes (LKCs and DKCs) and trafficked to a perinuclear region. Within 48 hours, confocal microscopy images suggest that LKCs display accelerated melanosome loss. This time-dependent decrease in carboxyfluorescein diacetate (CFDA) fluorescence was then quantitatively analyzed using flow cytometry. Consistent with the results of the confocal analysis, over a 48-hour time frame, LKCs appear to lose melanosomes more efficiently than DKCs. These experiments show that melanosomes are more rapidly lost in KCs derived from LS as opposed to DS.