Significance of melanin distribution in the epidermis for the protective effect against UV light.

Melanin, the most abundant skin chromophore, is produced by melanocytes and is one of the key components responsible for mediating the skin's response to ultraviolet radiation (UVR). Because of its antioxidant, radical scavenging, and broadband UV absorbing properties, melanin reduces the penetration of UVR into the nuclei of keratinocytes. Despite its long-established photoprotective role, there is evidence that melanin may also induce oxidative DNA damage in keratinocytes after UV exposure and therefore be involved in the development of melanoma. The present work aimed at evaluating the dependence of UV-induced DNA damage on melanin content and distribution, using reconstructed human epidermis (RHE) models. Tanned and light RHE were irradiated with a 233 nm UV-C LED source at 60 mJ/cm2 and a UV lamp at 3 mJ/cm2. Higher UV-mediated free radicals and DNA damage were detected in tanned RHE with significantly higher melanin content than in light RHE. The melanin distribution in the individual models can explain the lack of photoprotection. Fluorescence lifetime-based analysis and Fontana-Masson staining revealed a non-homogeneous distribution and absence of perinuclear melanin in the tanned RHE compared to the in vivo situation in humans. Extracellularly dispersed epidermal melanin interferes with photoprotection of the keratinocytes.

Skin optical properties from 200 to 300 nm support far UV-C skin-safety in vivo.

The growing threat of multi-drug resistant pathogens and airborne microbial diseases has highlighted the need to improve or develop novel disinfection methods for clinical environments. Conventional ultraviolet C (UV-C) lamps effectively inactivate microorganisms but are harmful to human skin and eyes upon exposure. The use of new 233 nm far UV-C LEDs as an antiseptic can overcome those limitations. In this research, the light penetration into the skin was elucidated for the UV-C region (<300 nm) by measuring the scattering and absorption of skin layers and inverse Monte Carlo simulation, and further confirmed by the first clinical pilot trial in which healthy volunteers were irradiated with a dose of 60 mJ/cm2 at 233 nm. The radiation is strongly absorbed in the stratum corneum, resulting in minimal skin damage without inducing inflammatory responses. The results suggest that 233 nm far UV-C light emitting diodes (LEDs) could effectively inactivate microorganisms, while being safe and soft for the skin.

Evaluation of DNA lesions and radicals generated by a 233 nm far-UVC LED in superficial ex vivo skin wounds.

The application of a far-ultraviolet C (UVC) light emitting diode (LED) of 233 nm showed significant bactericidal efficacy at an applied dose between 20 and 80 mJ cm-2 as reported recently. In addition, only minor epidermal DNA lesions were observed in ex vivo human skin and in vitro epidermal models <10% of the minimal erythema dose of UVB radiation. To broaden the potential range of applications of such systems, e.g. to include postoperative application on wounds for the purpose of decontamination, we assessed how a disruption of normal anatomic skin structure and function influences the skin damage induced by light from 233 nm far-UVC LEDs. Thus, we induced superficial skin wounds by mechanical detachment of the stratum corneum in ex vivo human skin. Barrier-disruption of the skin could be successfully determined by measuring an increase in the transepidermal water loss (TEWL) and the stratum corneum loss could be determined morphologically by 2-photon microscopy (2-PM). After far-UVC irradiation of the skin, we screened the tissue for the development of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). The abundance of DNA lesions was elevated in wound skin in comparison to intact skin after irradiation with far-UVC. However, no increase in DNA lesions was detected when artificial wound exudate consisting of cell culture medium and serum was applied to the disrupted skin surface prior to irradiation. This effect agrees with the results of ray tracing simulations of the absorption of far-UVC light incident on a superficial skin wound. Interestingly, no significant deviations in radical formation between intact skin and superficially wounded skin were detected after far-UVC irradiation as analyzed by electron paramagnetic resonance (EPR) spectroscopy. In conclusion, 233 nm LED light at a dose of 60 mJ/cm2 could be applied safely on superficial wounds for the purpose of skin antisepsis as long as the wounds are covered with wound fluid.

Revealing the Meissner Corpuscles in Human Glabrous Skin Using In Vivo Non-Invasive Imaging Techniques.

The presence of mechanoreceptors in glabrous skin allows humans to discriminate textures by touch. The amount and distribution of these receptors defines our tactile sensitivity and can be affected by diseases such as diabetes, HIV-related pathologies, and hereditary neuropathies. The quantification of mechanoreceptors as clinical markers by biopsy is an invasive method of diagnosis. We report the localization and quantification of Meissner corpuscles in glabrous skin using in vivo, non-invasive optical microscopy techniques. Our approach is supported by the discovery of epidermal protrusions which are co-localized with Meissner corpuscles. Index fingers, small fingers, and tenar palm regions of ten participants were imaged by optical coherence tomography (OCT) and laser scan microscopy (LSM) to determine the thickness of the stratum corneum and epidermis and to count the Meissner corpuscles. We discovered that regions containing Meissner corpuscles could be easily identified by LSM with an enhanced optical reflectance above the corpuscles, caused by a protrusion of the strongly reflecting epidermis into the stratum corneum with its weak reflectance. We suggest that this local morphology above Meissner corpuscles has a function in tactile perception.

Microdialysis on Ex Vivo Porcine Ear Skin Can Validly Study Dermal Penetration including the Fraction of Transfollicular Penetration-Demonstrated on Caffeine Nanocrystals.

Common ex vivo methods for penetration investigations often fail to monitor transfollicular penetration appropriately. In the present investigation, the validity of dermal microdialysis on the ex vivo porcine ear skin to investigate penetration kinetics, including transfollicular penetration, was studied. In setup A, a caffeine nanocrystal formulation was compared to a non-particular caffeine gel formulation. In setup B, two caffeine nanocrystal formulations of different sizes (200 nm, 700 nm) were compared to each other. Microdialysis samples were collected for 46 h. After sampling, the skin layers were separated, homogenized, and caffeine was quantified in all samples. In setup A the area under the curve (AUC) after crystal gel formulation application was 12 times higher than after non-particular formulation application. Setup B showed an increased AUC of 42% in the microdialysis data when the 700 nm caffeine crystals were applied compared to the 200 nm crystals. The microdialysis data was supported by the separation, homogenization and extraction data. Microdialysis performed on ex vivo porcine ear skin is a novel experimental setup. It is of high interest for further investigations since it is able to also capture the impact of follicular and transfollicular penetration kinetics as no other ex vivo setup can.

Primary Human Renal Proximal Tubular Epithelial Cells (pHRPTEpiCs): Shiga Toxin (Stx) Glycosphingolipid Receptors, Stx Susceptibility, and Interaction with Membrane Microdomains.

Tubular epithelial cells of the human kidney are considered as targets of Shiga toxins (Stxs) in the Stx-mediated pathogenesis of hemolytic-uremic syndrome (HUS) caused by Stx-releasing enterohemorrhagic Escherichia coli (EHEC). Analysis of Stx-binding glycosphingolipids (GSLs) of primary human renal proximal tubular epithelial cells (pHRPTEpiCs) yielded globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer) with Cer (d18:1, C16:0), Cer (d18:1, C22:0), and Cer (d18:1, C24:1/C24:0) as the dominant lipoforms. Investigation of detergent-resistant membranes (DRMs) and nonDRMs, serving as equivalents for the liquid-ordered and liquid-disordered membrane phase, respectively, revealed the prevalence of Gb3Cer and Gb4Cer together with cholesterol and sphingomyelin in DRMs, suggesting lipid raft association. Stx1a and Stx2a exerted strong cellular damage with half-maximal cytotoxic doses (CD50) of 1.31 × 102 pg/mL and 1.66 × 103 pg/mL, respectively, indicating one order of magnitude higher cellular cytotoxicity of Stx1a. Surface acoustic wave (SAW) real-time interaction analysis using biosensor surfaces coated with DRM or nonDRM fractions gave stronger binding capability of Stx1a versus Stx2a that correlated with the lower cytotoxicity of Stx2a. Our study underlines the substantial role of proximal tubular epithelial cells of the human kidney being associated with the development of Stx-mediated HUS at least for Stx1a, while the impact of Stx2a remains somewhat ambiguous.

Analysis of the morphometric parameters of pig ear hair follicles.

BACKGROUND: Porcine ear skin is used in studies of percutaneous penetration as a substitute for human skin. The objective of the present study was to determine the structure of the hair follicles on the dorsal area of porcine ear skin and make a morphometric comparison with the hair follicles of human skin. MATERIALS AND METHODS: Sections of frozen biopsies were cut vertically to the skin surface in longitudinal sections using a cryotome and were investigated using microscopy. For each hair follicle, various parameters were determined. RESULTS: The follicular density in porcine ear skin varies according to the area studied, and the length of most of the follicles was approximately 1458 ± 286 µm. The size of the follicular orifice was also determined in a total of 305 follicles. It showed a diameter of roughly 113 ± 43 µm. CONCLUSION: The results showed a very good similarity between human and pig hair follicles. Therefore, porcine ear skin can be considered as a very suitable model of human skin in dermal and especially follicular penetration studies.

Investigation of transfollicular caffeine penetration using microdialysis on ex vivo porcine ear skin.

The aim of this study was to develop an ex vivo method that allows to quantify the transfollicular penetration of topically applied substances by combining microdialysis and selective follicular closure with varnish. An experimental setup with three skin areas on ex vivo intact porcine ear skin was designed (varnish on hair follicle, varnish next to hair follicle, no varnish). On each area, 10 µl/cm2 caffeine-hydroxyethyl-cellulose-gel was applied. Samples were collected for 22 h by microdialysis. After sampling, the skin layers were separated, homogenized and caffeine was quantified by high pressure liquid chromatography (HPLC) in all samples. Potential impact of the varnish placed next to the follicle by tension on the follicle during the drying process was monitored by a microscopic setup and could be excluded. The microdialysis and homogenization study showed a significantly reduced penetration of caffeine when the hair follicles were closed. In areas with open hair follicles caffeine was detected already in the first ten minutes after application. The reported novel combination of two methods is suitable to investigate ex vivo transfollicular penetration. Possible impact of the closure material in the control area can be ruled out by adjusting the design of the control area in future studies.

Solvent-Containing Closure Material Can Be Used to Prevent Follicular Penetration of Caffeine and Fluorescein Sodium Salt on Porcine Ear Skin.

AIM: The skin represents a drug delivery portal. The establishment of a skin model capable of distinguishing between the follicular and intercellular penetration pathways remains a challenge. The study described herein was aimed at showing the influence of two nail varnishes as closure material and four application techniques to spread the active pharmaceutical ingredient (API) on a successful follicular closure without inducing penetration-enhancing effects. MATERIALS AND METHODS: For all experiments, ex vivo porcine ear skin was used. In study design A, a standard and a solvent-free nail varnish were compared. It was tested whether the different application techniques (spreading with pipette, careful finger massage, 5-Hz finger massage, 5-Hz automatic massage) potentially destroy an intact follicular closure. Laser scanning microscopy imaging was used to measure if the model drug (fluorescein sodium salt) penetrated into the hair follicles. Study design B investigated how the penetration is affected when applying standard nail varnish containing solvents to skin. It was tested if the varnish blocks the API (caffeine) on completely covered areas and if adjacent areas show increased penetration. Furthermore, lateral diffusion of the API was investigated. After 20 h, the skin layers were separated by tape stripping and heat separation. The tissue samples were homogenized. Caffeine was quantified by chromatography. RESULTS: In study design A, the standard nail varnish showed a secure follicular closure, while the solvent-free nail varnish was not able to prevent follicular penetration. Moreover, rapid application techniques were found to destroy an intact follicular closure. Only the two most gentle application techniques kept the follicular closing intact. In study design B, no caffeine was detected in both skin areas that were completely covered. Since no significant difference in caffeine penetration between the two uncovered groups was found, any influence of the applied closure material on adjacent areas was excluded. CONCLUSION: This study clearly demonstrates that a standard nail varnish in combination with a gentle application technique of the API provides a secure follicular closure. The presented study only investigated the closure for the substances caffeine and fluorescein sodium salt. The results might not be transferable to all kinds of APIs.