EGFR inhibitors switch keratinocytes from a proliferative to a differentiative phenotype affecting epidermal development and barrier function.

BACKGROUND: Cutaneous adverse drug reactions (CADR) associated with oncology therapy involve 45-100% of patients receiving kinase inhibitors. Such adverse reactions may include skin inflammation, infection, pruritus and dryness, symptoms that can significantly affect the patient's quality of life. To prevent severe skin damages dose adjustment or drug discontinuation is often required, interfering with the prescribed oncology treatment protocol. This is particularly the case of Epidermal Growth Factor Receptor inhibitors (EGFRi) targeting carcinomas. Since the EGFR pathway is pivotal for epidermal keratinocytes, it is reasonable to hypothesize that EGFRi also affect these cells and therefore interfere with the epidermal structure formation and skin barrier function. METHODS: To test this hypothesis, the effects of EGFRi and Vascular Endothelial Growth Factor Receptor inhibitors (VEGFRi) at therapeutically relevant concentrations (3, 10, 30, 100 nM) were assessed on proliferation and differentiation markers of human keratinocytes in a novel 3D micro-epidermis tissue culture model. RESULTS: EGFRi directly affect basal keratinocyte growth, leading to tissue size reduction and switching keratinocytes from a proliferative to a differentiative phenotype, as evidenced by decreased Ki67 staining and increased filaggrin, desmoglein-1 and involucrin expression compared to control. These effects lead to skin barrier impairment, which can be observed in a reconstructed human epidermis model showing a decrease in trans-epidermal water loss rates. On the other hand, pan-kinase inhibitors mainly targeting VEGFR barely affect keratinocyte differentiation and rather promote a proliferative phenotype. CONCLUSIONS: This study contributes to the mechanistic understanding of the clinically observed CADR during therapy with EGFRi. These in vitro results suggest a specific mode of action of EGFRi by directly affecting keratinocyte growth and barrier function.

Visualisation of penetration of topical antifungal drug substances through mycosis-infected nails by matrix-assisted laser desorption ionisation mass spectrometry imaging.

BACKGROUND: Matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI) is a mass spectrometry-based technique, which can be applied for compound-specific imaging of pharmaceuticals in tissues samples. MALDI-MSI technology is widely used to visualise penetration and distribution profile through different tissues but has never been used with nail tissue. OBJECTIVES: This study used MALDI-MSI technology to visualise distribution profile and penetration into ex vivo human mycosis-infected toenails of three antifungal active ingredients amorolfine, ciclopirox and naftifine contained in topical onychomycosis nail treatment preparations, marketed as Loceryl® , Ciclopoli® and Exoderil® . METHODS: Three mycosis-infected toenails were used for each treatment condition. Six and twenty-four hours after one single topical application of antifungal drugs, excess of formulation was removed, nails were cryo-sectioned at a thickness of 20 µm, and MALDI matrix was deposited on each nail slice. Penetration and distribution profile of amorolfine, ciclopirox and naftifine in the nails were analysed by MALDI-MSI. RESULTS: All antifungal actives have been visualised in the nail by MALDI-MSI. Ciclopirox and naftifine molecules showed a highly localised distribution in the uppermost layer of the nail plate. In comparison, amorolfine diffuses through the nail plate to the deep layers already 6 hours after application and keeps diffusing towards the lowest nail layers within 24 hours. CONCLUSIONS: This study shows for the first-time distribution and penetration of certain antifungal actives into human nails using MALDI-MSI analysis. The results showed a more homogeneous distribution of amorolfine to nail and a better penetration through the infected nails than ciclopirox and naftifine.

MicroRNA-23b-3p regulates human keratinocyte differentiation through repression of TGIF1 and activation of the TGF-ß-SMAD2 signalling pathway.

MicroRNAs (miRNAs) are a class of short non-coding RNAs capable of repressing gene expression at the post-transcriptional level. miRNAs participate in the control of numerous cellular mechanisms, including skin homeostasis and epidermal differentiation. However, few miRNAs involved in these processes have been identified so far in human skin, and the gene networks they control remain largely unknown. Here, we focused on miR-23b-3p, a miRNA that is expressed during the late step of human keratinocyte differentiation. We report that miR-23b-3p silencing modulates epidermal differentiation in human skin reconstructs. The SMAD transcriptional corepressor TGIF1 was identified on bioinformatic analysis as a potential target of miR-23b-3p. Expression analysis and reporter gene assays confirmed direct regulation of TGIF1 expression by miR-23b-3p. Finally, we showed that miR-23-3p was able to activate TGF-ß signalling in human keratinocytes by increasing SMAD2 phosphorylation through TGIF1 repression. Taken together, these data identify miR-23b-3p as a new regulator of human epidermal differentiation in line with TGF-ß signalling.

Melanin fate in the human epidermis: a reassessment of how best to detect and analyse histologically.

Melanin is the predominant pigment responsible for skin colour and is synthesized by the melanocyte in the basal layer of the epidermis and then transferred to surrounding keratinocytes. Despite its optical properties, melanin is barely detectable in unstained sections of human epidermis. However, identification and localization of melanin is of importance for the study of skin pigmentation in health and disease. Current methods for the histologic quantification of melanin are suboptimal and are associated with significant risk of misinterpretation. The aim of this study was to reassess the existing literature and to develop a more effective histological method of melanin quantification in human skin. Moreover, we confirm that Warthin-Starry (WS) stain provides a much more sensitive and more specific melanin detection method than the commonplace Fontana-Masson (FM) stain. For example, WS staining sensitivity allowed the visualization of melanin even in very pale Caucasian skin that was missed by FM or Von Kossa (VK) stains. From our reassessment of the histology-related literature, we conclude that so-called melanin dust is most likely an artifact of discoloration due to non-specific silver deposition in the stratum corneum. Unlike FM and VK, WS was not associated with this non-specific stratum corneum darkening, misinterpreted previously as 'degraded' melanin. Finally, WS melanin particle counts were largely similar to previously reported manual counts by transmission electron microscopy, in contrast to both FM and VK. Together these findings allow us to propose a new histology/Image J-informed method for the accurate and precise quantification of epidermal melanin in skin.

Penetration Profiles of Four Topical Antifungals in Mycotic Human Toenails Quantified by Matrix-Assisted Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Imaging.

INTRODUCTION: Onychomycosis is a fungal infection of the nails that can be challenging to treat. Here, matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging was applied to the quantitative analysis of the penetration profile of the antifungal compound, amorolfine, in human mycotic toenails. The amorolfine profile was compared with those of three other antifungals, ciclopirox, naftifine, and tioconazole. METHODS: Antifungal compounds (amorolfine 5% lacquer, ciclopirox 8% lacquer, naftifine 1% solution, and tioconazole 28% solution) were applied to mycotic nails (n = 42). Nail sections were prepared, and MALDI-FTICR analysis was performed on the sections at a spatial resolution of 70 µm to compare the distribution profiles. Based on the minimum inhibitory concentrations of the four test compounds needed to kill 90% (MIC90) of the fungal organism, Trichophyton rubrum, the fold differences between the MIC90 and the antifungal concentrations in the nails (termed the multiplicity of the MIC90) were calculated for each. RESULTS: The penetration profiles indicated higher concentrations of amorolfine and ciclopirox in the deeper layers of the nails 3 h after treatment, compared with naftifine and tioconazole. The mean concentrations across the entire nail sections at 3 h were significantly different among the four antifungals: amorolfine, 2.46 mM; ciclopirox, 0.95 mM; naftifine, 0.63 mM; and tioconazole, 1.36 mM (p = 0.016; n = 8 per compound). The median multiplicity of the MIC90 at 3 h was 191-fold for amorolfine, tenfold for ciclopirox, 52-fold for naftifine, and 208-fold for tioconazole. CONCLUSION: In this study, MALDI-FTICR was successfully applied to the quantitative analysis of antifungal distribution in human mycotic nails. The findings suggest that amorolfine penetrates deeper layers of the nail and accumulates at concentrations far exceeding the MIC needed to exert antimycotic activity.

Penetration Profile of Terbinafine Compared to Amorolfine in Mycotic Human Toenails Quantified by Matrix-Assisted Laser Desorption Ionization-Fourier Transform Ion Cyclotron Resonance Imaging.

INTRODUCTION: Amorolfine 5% lacquer is an established topical treatment for fungal infection of the nails. The success of topical therapy for onychomycosis depends on whether the permeated drug concentration in the deep nail bed is retained above the effective antifungal minimum inhibitory concentration (MIC). We compared the penetration profile of amorolfine and a new topical formula of terbinafine in human mycotic toenails using matrix-assisted laser desorption ionization mass spectrometry imaging-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging. METHODS: Amorolfine 5% lacquer and terbinafine 7.8% lacquer were applied to mycotic nails (n = 17); nail sections were prepared, and MALDI-FTICR analysis was performed. Based on the MICs of amorolfine and terbinafine needed to kill 90% (MIC90) of Trichophyton rubrum, the fold differences between the MIC90 and the antifungal concentrations in the nails (the multiplicity of the MIC90) were calculated overall and for the keratin-unbound fractions. RESULTS: Both amorolfine and terbinafine penetrated the entire thickness of the nail. The mean concentration across the entire nail section 3 h following terbinafine treatment was 1414 µg/g of tissue (equivalent to 4.9 mM) compared with 780 µg/g (2.5 mM) following amorolfine treatment (not significantly different; p = 0.878). The median multiplicity of the MIC90 was significantly higher in amorolfine- than terbinafine-treated nails overall (191 vs. 48; p = 0.010) and for the keratin-unbound fractions only (7.4 vs. 0.8; p = 0.002). CONCLUSION: In this ex vivo study, MALDI-FTICR demonstrated that, although amorolfine 5% and terbinafine 7.8% had similar distribution profiles, both penetrating from the surface to the nail bed, the concentration of amorolfine in the nail was significantly higher than that of terbinafine relative to their respective MIC90 values. Clinical studies are required to determine whether these effects translate to a clinical difference in treatment success.

Differential miRNA expression profiles in proliferating or differentiated keratinocytes in response to gamma irradiation.

BACKGROUND: MicroRNAs (miRNAs), a group of short non-coding RNAs that negatively regulate gene expression, have recently emerged as potential modulators of cellular response to ionizing radiations both in vitro and in vivo in various cell types and tissues. However, in epidermal cells, the involvement of the miRNA machinery in the cellular response to ionizing radiations remains to be clarified. Indeed, understanding the mechanisms of cutaneous radiosensitivity is an important issue since skin is the most exposed organ to ionizing radiations and among the most sensitive. RESULTS: We settled up an expression study of miRNAs in primary human skin keratinocytes using a microfluidic system of qPCR assay, which permits to assess the expression of almost 700 annotated miRNAs. The keratinocytes were cultured to a proliferative or a differentiated state mimicking basal or suprabasal layers of human epidermis. These cells were irradiated at 10 mGy or 6 Gy and RNA was extracted 3 hours after irradiation. We found that proliferative cells irradiated at 6 Gy display a global fall of miRNA expression whereas differentiated cells exposed to the same dose display a global increase of miRNAs expression. We identified twenty miRNAs weakly but significantly modulated after 6 Gy irradiation, whereas only 2 miRNAs were modulated after low-dose irradiation in proliferating cells. To go further into the biological meaning of this miRNA response, we over-expressed some of the responding miRNA in proliferating cells: we observed a significant decrease of cell viability 72 hours after irradiation. Functional annotation of their predicted targets revealed that G-protein related pathways might be regulated by these responding miRNAs. CONCLUSIONS: Our results reveal that human primary keratinocytes exposed to ionizing irradiation expressed a miRNA pattern strongly related to the differentiation status of irradiated cells. We also demonstrate that some miRNAs play a role in the radiation response to ensure the short-term survival of irradiated keratinocytes.

Glycerol and natural sugar-derived complex modulate differentially stratum corneum water-binding properties and structural parameters in an in vitro Raman-desorption model.

The epidermal protective functions are closely associated with skin hydration homeostasis. The understanding of different states of water binding is a rising concept in assessing topically applied formulations and their interaction within the stratum corneum (SC). In addition to global water content, primary bound water, partially bound water, and unbound water and barrier-related lipid lateral packing and protein secondary structure can be measured by Raman spectroscopy. This study aimed to establish an in vitro SC model to evaluate differences in the efficacy of a natural sugar-derived complex in combination with glycerol and a botanical extract in modulating SC water binding and structural proteins and barrier lipids. These compounds were selected due to their water-binding and soothing properties. The SC water profiles were assessed at the surface and in 8 µm SC depth. After a 12-hour hyperhydration and subsequent product incubation the measurements were performed during a 6 hours desiccation phase. The maximal water caption and the time until reaching a steady state are measured as well as water retention and resistance against water loss. Global water content, partially bound, and unbound water, as well as lipid and protein structures were assessed with confocal Raman microspectroscopy. Both the natural sugar-derived mixture and more pronounced, the same mixture with additional glycerol increased all three water-binding parameters at the surface and in 8 µm SC depth at the beginning and during the desiccation phase. Further addition of botanical extract did not result in an additional increase of the water-binding. All three formulations showed an increase in the lipid lateral packing values prevented the protein alteration as measured by ß-sheets signal compared to blank. The present model is suited for screening studies comparing the specific effects of different compounds on hydration states. The natural sugar-derived mixture Aquaxyl showed evidence for an improvement of all SC hydration states, lipid and protein structure which was further enhanced by the addition of glycerol 5%. This improvement was evidenced at the surface and within the SC for all hydration-related parameters, and the lipid as well the protein structures. The addition of botanical extract phytoessence blue daisy did not show further improvement.