Normal Human Skin Samples Research Articles

Langerhans Cells Directly Interact with Resident T Cells in the Human Epidermis

Adult human skin contains nearly twice as many T cells as the peripheral blood, which include tissue-resident memory T (TRM) cells. However, the precise mechanisms maintaining TRM cells in the healthy skin remain unclear. Using normal human skin samples, we find that Langerhans cells (LCs) contact T cells in the epidermis of the elderly. LCs with high human leukocyte antigen (HLA)-II, CD86, and PD-L2 expression directly contacted PD-1+ TRM cells and CTLA-4+ regulatory T cells in the epidermis, indicating an axis of peripheral tolerance in a steady state. Environmental insults, ultraviolet B radiation and hapten, downregulated HLA-II and CD86 on LCs in the epidermis, suggesting that disruption of LC-T cell tolerogenic axis contributes to skin inflammation. Interestingly, immune checkpoint blockade therapy was associated with decreased epidermal LC-T cell contact in the normal skin of cancer patients affected by cutaneous immune-related adverse events. Collectively, our findings indicate that LCs may contribute to T cell tolerance in the epidermis.

Comparison of Normal Human Skin and Hypertrophic Scar Tissue Samples of Different Ages, Locations, and Stages of Maturity.

Scars disrupt the normal structure and function of the skin. The primary goal of plastic surgery is to prevent and reduce scarring. Therefore, we aimed to establish a comparison scheme between normal skin (NS) tissues of different ages and locations; hypertrophic scars (HTS) of different ages, locations, and maturities; and NS and HTS tissues to provide evidence on scar severity for improving treatment evaluation. Various methods including histology, immunohistochemistry, and immunofluorescence were employed to compare the general appearance, macrophage infiltration, fibroblast activity, degree of angiogenesis, and collagen fiber type and arrangement in human-sourced NS and HTS tissues of different ages, locations, and maturities in seven patients (three with NS and four with HTS) from the Department of Burn and Plastic Surgery of the Shandong Provincial Hospital from January 2019 to December 2020. The thicknesses of the epidermis and dermis of NS tissues varied with age and location. The epidermis of the upper arms, face, and upper eyelids of NS tissues sequentially thickened, whereas the dermis was sequentially thinner. Several glandular structures were identified in the upper eyelids but rarely in the face and upper arms. Histological changes in HTS tissue of different ages, locations, and maturity occur as scar formation time is prolonged, accompanied by increased CD86 levels and fibrosis. As the scar matured, connexin and VEGFR2 expression decreased, indicating reduced inflammation, fibroblast activity, and angiogenesis. The comparison between NS and HTS tissue also revealed significant differences; the positive expression of VEGFR2 and total collagen in HTS tissue was higher than that in NS tissue. We discovered significant differences among NS, HTS, and NS and HTS tissues of different ages, locations, and maturities. Further, this study may provide a basis for clarifying the treatment effect of different methods for HTS compared with those for NS, efficiently individualizing patients' treatment plans and ultimately shortening the scar treatment process.

Function of human skin T cells in wound healing in the in vitro experimental setting

Currently, the treatment of persistent non-healing wounds is among the most difficult clinical issues. We studied 20 samples of normal human skin, 10 specimens from patients with acute trauma, and 9 samples from the patients with chronic wounds that did not heal within 2 months. Using multicolor flow cytometry, we found that the resident T lymphocytes (CD3++ and CD3++) are able to locally produce biologically active substances, normalize human skin homeostasis, thus promoting the wound healing. The data obtained indicate that the blood contains mainly +T lymphocytes (p 0.001), while the +T cells detected in wounds represent a population similar to skin cells. We found no difference in the ratio of resident T cells in chronic and acute wounds, and healthy epithelium. Accordingly, non-healing of wounds and chronic clinical course may be caused by dysfunction of T cells. CD69 regulates T cell secretion of growth factors, IFN, IL-17 and IL-22. The relative number of CD69-expressing T cells from the patients with acute wounds was significantly increased, if compared with cells from normal epidermis and chronic wounds (10.5%2.3, 7.6%1.24, and 3.0%1.05, respectively. p 0.001). The number of cells with the CD3++CD69+ phenotype did not differ significantly between all three groups under comparison. Dysregulation of T cell-mediated healing in chronic wounds is caused by reduced production of IGF-1 by resident CD3++T lymphocytes (1.7%0.9 (p 0.001), and CD3++ (0.44%0.02, p 0.001) compared to CD3++T cells derived from acute wounds (13.6%5.6) and CD3++ (8.9%3.1). The + and + T cells isolated from non-healing chronic wounds did not respond to mitogenic stimuli, unlike the cells obtained from acute wounds and healthy skin. In vitro analysis of cytokine secretion by the CD69-deficient dermal T cells showed a lower spontaneous secretion of IL-22 (4.56%2.3 and 23.9%1.05 and 10.6%1.24, respectively; p 0.001) and IL-2 (0.9%0.08 and 22.6%2.5 and 3.9%1.0, and respectively; p 0.01). When analyzing the number of resident skin T cells secreting IL-17, we obtained the following differences for healthy skin (1.4%0.08), acute wounds (11.3%3.2) and chronic wounds (31.7%11.8), thus showing a significant intergroup difference (p 0.001). T lymphocytes in chronic wounds exhibit some functional disorders and are not able to produce biologically active substances that promote physiological tissue regeneration. The results suggest a role of resident T cells in human skin in wound healing processes and provide new insights into the pathogenesis of chronic wounds.

Identification of diagnostic molecules and potential therapeutic agents for atopic dermatitis by single-cell RNA sequencing combined with a systematic computing framework that integrates network pharmacology.

Atopic dermatitis (AD) is composed of highly flexible cellular participants. To better understand its pathobiology and molecular regulation mechanisms, it is necessary to combine single-cell RNA sequencing (scRNA-seq) with new computing frameworks or specific technologies, which may contribute to the development of better treatments for AD. The scRNA-seq data of GSE180885 and bulk RNA-seq data of GSE193309 were obtained from Gene Expression Omnibus (GEO) database, and the scRNA-seq data was analyzed by Seurat package to identify the cell types in AD. The genes related to the activity of AD topical drugs were obtained from the ChEMBL database, which provided a variety of bioactivity data such as multiple drugs and targets. AD-related genes were obtained from DisGeNET and CTD databases synthesizing human disease-related genes; the intersection of AD-related genes from these three sources with differentially expressed genes (DEGs) between non-diseased AD and normal human skin (NHS) samples and differential cell type marker genes was taken. The proximity analysis of drug gene network was performed based on the gene with the largest area of receiver operating characteristic (ROC) curve. Ten distinct cell types of AD and NHS were identified, except for phagocytes cells. Three hub genes, F10 and CALCRL and CTSB, were obtained. The area under the curve of ROC based on CTSB expression was the largest, which was 60.15%. By binding drug CTSB-related gene interaction network, we identified 145 potential drugs. Among them, the score of DB07045 and CTSB docking was the lowest, and molecular docking and molecular dynamics (MD) simulation confirmed the close and stable binding of DB07045 and cathepsin B. This work identified diagnostic molecules and potential therapeutic drugs of AD by scRNA-seq combined with a systematic computing framework of network pharmacology, which may provide valuable clues for drug design.

610 Myofibroblasts Are Not Characteristic Features of Keloid Lesions

IntroductionKeloids are disfiguring, fibroproliferative lesions that can result from an abnormal wound healing process. Keloids are characterized by excessive and abnormal deposition of extracellular matrix and unrestrained expansion beyond the original wound boundary. Keloids are challenging to treat, with low response rates to current interventions and high recurrence rates. Development of more effective, targeted therapies will require a deeper understanding of the mechanisms driving keloid pathology. It has been widely reported that keloids are characterized by the presence of myofibroblasts, which are specialized, contractile fibroblasts that express alpha-smooth muscle actin (αSMA) and play important roles during wound healing and tissue remodeling. However, the evidence supporting a role for myofibroblasts in keloid development is inconclusive, with conflicting reports in the literature. This complicates development of more effective therapies, as the benefit of interventions targeting myofibroblasts is unclear. The current study evaluated the presence of αSMA+ myofibroblasts in keloids, and analyzed αSMA expression in keloid-derived fibroblasts, to determine whether myofibroblast presence can be considered diagnostic of keloid lesions.MethodsKeloid, hypertrophic scar (HTS), and normal human skin samples were collected with Institutional Review Board approval and primary fibroblast cultures were established. Myofibroblasts in tissue sections were localized by αSMA immunostaining, and αSMA expression in pericytes of blood vessels was distinguished from αSMA+ myofibroblasts by collagen IV immunostaining of vessels. Expression of αSMA mRNA (ACTA2 gene) was measured using quantitative real-time PCR. Statistical analysis via t test was performed using SigmaPlot.ResultsNormal human skin samples did not exhibit αSMA+ myofibroblasts (0/8), but myofibroblasts were identified in 53% of keloids (8/15) and 60% of HTS (3/5) tissue samples. When present in keloids or HTS, the αSMA+ cells were not uniformly distributed, but were often observed in clusters in the deeper regions of the lesions. Comparison of ACTA2 mRNA expression did not reveal any significant differences between normal skin and keloid tissues (N=3/group), or between normal fibroblasts (N=7 donors) and keloid fibroblasts (N=9 donors).ConclusionsThe results suggest that although many keloids contain αSMA+ myofibroblasts, these cells do not appear to be diagnostic for keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloid lesions. However, a limitation of this study is that only excised keloid and HTS tissues, which tend to be larger, more mature lesions, were examined; thus we cannot rule out a potential role for myofibroblasts in early development of abnormal scars.

Retraction notice to "MicroRNA-16 inhibits cell proliferation and migration by targeting heat shock protein 70 in heat-denatured dermal fibroblasts".

Background/Aims: This study aimed to investigate the precise mechanism and function of miR-16 in heat-denatured primary human dermal fibroblasts. Methods: Primary human dermal fibroblasts were separated from normal hu man skin samples. Under heat stress, the levels of miR-16 and heat shock protein 70 (HSP70) were detected in primary human dermal fibroblasts by quantitative real-time polymerase chain reaction (qRT-PCR). Next, heat-denatured cells were transfected with synthetic scrambled negative control (NC) RNA (NC group), miR-16 mimics, miR-16 inhibitor or miR-16 inhibitor accompanied by small in terfering RNA targeting HSP70, then the mRNA level of HSP70 was detected by qRT-PCR, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-di phenyltetrazolium bromide (MTT) and AlamarBlue assay, cell migration was examined by Transwell assay and cell apoptosis was assessed by transferase dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) assay. In addition, cell apoptosis-related proteins, Bax and Bcl-2, were detected by Western blotting. Results: Heat stress significantly reduced miR-16 level and increased the mRNA level of HSP70 compared with untreated cells (p < 0.05). Overexpressed miR-16 reduced the mRNA level of HSP70, suppressed cell proliferation (p < 0.05 or p < 0.01), migration (p < 0.05), and promoted cell apoptosis (p < 0.001) compared with the NC group. Down-regulated miR-16 exerted an opposite effect on primary human dermal fibroblasts with heat-denaturation. Furthermore, effects of miR- 16 down-regulation on cell proliferation and migration were reversed by HSP70 silence. Conclusions: MiR-16 might have an inhibitory effect on cell proliferation and migration in heat-denatured human dermal fibroblasts, and HSP70 might be as sociated with the cell proliferation and migration as a target gene of miR-16.

117 Light or Dark Pigmentation of Engineered Skin Substitutes Containing Melanocytes Protects Against UV-Induced DNA Damage in Vivo

Abstract Introduction Engineered skin substitutes (ESS) were developed to meet the need for prompt wound closure in patients with large full thickness burns. ESS containing autologous fibroblasts and keratinocytes were shown to provide stable wound closure in burn patients, but are limited by hypopigmentation. DNA damage caused by ultraviolet (UV) radiation is a known risk factor for development of skin cancer. In normal human skin, epidermal melanocytes provide pigmentation, helping to shield skin from UV-induced DNA damage. The current study investigated inclusion of human melanocytes (hM) and their role in the response of ESS to UV light in vivo. Methods Primary cells were isolated from skin of healthy de-identified human donors with IRB approval. Three groups of ESS were prepared with fibroblasts and keratinocytes, +/- hM, and were grafted orthotopically to immunodeficient mice: ESS without hM; ESS with light skin-derived (Caucasian) hM (ESS+hML); and ESS with dark skin-derived (African American) hM (ESS+hMD). After 8 weeks in vivo, grafts were irradiated with 135 mJ/cm2 UV, and mice were euthanized after 2 or 24 hours; non-UV treated mice served as controls. Pigmentation and erythema were measured with a Mexameter. Melanocytes and cyclobutane pyrimidine dimers (CPDs) were quantified by immunostaining with anti-TYRP1 and anti-CPD antibodies, respectively, followed by image analysis (Nikon Elements). Statistical analyses (SigmaPlot) utilized t-test or one-way ANOVA; P&amp;lt; 0.05 was considered significant. Results At 8 weeks post-grafting, mean hM density in ESS+hML and ESS+hMD was not significantly different from normal human skin samples. Pigmentation (in Mexameter units) before UV irradiation was significantly different among groups (ESS+hMD &amp;gt; ESS+hML &amp;gt; ESS no hM). UV irradiation did not increase erythema in any group, but resulted in significantly increased pigmentation in ESS+hML and ESS+hMD at 2 hours, but not 24 hours, post-UV. CPDs, the most prevalent form of UV-induced DNA damage, were significantly elevated 24 hours post-UV in ESS without hM. DNA damage was significantly lower 24 hours post-UV in ESS+hML and ESS+hMD compared with ESS without hM. No differences in DNA damage were observed between ESS+hML and ESS+hMD. Conclusions Pigmentation of ESS+hML and ESS+hMD in vivo varied according to the skin phototype of the hM donor, with no difference in melanocyte density, which was similar to normal human skin. Inclusion of either light or dark hM decreased UV-induced DNA damage, suggesting that hM in ESS play a photoprotective role, as in normal human skin. Applicability of Research to Practice Protection against UV-induced DNA damage may reduce the risk of skin cancer in patients grafted with ESS containing melanocytes.

509 Decline in Langerhans cell precursor recruitment into the human skin explains the reduction in epidermal Langerhans cells with age

Aging is accompanied by immunosenescence and increased susceptibility to bacterial and viral infections. Skin provides the first line of physical and immunological defense against environmental insults. However, the age-related changes in the immune function of the human skin are unclear. Here,we investigated the age-related changes in epidermal Langerhans cells (LCs), which play a sentinel role in the initiation of the immune responses in the skin. We screened over 400 normal human skin samples to identify cohorts of young and old sun-protected skin from the same anatomical site. There was a significant reduction in the number of epidermal LCs with age. Among the possible explanations for this reduction, we found a significant reduction in the number of CD14+ CD207+ CCR6+dermal LC precursors with age. In addition, the expression of LC precursor-recruiting chemokine, CXCL14, was markedly down-regulated in the basal layer keratinocytes of the old skin. Importantly, we discovered that young skin recruited more LC precursor-like THP1 cells compared to old skin ex vivo, and this recruitment was inhibited by blocking CXCL14 signaling.Overall, our findings demonstrate an age-related decline in CXCL14-LC precursor axis, which results in the reduction of epidermal LCs under steady-state condition. CXCL14 may provide a novel therapeutic target for prevention of age-related LC reduction.

MicroRNA-16 inhibits cell proliferation and migration by targeting heat shock protein 70 in heat-denatured dermal fibroblasts.

Background/AimsThis study aimed to investigate the precise mechanism and function of miR-16 in heat-denatured primary human dermal fibroblasts.MethodsPrimary human dermal fibroblasts were separated from normal human skin samples. Under heat stress, the levels of miR-16 and heat shock protein 70 (HSP70) were detected in primary human dermal fibroblasts by quantitative real-time polymerase chain reaction (qRT-PCR). Next, heat-denatured cells were transfected with synthetic scrambled negative control (NC) RNA (NC group), miR-16 mimics, miR-16 inhibitor or miR-16 inhibitor accompanied by small interfering RNA targeting HSP70, then the mRNA level of HSP70 was detected by qRT-PCR, cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and AlamarBlue assay, cell migration was examined by Transwell assay and cell apoptosis was assessed by transferase dUTP (deoxyuridine triphosphate) nick end labeling (TUNEL) assay. In addition, cell apoptosis-related proteins, Bax and Bcl-2, were detected by Western blotting.ResultsHeat stress significantly reduced miR-16 level and increased the mRNA level of HSP70 compared with untreated cells (p < 0.05). Overexpressed miR-16 reduced the mRNA level of HSP70, suppressed cell proliferation (p < 0.05 or p < 0.01), migration (p < 0.05), and promoted cell apoptosis (p < 0.001) compared with the NC group. Down-regulated miR-16 exerted an opposite effect on primary human dermal fibroblasts with heat-denaturation. Furthermore, effects of miR16 down-regulation on cell proliferation and migration were reversed by HSP70 silence. ConclusionsMiR-16 might have an inhibitory effect on cell proliferation and migration in heat-denatured human dermal fibroblasts, and HSP70 might be associated with the cell proliferation and migration as a target gene of miR-16.

A proinflammatory microenvironment induces NFkB activation and beta-defensin expression through specific Toll Like Receptors in a 3D human skin model

Psoriasis is an autoimmune skin disease characterized by the formation and the progression of silvery plaques on the extensory surfaces of our body. Proinflammatory cytokines as Tumor Necrosis Factor (TNF)-alpha, interleukin (IL)-17, IL-22 and IL-23 represent for the normal skin a psoriatic microenvironment. In the 3D human skin model standardized in our lab in the last decade, we were able to dissect the events in which each cytokine exerts a specific effect, e.g. keratinocyte proliferation, Langerhans cell activation, cytoskeleton arrangement, and, more recently, the epidermal expression of Toll like Receptors (TLRs) 2, 7, 9. Several experimental studies reported that in psoriasis TLRs are expressed and their activation triggers i) NFkB translocation from the cytoplasm to the nucleus and ii) the release of beta defensins (HBDs). The present study was aimed at investigating the intracellular NFkB activation and HBD1 and HBD2 expression induced by a cytokine mix (TNF-alpha, IL-17, IL-22, IL-23) by indirect immunofluorescence. Bioptic samples of normal human skin were obtained after aesthetic surgery of young healthy informed women (n=7). After overnight incubation to reduce mechanical and termical stress, skin fragments were incubated in a Transwell system for 5 (T5), 24 (T24), and 48 (T48) hours with the cytokine mix. Parallel control samples were carried out and each patient was represented at all time points. In controls at all time-points NFkB was localized only in the cytoplasm, while, starting from T5, scattered basal nuclei were observed in the cytokine-incubated samples. At later time points, in the upper spinous and granular layers, NFkB nuclear immunostaining was evident. HBD2 expression was affected after cytokine mix exposure, while HBD1 distribution was similar to controls. Thanks to this simple but effective model, a deep knowledge of the early events occurring in the normal epidermis exposed to cytokines can be achieved, excluding the contribution of the blood and lymphatic vessels herein absent. This basic research can thus represent an important tool for targeting and counteracting single phenomenon leading to the formation/progression with the most innovative biological drugs.

Topical Fibronectin Improves Wound Healing of Irradiated Skin

Wound healing is significantly delayed in irradiated skin. To better understand global changes in protein expression after radiation, we utilized a reverse phase protein array (RPPA) to identify significant changes in paired samples of normal and irradiated human skin. Of the 210 proteins studied, fibronectin was the most significantly and consistently downregulated in radiation-damaged skin. Using a murine model, we confirmed that radiation leads to decreased fibronectin expression in the skin as well as delayed wound healing. Topically applied fibronectin was found to significantly improve wound healing in irradiated skin and was associated with decreased inflammatory infiltrate and increased angiogenesis. Fibronectin treatment may be a useful adjunctive modality in the treatment of non-healing radiation wounds.

Identification of candidate genes in atopic dermatitis based on bioinformatic methods.

Atopic dermatitis (AD) is a chronic or relapsing inflammatory disorder of the skin that frequently precedes asthma and allergic disorders. This study aimed to identify candidate genes related to AD using bioinformatic methods. The microarray data of GSE32924, including 12 nonlesional AD (ANL) and 13 lesional AD (AL) skin samples obtained from 14 patients with AD as well as eight other normal human skin samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were screened in ANL and AL skin samples compared with normal controls, followed by gene ontology (GO) and pathway enrichment analyses. Furthermore, the selected overlapping DEGs were analyzed to identify co-expressed genes, and a co-expression network was established. GeneCodis database was selected for functional annotation of the differentially co-expressed genes and a regulatory network was constructed. Compared with normal controls, 438 DEGs were identified in ANL skin samples and were mainly enriched in two pathways and the GO terms associated with epidermis development. Besides, 779 DEGs were identified in AL skin samples and were mainly enriched in four pathways and GO terms associated with immune response. Stimulated by retinoic acid 13 (STRA13), presenilin enhancer gamma secretase subunit (PSENEN), and nucleosome assembly protein 1-like 2 (NAP1L2) were significantly enriched by integration analysis of the co-expression and regulatory network. Genes STRA13, PSENEN, and NAP1L2 were presumed to play critical roles in AD and they may serve as potential targets for the treatment of AD.

In vitro 3-D model based on extending time of culture for studying chronological epidermis aging

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors.The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging.By using prolonged culture as a proxy for cellular aging, we extended to 120days the culture time of a skin equivalent model based on collagen–glycosaminoglycan–chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages.We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model.To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.

Selective susceptibility of human skin antigen presenting cells to productive dengue virus infection.

Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14+ and CD1c+ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response.

Comparison of xenobiotic metabolizing enzyme activities in ex vivo human skin and reconstructed human skin models from SkinEthic

Skin function is not limited to a physical barrier. According to its total surface area, it is also considered as an extra-hepatic metabolizing organ. In vitro engineered human skins have been developed to replace limited ex vivo normal human skin samples (NHS). Thus, assessing and comparing skin models from SkinEthic [Episkin™, RHE™ and the full thickness model (FTM)] with NHS in terms of metabolic capability are essential. The apparent activities of main cutaneous isoforms of cytochrome P450-dependent monooxygenases (CYP1A1/1B1, 2B6/2C18/2E1, 3A5/3A7), esterase, glutathione-S-[(GST), A, M, P, T], N-acetyl-(NAT1), uridinyl-diphosphate glucuronyl-(UDPGT 1A family) and sulfo-(SULT1A1) transferases were determined using probe substrates. Mean activities indicative of CYP1A1/1B1 (expressed as pmol/mg protein/6 h) in RHE™ (2.8) and FTM (2.6) were very similar to NHS (3.0) while Episkin™ showed a higher activity (9.1). Activities of CYP3A5/3A7 in FTM (3.3) and Episkin™ (3.6) were similar to NHS (3.8) while activity in RHE™ (13.3) was higher. CYP2B6/2C18/2E1 activity was below LOQ (0.5) in all skin models and NHS. Comparable intrinsic metabolic clearances were measured between NHS and skin models for esterase, UDPGT, GST and NAT1 activities. SULT1A1 activity toward probe substrates was not detected in skin models and observed at the limit of detection in NHS. Weak cytochrome P450-dependent monooxygenases, high esterase and transferase activities suggested that NHS and skin models exhibited limited functionalization and much greater detoxification (hydrolytic and conjugating) capacities. These results demonstrate that skin models are similar to NHS in terms of metabolic functionality toward xenobiotics investigated and useful tools to assess both the local efficiency and safety of cosmetics.

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

Elevated YAP and Its Downstream Targets CCN1 and CCN2 in Basal Cell Carcinoma: Impact on Keratinocyte Proliferation and Stromal Cell Activation

A Signal Transduction Pathway from TGF-β1 to SKP2 via Akt1 and c-Myc and its Correlation with Progression in Human Melanoma

Both SKP2 (S-phase kinase-associated protein 2) and transforming growth factor-β1 (TGF-β1) play important roles in cancer metastasis through different mechanisms: TGF-β1 via induction of epithelial-mesenchymal transition (EMT) and SKP2 via downregulating p27(kip1). Recent studies indicated that c-Myc and Akt1 were active players in metastasis. In this study we demonstrated a crosstalk between these pathways. Specifically, we found that TGF-β1 treatment increased SKP2 expression accompanied with increased phosphorylation of Akt1 and c-Myc protein accumulation during EMT. We demonstrated that Akt1 was required for TGF-β1-mediated SKP2 upregulation and that c-Myc transcription factor specifically bound to the promoter of SKP2 for its enhanced transcription. Analysis of 25 samples of normal human skin, nevi, and melanomas revealed a positive correlation between c-Myc and SKP2 accumulation. Furthermore, accumulation of SKP2 and c-Myc proteins was significantly higher in metastatic melanoma samples as compared with that in primary melanomas, which again was higher than that in normal skin or nevi. In summary, our results integrated TGF-β1 signals to SKP2 via Akt1 and c-Myc during EMT, and provided, to our knowledge, a previously unreported mechanistic molecular event for TGF-β1-induced metastasis in human melanoma.

Expression and Function of Macrophage Migration Inhibitory Factor in the Pathogenesis of UV‐Induced Cutaneous Nonmelanoma Skin Cancer†

Chronic skin exposure to ultraviolet light stimulates the production of cytokines known to be involved in the initiation of skin cancer. Recent studies in mouse models suggested a role for macrophage migration inhibitory factor (MIF) in the UVB-induced pathogenesis of nonmelanoma skin cancer (NMSC). Our studies aimed at defining the pathophysiological function of MIF in cutaneous inflammatory reactions and in the development and progression of NMSC. Immunohistochemical analysis revealed a moderate expression of MIF in normal human skin samples but an enhanced expression of this cytokine in lesional skin of patients with actinic keratosis or cutaneous SCC. Enzyme-linked immunosorbent assay studies showed a time-dependent increase in MIF secretion after a moderate single-dose UVB irradiation in NHEKs and SCC tumor cells. MIF is known to interact with CXCR2, CXCR4 and CD74. These receptors are not constitutively expressed in keratinocytes and HaCaT cells and their expression is not induced by UVB irradiation either. However, stimulation with IFNγ upregulated CD74 surface expression in these cells. Affymetrix(®) Gene Chip analysis revealed that only keratinocytes prestimulated with IFNγ are responsive to MIF. These findings indicate that MIF may be an important factor in the pathogenesis of NMSC tumorigenesis and progression in an inflammatory environment.

Differential diagnosis in primary and metastatic cutaneous melanoma by FT-Raman spectroscopy

To qualify the FT-Raman spectral data of primary and metastatic cutaneous melanoma in order to obtain a differential diagnosis. Ten normal human skin samples without any clinical or histopathological alterations, ten cutaneous melanoma fragments, and nine lymph node metastasis samples were used; 105, 140 and 126 spectra were obtained respectively. Each sample was divided into 2 or 3 fragments of approximately 2 mm³ and positioned in the Raman spectrometer sample holder in order to obtain the spectra; a monochrome laser light Nd:YAG at 1064 nm was used to excite the inelastic effect. To differentiate the three histopathological groups according to their characteristics extracted from the spectra, data discriminative analysis was undertaken. Phenylalanine, DNA, and Amide-I spectral variables stood out in the differentiation of the three groups. The percentages of correctly classified groups based on Phenylalanine, DNA, and Amide-I spectral features was 93.1%. FT-Raman spectroscopy is capable of differentiating melanoma from its metastasis, as well as from normal skin.

Aminolaevulinic acid diffusion characteristics in ‘in vitro’ normal human skin and actinic keratosis: implications for topical photodynamic therapy

The response rate of aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) in certain subtypes of actinic keratosis (AK), such as hypertrophic and hyperkeratotic lesions, is variable, an effect attributable to a supposed lack of ALA penetration. A detailed and depth-related profile of spatial ALA permeation in AK following drug administration would lead to a greater understanding of concentrations achievable before protoporphyrin IX biosynthesis and subsequent PDT. ALA penetration through excised normal human skin (NS) and AK lesions was evaluated using a cryostatic sectioning technique and radio-isotope counting following drug delivery using a novel, bioadhesive patch, loaded with 19, 38 or 50 mg/cm(2) ALA. Distinct differences in ALA concentration with respect to depth between AK and NS samples were shown, particularly within the superficial layers of the tissue structure, down to a depth of 1.0 mm. Patch application times were shown to influence ALA concentrations in tissue, but there was no clear correlation between ALA penetration in AK lesions taken from different body locations and from patients of different age. Similarly, the thickness of stratum corneum was not related to the ALA distribution profiles. Sizable variation in ALA concentration was a prominent feature of profiles through AK lesions, which may explain the variation of observed protoporphyrin IX production seen in the clinical implementation of AK PDT. That said, the results of this study show sufficient ALA penetration to a depth of 1.0 mm, which should be satisfactory for successful treatment of the majority of non-hyperkeratotic, hypertrophic AK using patch-based delivery methods.