Macrophage migration inhibitory factor (MIF) and its homolog D-dopachrome tautomerase (D-DT) are significant promotors of UVB- but not chemically induced non-melanoma skin cancer.

Non-melanoma skin cancer (NMSC) is the most common cancer in Caucasians worldwide. We investigated the pathophysiological role of MIF and its homolog D-DT in UVB- and chemically induced NMSC using Mif-/-, D-dt-/- and Mif-/-/D-dt-/- mice on a hairless SKH1 background. Knockout of both cytokines showed similar attenuating effects on inflammation after acute UVB irradiation and tumor formation during chronic UVB irradiation, without additive protective effects noted in double knockout mice, indicating that both cytokines activate a similar signaling threshold. In contrast, genetic deletion of Mif and D-dt had no major effects on chemically induced skin tumors. To get insight into the contributing mechanisms, we used an in vitro 3D skin model with incorporated macrophages. Application of recombinant MIF and D-DT led to an accumulation of macrophages within the epidermal part that could be reversed by selective inhibitors of MIF and D-DT pathways. In summary, our data indicate that MIF and D-DT contribute to the development and progression of UVB- but not chemically induced NMSC, a role at least partially accounted by effects of both cytokines on epidermal macrophage accumulation. These data highlight that MIF and D-DT are both potential therapeutic targets for the prevention of photocarcinogenesis but not chemical carcinogenesis.

MMP-3 plays a major role in calcium pantothenate-promoted wound healing after fractional ablative laser treatment.

Ablative fractional laser treatment leads to a loss of matrix metalloproteinase-3 (MMP-3) expression; therefore, in the present in vitro study, we addressed the role of MMP-3 and its regulation by calcium pantothenate in wound healing processes at the molecular level. Utilizing confocal laser microscopy, we investigated MMP-3 protein expression in fractional ablative CO2 laser-irradiated skin models. In addition, we established full-thickness 3D skin models using fibroblasts and keratinocytes with a MMP-3 knockdown that were irradiated with a fractional ablative Er:YAG laser to set superficial injuries with standardized dimensions and minimal thermal damage to the surrounding tissue. We revealed an upregulation of MMP-3 protein expression in laser-irradiated skin models receiving aftercare treatment with calcium pantothenate. Skin models with MMP-3 knockdown exhibited a slower wound closure after laser treatment compared to controls. Gene expression profiling detected an MMP-3 knockdown-dependent upregulation of cytokines and chemokines (e.g. IL-36B, CXCL17, IL-37, CXCL5), antimicrobial peptides (e.g., S100A7, S100A12), epidermal crosslinking enzymes (TGM5), and differentiation markers (e.g., LOR, KRT1, FLG2). We also detected a downregulation of cathepsin V and MMP-10, both of which play a prominent role in wound healing processes. After fractional ablative laser injury, an aftercare treatment with calcium pantothenate accelerated wound closure in MMP-3 expressing models faster than in MMP-3 knockdown models. Our data substantiate a major role of MMP-3 in wound healing processes after ablative laser treatments. For the first time, we could show that calcium pantothenate exerts its wound healing-promoting effects at least partly via MMP-3.

Biological Effects of Hyaluronic Acid-Based Dermal Fillers and Laser Therapy on Human Skin Models.

Injection of dermal fillers is one of the most frequently performed aesthetic procedures. The aim of the present study was to investigate the biological effects of different stabilized hyaluronan (HA) and poly-l-lactic acid fillers with and without subsequent additional fractional laser co-treatment on skin morphology and gene expression. Intradermal injection resulted in a significant enhancement of epidermal thickness detected by histological analysis. Combining HA fillers with ablative fractional CO2- or Er:YAG laser irradiation enhanced this effect. Gene expression profiling revealed an upregulation of modulators of tissue remodeling (eg TIMP3, SERPIN E1) and collagens (COL11A1). On the other hand, we detected a downregulation of differentiation markers (eg FLG, LOR, KRT1) and proinflammatory cytokines (eg IL-36, IL-1β). Interestingly, HA-based fillers revealed a specific upregulation pattern of chemokines such as CXCL5 andCCL20 suggesting a secondary effect of these fillers on the immune cells of the skin, especially monocytes and macrophages. Taken together, our data show enhancing effects of dermal fillers on epidermal thickness and prove the proliferating effects of these products on epidermal cells on the molecular level. Moreover, our findings reveal synergistic effects of fractional ablative laser treatment and HA dermal filler injection suggesting a combination of both treatments. J Drugs Dermatol. 2020;19(9):897-899. doi:10.36849/JDD.2020.4856.

Inter-α-Trypsin Inhibitor Heavy Chain 5 (ITIH5) Is a Natural Stabilizer of Hyaluronan That Modulates Biological Processes in the Skin.

INTRODUCTION: Hyaluronan (HA) is a major component of the skin that exerts a variety of biological functions. Inter-α-trypsin inhibitor heavy chain (ITIH) proteins comprise a family of hyaladherins of which ITIH5 has recently been described in skin, where it plays a functional role in skin morphology and inflammatory skin diseases including allergic contact dermatitis (ACD). OBJECTIVE: The current study focused on the ITIH5-HA interaction and its potential clinical and functional impact in extracellular matrix (ECM) stabilization. METHODS: Studying the molecular effects of ITIH5 in skin, we established skin models comprising murine skin cells of Itih5 knockout mice and corresponding wild-type controls. In addition, human dermal fibroblasts with an ITIH5 knockdown as well as a murine recombinant Itih5 protein were established to examine the interaction between ITIH5 and HA using in vitro adhesion and HA degradation assays. To understand more precisely the role of ITIH5 in inflammatory skin diseases such as ACD, we generated ITIH5 knockout cells of the KeratinoSens® cell line. RESULTS: Using murine skin models, ITIH5 knockdown fibroblasts, and a reactive oxygen species (ROS)-mediated HA degradation assay, we proved that ITIH5 binds to HA, thereby acting as a stabilizer of HA. Moreover, microarray profiling revealed the impact of ITIH5 on biological processes such as skin development and ECM homeostasis. Performing the in vitro KeratinoSens skin sensitization assay, we detected that ITIH5 decreases the sensitizing potential of moderate and strong contact sensitizers. CONCLUSION: Taken together, our experiments revealed that ITIH5 forms complexes with HA, thereby on the one hand stabilizing HA and facilitating the formation of ECM structures and on the other hand modulating inflammatory responses.

Bifonazole Exerts Anti-Inflammatory Effects in Human Three-Dimensional Skin Equivalents after UVB or Histamine Challenge.

BACKGROUND: In addition to its role as a broad-spectrum imidazole antifungal drug, data from animal models as well as human clinical trials also demonstrated an anti-inflammatory efficacy of bifonazole (BFZ). In the histamine wheal test and after UV radiation, BFZ showed antiphlogistic effects that were comparable to those of hydrocortisone. However, the underlying molecular mechanisms of the anti-inflam-matory properties of BFZ are poorly understood. METHODS: Performing an in vitro study we used full-thickness three-dimensional (3D) skin models containing macrophages as mediators of inflammation. We conducted two sets of experiments. In a first set we exposed our models to UVB irradiation to provoke an inflammation. A second approach used the addition of histamine into the culture medium. In both approaches, models were treated topically with a BFZ-containing ointment or a placebo ointment for 24 h, and then the effects were examined histologically as well as with microarray and quantitative real-time PCR analyses. RESULTS: Histological examination showed that the BFZ-containing ointment reconstituted UVB- and histamine-mediated disorders within the skin models. Performing gene expression profiling in models that were treated with the BFZ-containing ointment after UVB irradiation, we detected an upregu-lation of differentiation markers (fillagrin, loricrin, and keratin 1), antimicrobial peptides (DEFB103A), and members of the cytochrome P450 family (CYP1A1 and CYP1B1) as well as a downregulation of genes that are involved in immune response (CCL22, CXCL12, CCL7, IRF1, ICAM1, TLR3, and RARRES3) and matrix metalloproteinases (MMP12 and MMP7). Models that were treated with the BFZ-containing ointment after histamine application showed an upregulation of members of the cytochrome P450 family (CAP1A1, CYP1B1, and CYP24A1) and a downregulation of immune response-associated genes (CXCL6, CXCL12, CCL8, IL6, and IL32). CONCLUSION: We present the first in vitro study showing anti-inflammatory effects of BFZ in human 3D skin models. To our knowledge, this is the first time that these effects could be translated from human clinical trials into an in vitro test system, allowing a more detailed examination of molecular mechanisms that were regulated by BFZ.

Novel Human Full-Thickness Three-Dimensional Nonkeratinized Mucous Membrane Model for Pharmacological Studies in Wound Healing.

INTRODUCTION: Efforts are increasingly aiming to develop in vitro models that can provide effective alternatives to in vivo experiments. The main aim of this study was the establishment of an in vitro model of the nonkeratinized mucous membrane that can be used as a standardized tool to evaluate biological and therapeutic effects of pharmaceuticals for mucosal wound healing. METHODS: We established a full-thickness in vitro model of the nonkeratinized mucous membrane. While histological examination was performed to assess morphological characteristics, we utilized gene expression profiling using microarray and qRT-PCR analyses to identify molecular effects of treatment with a dexpanthenol-containing ointment after laser wounding. RESULTS: Performing histological and immunofluorescence analyses we proved that our model mimics the two distinctive layers of the mucous membrane - the stratified squamous epithelium and the lamina propria. We used this model to investigate molecular effects of a dexpanthenol-containing ointment that is commonly used for the wound treatment of mucous membranes. For that purpose, our model exhibits a unique feature in that dexpanthenol and proliferation-enhancing additives that may interfere with our studies are not required for the maintenance of the model culture. After setting standardized lesions with a nonsequential fractional ultrapulsed CO2 laser, topical treatment with the dexpanthenol-containing ointment enhanced wound closure in the model compared to placebo and untreated controls. Furthermore, microarray analysis revealed that the treatment of the laser-wounded model with the dexpanthenol-containing ointment evoked an upregulated expression of various genes related to accelerated wound healing. CONCLUSION: Overall, we verified that this novel mucous membrane model can be utilized in future to monitor ex vivo effects of various topical therapies on mucosa morphology, physiology, and gene expression. Our findings confirm the potential of the model as an in vitro tool for the replacement of pharmacological in vivo studies regarding mucosal wound healing.

Accelerated wound healing with a dexpanthenol-containing ointment after fractional ablative CO2 laser resurfacing of photo-damaged skin in a randomized prospective clinical trial.

Background: Laser therapy with an ablative CO2 laser is a prominent treatment option for photo-damaged skin. The healing process and therefore the success of a laser can be supported by an appropriate postoperative treatment of the laser-treated skin. Objective: The effect of a dexpanthenol-containing ointment with petroleum jelly on wound healing after fractional ablative CO2 laser therapy of photo-damaged skin. Methods: A total of 38 patients with photo-damaged skin received fractional ablative CO2 laser treatment. Occlusive wound care was conducted for a period of 7 days. The complete wound area was divided into two sections: one that was treated with a dexpanthenol-containing ointment and a section that was treated with petroleum jelly. This study had three primary outcome measures: (1) Overview images as well as dermatoscopic images of the laser treated skin were taken immediately after laser treatment and on days 1, 2, 5 and 14 (study visits). Dermatoscopic images were analysed to determine changes of the diameter of the individual lesions between the study visits. (2) Wound-healing rate was visually assessed, based on the measure of re-epithelialization. (3) Cosmetic results were evaluated during study visits by patients and physicians applying a visual analogue scale (VAS). Results: Measuring the diameter of laser-generated lesions revealed a significantly faster cure of the lesions in wound sections that were treated with the dexpanthenol-containing ointment on days 1 and 2, in comparison to the lesions that were treated with petroleum jelly. Concordantly, visual evaluation of the wounds revealed significantly better cosmetic results and re-epithelialization on days 1, 2 and 5 after laser treatment in wound sites that were treated with the dexpanthenol-containing ointment. All patients exhibited a completed wound healing on day 14 after laser treatment. Conclusion: In this comparative study, post-operative treatment of laser-treated skin with a dexpanthenol-containing ointment led to a significantly faster wound closure in comparison to petroleum jelly, especially during the early phase of wound healing. Moreover, assessment of the cosmetic result exhibited beneficial effects of the dexpanthenol-containing ointment in the post-operative wound care after laser treatment. These results emphasize that the use of a dexpanthenol-containing ointment in the post-operative phase following CO2 laser therapy could be a promising alternative to the routinely used treatment with petroleum jelly.

Comprehensive molecular characterization of microneedling therapy in a human three-dimensional skin model.

BACKGROUND AND OBJECTIVES: Microneedling therapy is a widely used technique in dermatology. However, little is known about the underlying molecular effects of this therapy on extracellular matrix remodeling, wound healing, and inflammation. The aim of this study was to examine morphological and molecular changes caused by microneedling treatment in a standardized in vitro full-thickness 3D model of human skin. MATERIALS AND METHODS: A microneedling device was used to treat full-thickness 3D skin models. Specimens were harvested at specified time points and qRT-PCR and microarray studies were performed. Frozen sections were examined histologically. RESULTS: Microneedling treatment caused morphological changes in the skin model resulting in an almost complete recovery of the epidermis five days after treatment. Microarray analysis identified an upregulation of genes that are associated with tissue remodeling and wound healing (e.g. COL3A1, COL8A1, TIMP3), epithelial proliferation and differentiation (KRT13, IGF1), immune cell recruitment (CCL11), and a member of the heat shock protein family (HSPB6). On the other hand, we detected a downregulation of pro-inflammatory cytokines (e.g. IL1α, IL1ß, IL24, IL36γ, IL36RN), and antimicrobial peptides (e.g. S100A7A, DEFB4). These data were confirmed by independent RT-PCR analyses. CONCLUSION: We present for the first time the direct molecular effects of microneedling therapy on epidermal keratinocytes and dermal fibroblasts using a standardized 3D skin model. Treatment resulted in histological alterations and changed the expression of various genes related to epidermal differentiation, inflammation, and dermal remodeling. This data suggests that skin microneedling plays a role in dermal remodeling, increases epidermal differentiation, and might also have a direct effect on collagen synthesis. These findings may increase our understanding of the molecular mechanisms of human skin repair induced by microneedling therapy and will allow comparisons with competing applications, such as ablative laser therapies.

Expression of CYP1A1, CYP1B1 and IL-1ß in PBMCs and skin samples of PCB exposed individuals.

Polychlorinated biphenyls (PCBs) are well- known man-made persistent environmental pollutants and endocrine disruptors. As a result of mass production in the past, background levels of these compounds can be measured in human blood worldwide. In 2010 high internal levels of PCBs were discovered in workers of a transformer-recycling company in Germany. Our aim was to measure, whether the expression of CYP1A1, CYP1B1, and IL-1ß is dysregulated in peripheral blood mononuclear cells (PBMCs) of the exposed individuals (n = max 308). Further, we measured the regulation of CYP1A1, CYP1B1, AHRR (aromatic hydrocarbon receptor repressor) and IL-1ß in skin samples of 25 workers with elevated plasma PCB levels using quantitative PCR (q-RT-PCR). We found a significant correlation between the regulation of IL-1ß in skin samples and lipid adjusted PCB levels. In the PBMCs, the expression levels of CYP1A1, CYP1B1 and IL-1ß decreased over time with decreasing PCB plasma levels. The upregulation of the cytokine IL-1ß in exposed individuals with higher PCB plasma levels warrants further investigation in order to examine its role in the pathophysiology of autoimmune disorders and tumor promotion.

Effects of a ceramide-containing water-in-oil ointment on skin barrier function and allergen penetration in an IL-31 treated 3D model of the disrupted skin barrier.

Atopic dermatitis (AD) is a chronically relapsing, pruritic inflammation of the skin with dryness and disturbed skin barrier function. Recently, we established that IL-31 treatment of human 3D skin models resulted in a disrupted skin barrier phenotype resembling AD. In this model, we found that IL-31 interferes with the differentiation of keratinocytes and inhibits the expression of terminal differentiation markers. In the present study, we investigated the effects of a ceramide-containing water-in-oil skin care ointment on the physical skin barrier structure and function in disrupted skin barrier models, generated either by using primary normal human epidermal keratinocytes (NHEK) or HaCaT cells. We observed that the physical skin barrier of the models recovered after daily topical treatment with the ceramide-containing ointment. Topical application of the ointment prevented downregulation of filaggrin and disorganization of other differentiation markers, such as keratin 10 and ß4-integrin, as demonstrated by immunohistological analysis. The expression of Ki67 was also upregulated in response to the ointment. Furthermore, functional studies revealed that local application of the ointment diminished the increased uptake of fluorescently labelled recombinant allergens of timothy grass (phl p1) in our model. In conclusion, our data revealed that topical application of a ceramide-containing skin care ointment reduced IL-31 induced impairments of the physical skin barrier and skin barrier function in an in vitro model of the disrupted skin barrier. This standardized model can be utilized in the future to monitor ex vivo effects of various topical therapies on skin morphology, physiology, and gene expression.

Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

The response of the skin to harmful environmental agents is shaped decisively by the status of the immune system. Keratinocytes constitutively express and secrete the chemokine-like mediator, macrophage migration inhibitory factor (MIF), more strongly than dermal fibroblasts, thereby creating a MIF gradient in skin. By using global and epidermis-restricted Mif-knockout (Mif-/- and K14-Cre+/tg; Miffl/fl) mice, we found that MIF both recruits and maintains antigen-presenting cells in the dermis/epidermis. The reduced presence of antigen-presenting cells in the absence of MIF was associated with accelerated and increased formation of nonmelanoma skin tumors during chemical carcinogenesis. Our results demonstrate that MIF is essential for maintaining innate immunity in skin. Loss of keratinocyte-derived MIF leads to a loss of control of epithelial skin tumor formation in chemical skin carcinogenesis, which highlights an unexpected tumor-suppressive activity of MIF in murine skin.-Brocks, T., Fedorchenko, O., Schliermann, N., Stein, A., Moll, U. M., Seegobin, S., Dewor, M., Hallek, M., Marquardt, Y., Fietkau, K., Heise, R., Huth, S., Pfister, H., Bernhagen, J., Bucala, R., Baron, J. M., Fingerle-Rowson, G. Macrophage migration inhibitory factor protects from nonmelanoma epidermal tumors by regulating the number of antigen-presenting cells in skin.

Synthesis of a Sulfonimidamide-Based Analog of Tasisulam and Its Biological Evaluation in the Melanoma Cell Lines SKMel23 and A375.

Tasisulam is a promising antitumor agent with complex pharmacology, which is used as an antiproliferative agent in patients with metastatic melanoma and other solid tumors. Phase 2 melanoma studies showed promising results but had to be stopped because of insufficient tasisulam clearance leading to toxic side effects. To reduce the negative effects of tasisulam, we synthesized a novel sulfonimidamide-based analog to evaluate its antiproliferative effects in comparison to the original compound by performing a cell proliferation assay in melanoma cell lines SKMel23 and A375. The results revealed that the analog had inhibitory effects on the proliferation comparable to tasisulam in both investigated cell lines. These results could contribute to a reduced toxicity of tasisulam and lead to further clinical trials in metastatic melanoma.

Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma.

INTRODUCTION: Interferon alpha (IFNα) is routinely used in the clinical practice for adjuvant systemic melanoma therapy. Understanding the molecular mechanism of IFNα effects and prediction of response in the IFNα therapy regime allows initiation and continuation of IFNα treatment for responder and exclusion of non-responder to avoid therapy inefficacy and side-effects. The transporter protein associated with antigen processing-1 (TAP1) is part of the MHC class I peptide-loading complex, and important for antigen presentation in tumor and antigen presenting cells. In the context of personalized medicine, we address this potential biomarker TAP1 as a target of IFNα signalling. RESULTS: We could show that IFNα upregulates TAP1 expression in peripheral blood mononuclear cells (PBMCs) of patients with malignant melanoma receiving adjuvant high-dose immunotherapy. IFNα also induced expression of TAP1 in mouse blood and tumor tissue and suppressed the formation of melanoma metastasis in an in vivo B16 tumor model. Besides its expression, TAP binding affinity and transport activity is induced by IFNα in human monocytic THP1 cells. Furthermore, our data revealed that IFNα clearly activates phosphorylation of STAT1 and STAT3 in THP1 and A375 melanoma cells. Inhibition of Janus kinases abrogates the IFNα-induced TAP1 expression. These results suggest that the JAK/STAT pathway is a crucial mediator for TAP1 expression elicited by IFNα treatment. CONCLUSION: We suppose that silencing of TAP1 expression provides tumor cells with a mechanism to escape cytotoxic T-lymphocyte recognition. The observed benefit of IFNα treatment could be mediated by the shown dual effect of TAP1 upregulation in antigen presenting cells on the one hand, and of TAP1 upregulation in 'silent' metastatic melanoma cells on the other hand. In conclusion, this work contributes to a better understanding of the mode of action of IFNα which is essential to identify markers to predict, assess and monitor therapeutic response of IFNα treatment in the future.

Effects of non-ablative fractional erbium glass laser treatment on gene regulation in human three-dimensional skin models.

Clinical experiences with non-ablative fractional erbium glass laser therapy have demonstrated promising results for dermal remodelling and for the indications of striae, surgical scars and acne scars. So far, molecular effects on human skin following treatment with these laser systems have not been elucidated. Our aim was to investigate laser-induced effects on skin morphology and to analyse molecular effects on gene regulation. Therefore, human three-dimensional (3D) organotypic skin models were irradiated with non-ablative fractional erbium glass laser systems enabling qRT-PCR, microarray and histological studies at same and different time points. A decreased mRNA expression of matrix metalloproteinases (MMPs) 3 and 9 was observed 3 days after treatment. MMP3 also remained downregulated on protein level, whereas the expression of other MMPs like MMP9 was recovered or even upregulated 5 days after irradiation. Inflammatory gene regulatory responses measured by the expression of chemokine (C-X-C motif) ligands (CXCL1, 2, 5, 6) and interleukin expression (IL8) were predominantly reduced. Epidermal differentiation markers such as loricrin, filaggrin-1 and filaggrin-2 were upregulated by both tested laser optics, indicating a potential epidermal involvement. These effects were also shown on protein level in the immunofluorescence analysis. This novel standardised laser-treated human 3D skin model proves useful for monitoring time-dependent ex vivo effects of various laser systems on gene expression and human skin morphology. Our study reveals erbium glass laser-induced regulations of MMP and interleukin expression. We speculate that these alterations on gene expression level could play a role for dermal remodelling, anti-inflammatory effects and increased epidermal differentiation. Our finding may have implications for further understanding of the molecular mechanism of erbium glass laser-induced effects on human skin.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is overexpressed in inflammatory skin diseases and affects epidermal morphology in constitutive knockout mice and murine 3D skin models.

Inter-α-trypsin inhibitors are protease inhibitors that are thought to be important regulators in various acute-phase processes. They are composed of one light chain (bikunin) and different heavy chains (ITIHs). The only function known so far of ITIHs is the covalent linkage to hyaluronan (HA). As there is virtually no knowledge on the distribution and function of ITIH proteins in skin tissue, we performed a systematic characterization of ITIH expression in healthy and diseased skin. Using GeneChip(®) Human Exon 1.0 ST expression profiling, we found that ITIH5 represents the major ITIH family member expressed in human skin. Moreover, the use of quantitative reverse transcription PCR and a customized ITIH5-specific antibody indicated that ITIH5 is predominantly produced by dermal fibroblasts. Immunohistochemical analysis revealed a clearly detectable ITIH5 protein expression in normal skin. Interestingly, ITIH5 expression was significantly up-regulated in inflammatory skin diseases. Furthermore, 3D skin models employing murine Itih5(-/-) epidermal keratinocytes and dermal fibroblasts as well as skin specimens of Itih5(-/-) mice revealed a significantly altered epidermal structure compared to wild-type controls. Hence, we can strengthen the presumption that ITIH5 may constitute a novel regulatory molecule of the human skin that could play an important role in inflammation via its interaction with HA.

Characterization of a novel standardized human three-dimensional skin wound healing model using non-sequential fractional ultrapulsed CO2 laser treatments.

BACKGROUND AND OBJECTIVE: At present, there is no standardized in vitro human skin model for wound healing. Therefore, our aim was to establish and characterize an in vitro/ex vivo three-dimensional (3D) wound healing model, which we employed to analyze the effects of dexpanthenol on wound healing and gene regulation. MATERIALS AND METHODS: The novel human 3D skin wound healing model using scaffold and collagen 3D organotypic skin equivalents was irradiated with a non-sequential fractional ultrapulsed CO2 laser. These standardized injured full-thickness skin equivalents enable qRT-PCR, microarray, and histological studies analyzing the effect of topically or systemically applied compounds on skin wound healing. RESULTS: These human laser-irradiated skin models were found to be appropriate for in vitro wound healing analysis. Topical treatment of skin wounds with a 5% dexpanthenol water-in-oil emulsion or two different 5% dexpanthenol oil-in-water emulsions clearly enhanced wound closure compared to laser-irradiated untreated control models. To find out whether this positive effect is caused by the active substance dexpanthenol, laser-irradiated skin models were cultured in calciumpantothenate containing medium (20 µg/ml) compared to skin equivalents cultured without calciumpantothenate. 3D models cultured in calciumpantothenate revealed considerably faster wound closure compared to the control models. Quantitative RT-PCR studies showed enhanced mRNA expression of MMP3, IL1α, keratin-associated protein 4-12 (KRTAP4-12), and decreased expression of S100A7 in laser-irradiated skin models cultured in medium containing calciumpantothenate. CONCLUSION: This novel standardized human 3D skin wound healing model proves useful for topical pharmacological studies on wound healing and reveals new insights into molecular mechanisms of dexpanthenol-mediated effects on wound healing. In addition, these novel 3D model systems can be used to monitor ex vivo effects of various laser systems on gene expression and morphology of human skin.

Knockdown of lecithin retinol acyltransferase increases all-trans retinoic acid levels and restores retinoid sensitivity in malignant melanoma cells.

Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.

Downregulation of STRA6 expression in epidermal keratinocytes leads to hyperproliferation-associated differentiation in both in vitro and in vivo skin models.

Retinoids are known to affect skin cell proliferation and differentiation and are key molecules that target retinoid and retinoic acid receptors (RXRs and RARs), leading to physiological and pharmacologic effects. Our aim was to elucidate the role of the retinol-binding protein receptor STRA6, mediating cellular uptake of retinol, on skin structure and function. Our results indicate that STRA6 is constitutively expressed in human epidermal keratinocytes and dermal fibroblasts and is regulated via RAR/RXR-mediated pathways. HaCaT (Human adult low Calcium high Temperature) cells with stable STRA6 knockdown (STRA6KD) showed increased proliferation. Consistently, human organotypic 3D skin models using stable STRA6KD HaCaT cells showed a significantly thicker epidermis and enhanced expression of activation, differentiation, and proliferation markers. The effects were reversible after treatment with free retinol. Human skin reconstitution employing STRA6KD HaCaT cells leads to massive epithelial thickening under in vivo conditions in SCID mice. We propose that STRA6KD could lead to cellular vitamin A deficiency in keratinocytes. Consequently, STRA6 has a role for regulating retinoid homeostasis and in helping to program signaling that drives proliferation and differentiation of human skin cells. By its influence on hyperproliferation-associated differentiation, STRA6 could also have a role in skin regeneration and could be a target for pharmacological approaches to improve wound healing.

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.

Active transport of contact allergens in human monocyte-derived dendritic cells is mediated by multidrug resistance related proteins.

The multidrug resistance related proteins (MRPs) function as efflux transporters of a variety of large organic anions or their conjugates. In recent studies we demonstrated that antigen-presenting cells express a specific pattern of MRPs. MRP-mediated efflux activity of human monocyte-derived dendritic cells (moDCs) was analyzed using an in vitro transport assay. The efflux transport of radiolabeled contact allergens was inhibited using the specific MRP inhibitor indomethacin. Treatment with indomethacin increased intracellular concentration of [³H] eugenol and [³H] isoeugenol in moDCs. In addition by using MRP1 expressing inside-out membrane vesicles we revealed that the transport of eugenol is mediated by MRP1. Human DCs were employed to assess the sensitizing potential of contact allergens and alters their cytokine gene expression profile. Hence, to survey the functionality of indomethacin after stimulation with contact allergens IL-8 and TRIM16 regulation was measured by a DC-based in vitro assay. Incubation with isoeugenol after pre-treatment with indomethacin leads to increased IL-8 and TRIM16 gene expression. These results strongly support the functional role of MRPs in the active efflux of contact allergens also in antigen-presenting cells like moDCs, a novel mechanism which could possibly play a role in the pathogenesis of contact allergy.