TSLP is a direct trigger for T cell migration in filaggrin-deficient skin equivalents

Mutations in the gene encoding for filaggrin (FLG) are major predisposing factors for atopic dermatitis (AD). Besides genetic predisposition, immunological dysregulations considerably contribute to its pathophysiology. For example, thymic stromal lymphopoietin (TSLP) is highly expressed in lesional atopic skin and significantly contributes to the pathogenesis of AD by activating dendritic cells that then initiate downstream effects on, for example, T cells. However, little is known about the direct interplay between TSLP, filaggrin-deficient skin and other immune cells such as T lymphocytes. In the present study, FLG knockdown skin equivalents, characterised by intrinsically high TSLP levels, were exposed to activated CD4+ T cells. T cell exposure resulted in an inflammatory phenotype of the skin equivalents. Furthermore, a distinct shift from a Th1/Th17 to a Th2/Th22 profile was observed following exposure of T cells to filaggrin-deficient skin equivalents. Interestingly, TSLP directly stimulated T cell migration exclusively in filaggrin-deficient skin equivalents even in the absence of dendritic cells, indicating a hitherto unknown role of TSLP in the pathogenesis of AD

Small, Cationic Antifungal Proteins from Filamentous Fungi Inhibit Candida albicans Growth in 3D Skin Infection Models

Candida albicans represents one of the most prevalent opportunistic fungal pathogens, causing superficial skin and mucosal infections in humans with certain predisposing health conditions and life-threatening systemic infections in immunosuppressed patients. The emerging drug resistance of this human-pathogenic yeast and the limited number of antifungal drugs for prevention and treatment of infections urgently demands the identification of new antifungal compounds with novel mechanisms of action. The emerging resistance of human-pathogenic fungi to antifungal drugs urges the development of alternative therapeutic strategies. The small, cationic antifungal proteins (AFPs) from filamentous ascomycetes represent promising candidates for next-generation antifungals. These bio-molecules need to be tested for tolerance in the host and efficacy against fungal pathogens before they can be safely applied in humans. Testing of the efficacy and possible adverse effects of new drug candidates in three-dimensional (3D) human-cell based models represents an advantageous alternative to animal experiments. In, this study, as a proof-of-principle, we demonstrate the usefulness of 3D skin infection models for screening new antifungal drug candidates for topical application. We established a cutaneous infection with the opportunistic human-pathogenic yeast Candida albicans in a commercially available 3D full-thickness (FT) skin model to test the curative potential of distinct AFPs from Penicillium chrysogenum (PAFopt, PAFB, and PAFC) and Neosartorya (Aspergillus) fischeri (NFAP2) in vitro. All tested AFPs were comparably well tolerated by the skin models. The infected 3D models exhibited reduced epidermal permeability barriers, allowing C. albicans to colonize the epidermal and dermal layers, and showed increased secretion of the pro-inflammatory cytokine IL-6 and the chemokine IL-8. AFP treatment diminished the fungal burden and penetration depth of C. albicans in the infected models. The epidermal permeability barrier was restored and the secretion of IL-8 was decreased following AFP treatment. In summary, our study proves that the tested AFPs exhibit antifungal potential against cutaneous C. albicans infection in a 3D FT skin model. IMPORTANCE Candida albicans represents one of the most prevalent opportunistic fungal pathogens, causing superficial skin and mucosal infections in humans with certain predisposing health conditions and life-threatening systemic infections in immunosuppressed patients. The emerging drug resistance of this human-pathogenic yeast and the limited number of antifungal drugs for prevention and treatment of infections urgently demands the identification of new antifungal compounds with novel mechanisms of action. Small, cationic antifungal proteins (AFPs) from filamentous fungi represent promising candidates for next-generation antifungals for topical application. These bio-molecules need to be tested for tolerance by the host and efficacy in pathogen clearance prior to being involved in clinical trials. In a proof-of-principle study, we provide evidence for the suitability of 3D human-cell based models as advantageous alternatives to animal experiments. We document the tolerance of specific AFPs and their curative efficacy against cutaneous C. albicans infection in a 3D skin model

Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4+

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH4+. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH4+, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH4+ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH4+ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH4+ via TRPV3 might stimulate skin cornification or intestinal NH4+ transport are discussed

Alkaline poly(ethylene glycol)-based hydrogels for a potential use as bioactive wound dressings

The number of patients with chronic wounds is increasing constantly in today's aging society. However, little work is done so far tackling the associated disadvantageous shift of the wound pH. In our study, we developed two different approaches on pH-modulating wound dressing materials, namely, bioactive interpenetrating polymer network hydrogels based on poly(ethylene glycol) diacrylate/N-vinylimidazole/alginate (named VIx) and poly(ethylene glycol) diacrylate/2-dimethylaminoethyl methacrylate/N-carboxyethylchitosan (named DMAEMA(x)). Both formulations showed a good cytocompatibility and wound healing capacity in vitro. The developed dressing materials significantly increased the cell ingrowth in wounded human skin constructs; by 364% and 313% for the VIx and the DMAEMA(x) hydrogel formulation, respectively. Additionally, VIx hydrogels were found to be suitable scaffolds for superficial cell attachment. Our research on the material properties suggests that ionic interactions and hydrogen bonds are the driving forces for the mechanical and swelling properties of the examined hydrogels. High amounts of positively charged amino groups in DMAEMA(x) hydrogels caused increased liquid uptake (around 190%), whereas VIx hydrogels showed a 10-fold higher maximum compressive stress in comparison to hydrogels without ionizable functional groups. (c) 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3360-3368, 2017

pH-Modulating Poly(ethylene glycol)/Alginate Hydrogel Dressings for the Treatment of Chronic Wounds

The development of chronic wounds has been frequently associated with alkaline pH values. The application of pH-modulating wound dressings can, therefore, be a promising treatment option to promote normal wound healing. This study reports on the development and characterization of acidic hydrogel dressings based on interpenetrating poly(ethylene glycol) diacrylate/acrylic acid/alginate networks. The incorporation of ionizable carboxylic acid groups results in high liquid uptake up to 500%. The combination of two separate polymer networks significantly improves the tensile and compressive stability. In a 2D cell migration assay, the application of hydrogels (0% to 1.5% acrylic acid) results in complete "wound" closure; hydrogels with 0.25% acrylic acid significantly increase the cell migration velocity to 19.8 +/- 1.9 mu m h(-1). The most promising formulation (hydrogels with 0.25% acrylic acid) is tested on 3D human skin constructs, increasing keratinocyte ingrowth into the wound by 164%

Regenerative potential of adipocytes in hypertrophic scars is mediated by myofibroblast reprogramming

Abstract: Abnormal scarring is a major challenge in modern medicine. The central role of myofibroblasts and TGF-β signaling in scarring is widely accepted, but effective treatment options are missing. Autologous fat grafting is a novel approach that has led to significant improvements in the functionality and appearance of scar tissue. While the underlying mechanism is unknown, the potential role of paracrine effects of adipocytes has been discussed. Hence, with the aim of unraveling the regenerative potential of adipocytes, their effects on in vitro differentiated myofibroblasts and on fibroblasts from hypertrophic scars were investigated. Exposure to adipocyte-conditioned medium significantly decreased the expression of the myofibroblast marker α-SMA and ECM components, indicating the occurrence of myofibroblast reprogramming. Further analysis demonstrated that myofibroblast reprogramming was triggered by BMP-4 and activation of PPARγ signaling initiating tissue remodeling. These findings may pave the way for novel therapeutic strategies for the prevention or treatment of hypertrophic scars. Key messages: Adipocytes induce distinct regenerative effects in hypertrophic scar tissue.Adipocytes secrete several proteins which are involved in wound healing and regeneration.Adipocytes secrete BMP-4 which activates myofibroblast reprogramming.Mediators secreted by adipocytes directly and indirectly activate PPARγ which exerts distinct anti-fibrotic effects.These findings may pave the way for novel therapeutic strategies for the prevention or treatment of hypertrophic scars

A naked eye-invisible ratiometric fluorescent microneedle tattoo for real-time monitoring of inflammatory skin conditions

The field of portable healthcare monitoring devices has an urgent need for the development of real-time, noninvasive sensing and detection methods for various physiological analytes. Currently, transdermal sensing techniques are severely limited in scope (i.e., measurement of heart rate or sweat composition), or else tend to be invasive, often needing to be performed in a clinical setting. This study proposes a minimally invasive alternative strategy, consisting of using dissolving polymeric microneedles to deliver naked eye-invisible functional fluorescent ratiometric microneedle tattoos directly to the skin for real-time monitoring and quantification of physiological and pathological parameters. Reactive oxygen species are overexpressed in the skin in association with various pathological conditions. Here, one demonstrates for the first time the microneedle-based delivery to the skin of active fluorescent sensors in the form of an invisible, ratiometric microneedle tattoo capable of sensing reactive oxygen species in a reconstructed human-based skin disease model, as well as an in vivo model of UV-induced dermal inflammation. One also elaborates a universal ratiometric quantification concept coupled with a custom-built, multiwavelength portable fluorescence detection system. Fully realized, this approach presents an opportunity for the minimally invasive monitoring of a broad range of physiological parameters through the skin