Lymphocyte surface markers and cytokines are suitable for detection and potency assessment of skin-sensitizing chemicals in an in vitro model of allergic contact dermatitis: the LCSA-ly.

Allergic contact dermatitis is a widespread health disorder and occupational skin disease. Hence, screening for contact-sensitizing chemicals is highly relevant to toxicology, dermatology, and occupational medicine. The use of animal tests for this purpose is constrained by ethical considerations, need for high-throughput screening, and legislation (e.g., for cosmetics in the European Union). T cell activation is the final and most specific key event of the "adverse outcome pathway" for skin sensitization and therefore a promising target for the development of in vitro sensitization assays. We present a novel in vitro sensitization assay with a lymphocyte endpoint as an add-on to the loose-fit coculture-based sensitization assay (LCSA): the LCSA-ly. While the LCSA measures dendritic cell activation, the LCSA-ly offers the option for an additional lymphocyte endpoint which can be measured concurrently. We incorporated lymphocytes in our previously established coculture of primary human keratinocytes and monocyte-derived dendritic cells and tested nine substances: five sensitizers [2,4-dinitrochlorobenzene (DNCB) 1.25-15 µmol/l, p-phenylenediamine (PPD) 15.6-125 µmol/l, 2-mercaptobenzothiazole (MBT) 50-1000 µmol/l, coumarin, and resorcinol (both: 250-1500 µmol/l)] and four non-sensitizers (monochlorobenzene, caprylic acid, glycerol, and salicylic acid (all: 125-1000 µmol/l)]. DNCB and MBT increased a subset of IL-23 receptor+/IFN-γ receptor 1 (CD119)+ lymphocytes. DNCB, PPD, and MBT enhanced a subunit of the IL-4 receptor (CD124) and a memory marker (CD44) on lymphocytes. Remarkably, DNCB, PPD, and MBT raised IL-4 concentrations in coculture supernatants while IFN-γ levels decreased, which might point to Th2 activation in vitro. Coumarin, resorcinol, and non-sensitizers did not alter any of the tested surface markers or cytokines. IL-17 was not affected by any of the substances. Relative strength of sensitizers according to lymphocyte markers was DNCB > PPD > MBT, which corresponds to earlier results from the LCSA without lymphocyte endpoint, the murine local lymph node assay, and human data. This study is the first to prove the suitability of lymphocyte surface markers for sensitization testing and potency assessment.

A novel method to generate monocyte-derived dendritic cells during coculture with HaCaT facilitates detection of weak contact allergens in cosmetics.

The in vitro sensitization assay LCSA (Loose-fit Coculture-based Sensitization Assay) has proved reliable for the detection of contact sensitizers in the past. However, the coculture of human monocyte-derived dendritic cells (DCs) with primary human keratinocytes (KCs) in serum-free medium is relatively complex compared to other sensitization assays which use continuous cell lines. To facilitate high-throughput screening of chemicals, we replaced KCs with the HaCaT cell line under various culture conditions. Coculture of HaCaT with peripheral blood mononuclear cells in serum-supplemented medium leads to generation of CD1a+/CD1c+ DCs after addition of GM-CSF, IL-4, and TGF-ß1 (as opposed to CD1a-/CD1c- DCs which arise in the "classic" LCSA coculture). These cells resemble monocyte-derived DCs generated in monoculture, but, unlike those, they show a marked upregulation CD86 after treatment with contact allergens. All of the nine sensitizers in this study were correctly identified by CD1a+/CD1c+ DCs in coculture with HaCaT. Among the substances were weak contact allergens such as propylparaben (which is false negative in the local lymph node assay in mice) and resorcinol (which was not detected by CD1a-/CD1c- DCs in the "classic" LCSA). The level of CD86 upregulation on CD1a+/CD1c+ DCs was higher for most allergens compared to CD1a-/CD1c- DCs, thus improving the assay's discriminatory power. Three out of four non-sensitizers were also correctly assessed by the coculture assay. A false-positive reaction to caprylic (octanoic) acid confirms earlier results that some fatty acids are able to induce CD86 on DC in vitro. In conclusion, change of the LCSA protocol led to reduction of time and cost while even increasing the assay's sensitivity and discriminatory power.

Application of single molecule fluorescence microscopy to characterize the penetration of a large amphiphilic molecule in the stratum corneum of human skin.

We report here on the application of laser-based single molecule total internal reflection fluorescence microscopy (TIRFM) to study the penetration of molecules through the skin. Penetration of topically applied drug molecules is often observed to be limited by the size of the respective drug. However, the molecular mechanisms which govern the penetration of molecules through the outermost layer of the skin are still largely unknown. As a model compound we have chosen a larger amphiphilic molecule (fluorescent dye ATTO-Oxa12) with a molecular weight >700 Da that was applied to excised human skin. ATTO-Oxa12 penetrated through the stratum corneum (SC) into the viable epidermis as revealed by TIRFM of cryosections. Single particle tracking of ATTO-Oxa12 within SC sheets obtained by tape stripping allowed us to gain information on the localization as well as the lateral diffusion dynamics of these molecules. ATTO-Oxa12 appeared to be highly confined in the SC lipid region between (intercellular space) or close to the envelope of the corneocytes. Three main distinct confinement sizes of 52 ± 6, 118 ± 4, and 205 ± 5 nm were determined. We conclude that for this amphiphilic model compound several pathways through the skin exist.

Serine Protease-Mediated Cutaneous Inflammation: Characterization of an Ex Vivo Skin Model for the Assessment of Dexamethasone-Loaded Core Multishell-Nanocarriers.

Standard experimental set-ups for the assessment of skin penetration are typically performed on skin explants with an intact skin barrier or after a partial mechanical or chemical perturbation of the stratum corneum, but they do not take into account biochemical changes. Among the various pathological alterations in inflamed skin, aberrant serine protease (SP) activity directly affects the biochemical environment in the superficial compartments, which interact with topically applied formulations. It further impacts the skin barrier structure and is a key regulator of inflammatory mediators. Herein, we used short-term cultures of ex vivo human skin treated with trypsin and plasmin as inflammatory stimuli to assess the penetration and biological effects of the anti-inflammatory drug dexamethasone (DXM), encapsulated in core multishell-nanocarriers (CMS-NC), when compared to a standard cream formulation. Despite a high interindividual variability, the combined pretreatment of the skin resulted in an average 2.5-fold increase of the transepidermal water loss and swelling of the epidermis, as assessed by optical coherence tomography, as well as in a moderate increase of a broad spectrum of proinflammatory mediators of clinical relevance. The topical application of DXM-loaded CMS-NC or DXM standard cream revealed an increased penetration into SP-treated skin when compared to untreated control skin with an intact barrier. Both formulations, however, delivered sufficient amounts of DXM to effectively suppress the production of interleukin-6 (IL-6), interleukin-8 (IL-8) and Thymic Stromal Lymphopoietin (TSLP). In conclusion, we suggest that the herein presented ex vivo inflammatory skin model is functional and could improve the selection of promising drug delivery strategies for anti-inflammatory compounds at early stages of development.

Core-multishell nanocarriers enhance drug penetration and reach keratinocytes and antigen-presenting cells in intact human skin.

In reconstructed skin and diffusion cell studies, core-multishell nanocarriers (CMS-NC) showed great potential for drug delivery across the skin barrier. Herein, we investigated penetration, release of dexamethasone (DXM), in excised full-thickness human skin with special focus on hair follicles (HF). Four hours and 16 h after topical application of clinically relevant dosages of 10 µg DXM/cm2 skin encapsulated in CMS-NC (12 nm diameter, 5.8% loading), presence of DXM in the tissue as assessed by fluorescence microscopy of anti-DXM-stained tissue sections as well as ELISA and HPLC-MS/MS in tissue extracts was enhanced compared to standard LAW-creme but lower compared to DXM aqueous/alcoholic solution. Such enhanced penetration compared to conventional cremes offers high potential for topical therapies, as recurrent applications of corticosteroid solutions face limitations with regard to tolerability and fast drainage. The findings encourage more detailed investigations on where and how the nanocarrier and drug dissociate within the skin and what other factors, e.g. thermodynamic activity, influence the penetration of this formulations. Microscopic studies on the spatial distribution within the skin revealed accumulation in HF and furrows accompanied by limited cellular uptake assessed by flow cytometry (up to 9% of total epidermal cells). FLIM clearly visualized the presence of CMS-NC in the viable epidermis and dermis. When exposed in situ a fraction of up to 25% CD1a+ cells were found within the epidermal CMS-NC+ population compared to approximately 3% CD1a+/CMS-NC+ cells after in vitro exposure in short-term cultures of epidermal cell suspensions. The latter reflects the natural percentage of Langerhans cells (LC) in epidermis suspensions and indicated that CMS-NC were not preferentially internalized by one cell type. The increased CMS-NC+ LC proportion after exposure within the tissue is in accordance with the strategic suprabasal LC-localization. More specifically we postulate that the extensive dendrite meshwork, their position around HF orifices and their capacity to modulate tight junctions facilitated a preferential uptake of CMS-NC by LC within the skin. This newly identified aspect of CMS-NC penetration underlines the potential of CMS-NC for dermatotherapy and encourages further investigations of CMS-NC for the delivery of other molecule classes for which intracellular delivery is even more crucial.

Selenoprotein W as biomarker for the efficacy of selenium compounds to act as source for selenoprotein biosynthesis.

Selenium is an essential trace element and, like all elements, present in many different compounds with unequivocal functions. This fact is only sporadically mentioned when recommended intake or supplementation is indicated just as "selenium." In mammals, selenium is an integral part of selenoproteins as selenocysteine. Selenocysteine is formed from serine at the respective tRNA((ser)sec), a reaction that requires selenophosphate formed from selenide and ATP. Thus, only compounds that can be metabolized into selenide can serve as sources for selenoprotein biosynthesis. We therefore tested the ability of selenium compounds such as sodium selenite, methylseleninic acid (MeSeA), Se-methyl selenocysteine, and selenomethionine to increase the activity, protein, or mRNA levels of commonly used biomarkers of the selenium status, glutathione peroxidase-1 (GPx1) and thioredoxin reductase, and of putatively new biomarkers, selenoprotein W1 (SepW1), selenoprotein H, and selenoprotein 15 in three different cell lines. Selenite and MeSeA were most efficient in increasing all markers tested, whereas the other compounds had only marginal effects. Effects were higher in the noncancerous young adult mouse colon cells than in the cancer cell lines HepG2 and HT-29. At the protein level, SepW1 responded as well as GPx1 and at the mRNA level, even better. Thus, the outcome of selenium treatment strongly depends on the chemical form, the cell type, and the biomarker used for testing efficacy.