Pia Houdek

Tight junctions form a barrier in human epidermis.

Tight junctions (TJ) are cell-cell junctions that have proved to form a paracellular barrier for solutes and water between cells of epithelia, including the stratum granulosum of the stratified epithelium of the epidermis of newborn mice. In mice lacking claudin-1, a major barrier-forming TJ component, this barrier was abolished. However, the role of TJ in human skin is controversially discussed as unambiguous data were missing so far. Here, we investigated TJ barrier function in healthy human skin as well as in skin samples from psoriatic lesions which are characterized by an altered localization of TJ proteins. We demonstrate for human skin that occludin- and claudin-1-positive sites in the stratum granulosum form a barrier for extracellular biotin-SH (557Da) and that in psoriatic skin the localization of the barrier and the TJ proteins are altered in parallel.

Evidence for distinct populations of human Merkel cells

Merkel cells (MCs) are neuroendocrine cells of unknown origin located in the skin. They are identified at electron microscopic level by electron dense granules, at light microscopic level by the presence of cytokeratins 8, 18, 19 and 20. Contradictory reports concerning the presence of other molecules of epithelial as well as neural origin prompted us to investigate whether there are distinct populations of human MCs. Here, we show the heterogeneous expression of villin, N-CAM, NGF-R, and neurofilaments in MCs. Synaptophysin is found in all MCs but with different intensity, nestin is absent. Expression patterns vary between interfollicular epidermis, hair follicles and glabrous epidermis. We conclude that there are distinct populations of MCs, but all populations contain markers for epithelial as well as neural cells. Putative functions of the distinct populations are discussed.

Diversity of desmosomal proteins in regenerating epidermis: immunohistochemical study using a human skin organ culture model.

Abstract We recently established a skin organ culture model for epithelial healing by creating a central defect in freshly excised human skin specimens and keeping them in culture for up to 7 days, either untreated or with transplantation of allogenic or autologous keratinocytes. In this study the molecular diversity of cell-cell junction proteins in the regenerating epidermis was analysed immunohistochemically using a broad spectrum of monoclonal antibodies against glycoproteins (cadherins) and plaque proteins of desmosomes. At all stages studied the entire set of desmosomal cadherins [desmogleins (Dsg) 1–3 and desmocollins (Dsc) 1–3] was detected, with Dsg3, Dsc2 and Dsc3 being the most prominent. In the disordered neoepithelium at day 3 (after transplantation) some desmosomal cadherins appeared in their respective stratum compartments. In regenerating epidermis on day 7, which exhibited a more ordered stratification and a compact horny layer, stratification-related patterns of desmosomal cadherins were more pronounced. However, some immaturity of the day-7 neoepidermis was reflected by relatively low levels of the maturation-associated Dsg1 and Dsc1 and a strong basal layer expression of Dsg2 which is sparse in normal epidermis. Desmosomal plaque proteins showed expression patterns similar to those in normal healthy epidermis. The adherens junction-related E-cadherin was also detected. Dendritic cells (melanocytes, Langerhans cells) were mainly present at the wound margins. In conclusion, this study demonstrated partial but not complete epidermal maturation and junction development during regeneration up to day 7. This model should also be useful in future studies to evaluate the effects of growth hormones to be used in therapeutic trials on chronic leg ulcers.

Expression Patterns of Tight Junction Proteins in Merkel Cell Carcinoma

Tight junctions (TJ) play a role in the barrier function of the epidermis. While Merkel cells are negative for Claudin 3, 4, 5, occludin and ZO-1, these TJ proteins are found in Merkel cell carcinoma. As TJ are able to connect neighbouring cells tightly and to control the paracellular pathway of substances, the formation of TJ might be a mechanism for a self-isolation of the tumour from its environment, especially from anti-tumorigenic cells and molecules. The synthesis of claudin 5, a marker for endothelial cells, in certain areas of Merkel cell carcinoma — in addition to blood vessels — gives a hint that a “vessel formation” might occur in this tumour.

Telomerase activity of Merkel cell carcinomas and Merkel cell carcinoma-derived cell cultures

Abstract Merkel cell carcinomas are rare malignant tumors of the skin, which are predominantly observed in elderly patients (mean age 65–70 years). It is believed but not yet proven that these tumors are derived from the Merkel cells of the epidermis and hair follicles. The Merkel cells themselves probably originate from an asymmetric cell division of basal keratinocytes and the resulting differentiated Merkel cells have presumably, at least in humans, lost their growth potential. The capability of indefinite cell division in germ line cells and in the great majority of malignant tumors as well as an increased growth potential in certain somatic cells (such as basal cells of renewable tissues) is correlated with cellular telomerase activity, which is absent in differentiated somatic cells. In this study the telomerase activity in cryostat sections of frozen Merkel cell tumor biopsies and in in vitro cultivated Merkel cell carcinoma cells was analyzed. We detected telomerase activity in four tumors and three of four cell cultures. These results show that despite their pronounced neuroendocrine differentiation and their occurrence in patients of advanced age, Merkel cell carcinomas possess telomerase activity similar to that of common carcinoma types.

Biphasic influence of Staphylococcus aureus on human epidermal tight junctions

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short‐term inoculation results in increased TJ barrier function—which could not be seen in HaCaT cells—hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin‐4 at cell membranes. Long‐term incubation resulted in decreased presence of claudin‐1 and claudin‐4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short‐term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs.

Major cell biological parameters of keratinocytes are predetermined by culture medium and donor source.

Major cell biological parameters of keratinocytes are predetermined by culture medium and donor source Michaela Zorn-Kruppa*, Thomas Volksdorf*, Christopher Ueck*, Eva Z€ oller, Konrad Reinshagen, Ina Ridderbusch, Guido Bruning, Pia Houdek, Ingrid Moll and Johanna Maria Brandner Department of Dermatology and Venerology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Department of Paediatric Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; Tabea GmbH und Co. KG, Center for Venous and Dermatosurgery, Hamburg, Germany Correspondence: Johanna M. Brandner, Department of Dermatology and Venerology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. Tel.: +49-40-7410-55819, Fax: +49-40-7410-52655, e-mail: brandner@uke.de *These authors contributed equally to the manuscript.

Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.

The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.

An ex-vivo oral mucosa infection model for the evaluation of the topical activity of antifungal agents

Although Nystatin has been used since 1950s as a non‐absorbable antifungal agent, there is still no reliable in‐vivo data available stating a dose–effect relationship of Nystatin‐suspension in the treatment of oropharyngeal infection with Candida albicans. Here, we studied the efficacy of a commercially available topical Nystatin suspension in a new ex‐vivo model of candidiasis using porcine oral mucosa. After 48 and 96 h of C. albicans infection, 230 IU Nystatin (standard dosage), 100 IU and 20 IU proved to be equally efficacious. Multiple applications of Nystatin were not superior compared with single application. In dosages of 10 and 0.1 IU the activity of Nystatin suspension against C. albicans was no longer confirmed. In an agar diffusion model, the minimal biocidal concentration of Nystatin proved to be 0.25 IU. Our results suggest that the proposed porcine ex‐vivo model is much closer to the in‐vivo situation compared with other established in‐vitro models of the treatment of muco‐cutaneous candidiasis and may provide a substitute for animal models in the investigation of antifungal agents. Additionally, it seems to be a valuable tool for further investigations of the pathogenesis of C. albicans infections.

Comparison of In-Vitro and Ex-Vivo Wound Healing Assays for the Investigation of Diabetic Wound Healing and Demonstration of a Beneficial Effect of a Triterpene Extract

Diabetes mellitus is a frequent cause for chronic, difficult-to-treat wounds. New therapies for diabetic wounds are urgently needed and in-vitro or ex-vivo test systems are essential for the initial identification of new active molecules. The aim of this study is to compare in-vitro and ex-vivo test systems for their usability for early drug screening and to investigate the efficacy of a birch bark triterpene extract (TE) that has been proven ex-vivo and clinically to accelerate non-diabetic wound healing (WH), in a diabetic context. We investigated in-vitro models for diabetic WH, i.e. scratch assays with human keratinocytes from diabetic donors or cultured under hyperglycaemic conditions and a newly developed porcine ex-vivo hyperglycaemic WH model for their potential to mimic delayed diabetic WH and for the influence of TE in these test systems. We show that keratinocytes from diabetic donors often fail to exhibit significantly delayed WH. For cells under hyperglycaemic conditions significant decrease is observed but is influenced by choice of medium and presence of supplements. Also, donor age plays a role. Interestingly, hyperglycaemic effects are mainly hyperosmolaric effects in scratch assays. Ex-vivo models under hyperglycaemic conditions show a clear and substantial decrease of WH, and here both glucose and hyperosmolarity effects are involved. Finally, we provide evidence that TE is also beneficial for ex-vivo hyperglycaemic WH, resulting in significantly increased length of regenerated epidermis to 188±16% and 183±11% (SEM; p<0.05) compared to controls when using two different TE formulations. In conclusion, our results suggest that microenvironmental influences are important in WH test systems and that therefore the more complex hyperglycaemic ex-vivo model is more suitable for early drug screening. Limitations of the in-vitro and ex-vivo models are discussed. Furthermore our data recommend TE as a promising candidate for in-vivo testings in diabetic wounds.