Thomas Welss

Expression of hurpin, a serine proteinase inhibitor, in normal and pathological skin: overexpression and redistribution in psoriasis and cutaneous carcinomas

Abstract:  Hurpin was identified by differential display analysis studying UV‐repressible genes in the keratinocyte cell line HaCaT. We have previously reported that hurpin mRNA is overexpressed in psoriatic skin compared to non‐lesional or normal skin; hurpin inhibits cathepsin L and that, after overexpression in keratinocytes, hurpin decreases UV‐induced apoptosis. To further study the expression of hurpin, we have isolated monoclonal antibodies against hurpin and analyzed its expression in normal and diseased skin by immunohistochemistry (IHC). In the epidermis of normal skin, we found hurpin to be mainly expressed in the stratum basale. In contrast, we found an enhanced expression of hurpin in the stratum spinosum and stratum granulosum in the majority of diseased skin samples. Within the dermis of normal and diseased skin, hurpin was detected in sebaceous and sweat glands, hair follicles, and endothelial cells of blood vessels. Hurpin was localized in the cytoplasm in normal and diseased skin. Additionally to IHC, we analyzed hurpin expression in selected skin diseases by semiquantitative reverse‐transcription polymerase chain reaction. We found overexpression of hurpin mRNA in psoriasis, squamous cell carcinoma (SCC), and actinic keratosis. In contrast, expression of hurpin in melanoma and basal cell carcinoma was comparable to that in normal skin. Overall, the strongest overexpression was observed in SCC and psoriasis. Individual differences in hurpin expression between patients were observed. The increased expression and redistribution of hurpin in diseased skin suggests its possible involvement in inflammatory processes or the regulation of endogenous or pathogen‐derived proteinase activity. Additional studies will elucidate the physiological role of hurpin.

A novel organotypic 3D sweat gland model with physiological functionality

Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.