Ola Rollman

Mastocytosis is a KIT‐related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult‐onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 μM. Flow cytometric analysis of purified dermal MCs showed that mefloquine‐induced cell death was mainly due to apoptosis and accompanied by caspase‐3 activation. However, caspase inhibition provided only partial protection against mefloquine‐induced cell death, indicating predominantly caspase‐independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug‐induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule‐localised serine proteases play major roles in the execution of mefloquine‐induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

Tryptase is a tetrameric serine protease located within the secretory granules of mast cells. In the secretory granules, tryptase is stored in complex with negatively charged heparin proteoglycans and it is known that heparin is essential for stabilizing the enzymatic activity of tryptase. However, recent findings suggest that enzymatically active tryptase also can be found in the nucleus of murine mast cells, but it is not known how the enzmatic activity of tryptase is maintained in the nuclear milieu. Here we hypothesized that tryptase, as well as being stabilized by heparin, can be stabilized by DNA, the rationale being that the anionic charge of DNA could potentially substitute for that of heparin to execute this function. Indeed, we showed that double-stranded DNA preserved the enzymatic activity of human β-tryptase with a similar efficiency as heparin. In contrast, single-stranded DNA did not have this capacity. We also demonstrated that DNA fragments down to 400 base pairs have tryptase-stabilizing effects equal to that of intact DNA. Further, we showed that DNA-stabilized tryptase was more efficient in degrading nuclear core histones than heparin-stabilized enzyme. Finally, we demonstrated that tryptase, similar to its nuclear localization in murine mast cells, is found within the nucleus of primary human skin mast cells. Altogether, these finding reveal a hitherto unknown mechanism for the stabilization of mast cell tryptase, and these findings can have an important impact on our understanding of how tryptase regulates nuclear events.

Skin mast cells are implicated as detrimental effector cells in various inflammatory skin diseases such as contact eczema, atopic dermatitis and psoriasis. Selective reduction of cutaneous mast cells, e.g. by inducing targeted apoptosis, might prove a rational and efficient therapeutic strategy in dermatoses negatively influenced by mast cells. The objective of the present study was to evaluate whether a lysosomotropic agent such as siramesine can cause apoptosis of mast cells present in psoriatic lesions. Punch biopsies were obtained from lesional and uninvolved skin in 25 patients with chronic plaque psoriasis. After incubation with siramesine, the number of tryptase-positive mast cells and their expression of interleukin (IL)-6 and IL-17 was analysed. Skin biopsies were digested to allow flow cytometric analysis of the drug's effect on cutaneous fibroblasts and keratinocytes. Siramesine caused a profound reduction in the total number of mast cells in both lesional and uninvolved psoriatic skin biopsies without affecting the gross morphology of the tissue. The drug reduced the density of IL-6- and IL-17-positive mast cells, and showed antiproliferative effects on epidermal keratinocytes but had no apparent cytotoxic effect on keratinocytes or dermal fibroblasts. Considering the pathophysiology of psoriasis, the effects of siramesine on cutaneous mast cells may prove favourable from the therapeutic aspect. The results encourage further studies to assess the usefulness of siramesine and other lysosomotropic agents in the treatment of cutaneous mastocytoses and inflammatory skin diseases aggravated by dermal mast cells.

Summary: Direct microscopy of keratinised specimens is a standard screening procedure that assists clinicians to differentiate true superficial mycoses from non‐fungal disorders of the skin, nail and hair. Most clinical dermatologists use bright‐field microscopy when searching for dermatophyte fungi in clinical samples while laboratory‐based mycologists increasingly favour fluorescence microscopy in order to optimise visualisation of fungal elements. This study compared the validity and speediness of fluorescence microscopy vs. conventional light microscopy when screening for fungi in 206 dermatological samples from dermatology outpatients. Both senior dermatologist and a less experienced investigator (medical student) attained high and comparable levels of specificity (91.7–93.8%), positive predictive value (77.1–81.4%) and negative predictive value (83.7–89.9%) using either method. Fluorostaining with Blankophor prior to fluorescence microscopy increased the sensitivity by 22 ± 1% as compared to light microscopy of unstained samples. For both investigators, the time required to identify fungal elements by the fluorescence‐based technique was reduced by at least 50%, thus improving the performance of direct microscopy in the clinical setting.

The family of human epidermal growth factor receptors (EGFR, HER2–4) exerts key functions in normal and malignant epithelial cells. Both EGFR and HER2 are valuable targets for anti-cancer drugs by interfering with ligand binding, receptor dimerization, or tyrosine kinase activity. A similar therapeutic strategy has been advocated for chronic psoriasis since plaque lesions overexpress EGFR and its ligands. Our aim was to characterize EGFR/HER2 protein expression in skin cultures and to evaluate the effects of tyrosine kinase inhibitors on epidermal outgrowth, morphology, and EGFR activation. Human skin explants were established on cell-free dermis and cultured at the air–liquid interface. The impact of small-molecule HER inhibitors on outgrowth was assayed by fluorescence-based image analysis and histometry. Effects of a dual EGFR/HER2 kinase inhibitor, PKI166, on neoepidermis were studied by immunohistochemistry and Western blot. Receptor immunostaining showed in vivo-like distributions with highest EGFR intensity in the proliferative layers whereas HER2 was mainly expressed by suprabasal keratinocytes. Reepithelialization was associated with EGFR autophosphorylation irrespective of exogenous ligand stimulation. PKI166 inhibited neoepidermal EGFR activation, keratinocyte proliferation, and outgrowth from normal and psoriatic skin explants. The rate of epidermalization in presence of other HER inhibitors varied suggesting that drug specificity, potency, and reversibility determine the dynamic outcome. Overall, agents predominantly targeting EGFR kinase were more efficient inhibitors of epidermal regeneration than an HER2-selective drug. The study illustrates the usefulness of a dynamic skin model and emphasizes the potential of HER-directed approaches to control epidermal growth in hyperproliferative skin disorders.

Reconstituted skin models are valuable tools in studies of cutaneous biology although they are not generally devised to visualize and quantify the time course of reepithelialization. We describe a skin explant culture technique coupled with vital microscopy allowing sequential imaging of epithelial outgrowth while maintaining the tissue in culture. Radial expansion of neo-epidermis was initiated from a 2-mm skin punch biopsy explanted onto acellular human dermis and maintained at the air-liquid interface in serum-containing keratinocyte medium. Microscopic viewings and surface area measurements of newly formed epidermis were performed repeatedly using fluorescein-based cell staining and subsequent image analysis. The labeling and visualization procedures did not interfere with neo-epidermal growth or tissue architecture. In order to appraise the versatility of the model, we studied the effect of epidermal growth factor supplementation on the course of skin resurfacing as related to tissue morphology and proliferative activity over a 10-day cultivation period. Exogenous epidermal growth factor at 10 ng/ml increased the radial outgrowth rate (mean, + 13.3 percent), papillomatosis index (+ 19.2 percent), epithelial thickness (+ 49.8 percent), and fraction of Ki-67 positive basal cells (+ 18.4 percent) in neo-epidermis produced from a biopsy of normal human skin. The contribution of this miniaturized and convenient format for concurrent studies of dynamic and qualitative features of neo-epidermis should be a useful complement to existing assays of epidermalization.

Psoriasisresponds favourably to treatment with retinoids but the cellularpathways mediating these ef...