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MRGPRX2 was recently uncovered as the "missing link" in clinically relevant mast cell (MC) activation explaining previously puzzling phenomena. It is the receptor for various endogenous ligands and exogenous compounds alike, whose binding evokes rapid degranulation much like allergen‐mediated exocytosis. While the perceivable outcomes are similar, the two activation routes differ regarding mechanism and regulation. We recently reported that acute SCF administration curbs responses evoked by MRGPRX2 in human skin MCs. Maintenance of MCs in culture requires the presence of MC supportive factors and renders the cells functionally and molecularly unequal to ex vivo counterparts. Here, we asked whether expansion in culture impacts the pseudo‐allergic route, and if so, what contribution SCF and IL‐4 play in this scenario. We report that the in vitro micromilieu dampens (but does not erase) pseudo‐allergic responses and that this is accompanied by strongly reduced MRGPRX2 expression. Withdrawal of SCF or IL‐4 individually, but most potently of both collectively, partially reinstates the MRGPRX2 pathway, revealing that SCF and IL‐4 make negative adjustments to the pseudo‐allergic pathway. Under all conditions, the FcεRI‐triggered route showed the inverse pattern of regulation, substantiating that allergic and pseudo‐allergic MC activation can obey opposite rules, hinting at possible competition between them. [ABSTRACT FROM AUTHOR]

Mast cells ( MCs) from human skin have been notoriously resistant to gene manipulation, and a method to knock-down gene expression in in situ differentiated MCs is highly desired. The Dharmacon Accell® transfection system proved successful on several 'difficult-to-transfect' cells. In the present work, we therefore tested this method on skin-derived MCs using different si RNA entities. The si RNA was readily taken up, followed by pronounced, specific reduction of gene and protein expression. Hence, we present the first efficient technique for the manipulation of gene expression in primary skin MCs ex vivo, which combines high transfection rates with retained cell viability. [ABSTRACT FROM AUTHOR]

In order to develop systems to express mammalian proteins in human skin-derived cells, we tested 6 different viral and 1 eukaryoktic promoter (pCMV, pRSV, pSV, pMMTV, pPoly E, pPoly L, pHMT) for their ability to drive the expression to the chloramphenicol acetyltransferase (CAT) enzyme in different human skin-derived cells. DNA was transfected in human keratinocytes derived from normal foreskin and cervix in the HPV-negative cervical cancer line HT3 and in malignant melanoma cell lines (SK-Mel 23, SK-Mel 37) using a liposome-based technique or calcium precipitation. Transfection efficacy was controlled by cotransfection of a β-galactosidase gene construct. The enzymatic activity of the CAT-gene expression was determined of the cell extract prepared from the transfected cell with 14 C-labeled chloramphenicol. The CMV promoter was highly active in all skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, was highly active in all the skin- or mucosal-derived cells. In contrast to the strong CMV-promoter, the RSV-. SV-, and HMT-promoter were less active and varied in dependence of the cell type. The pattern of the promoter activity differed between benign and transformed genital keratinocytes. Only The SV-promoter showed a comparable strong basal activity, which was restricted to the SK-Mel 37 cells, In conclusion, the promoter activity has to be tested for each cell type depending on the aims of the gene expression. [ABSTRACT FROM AUTHOR]

Abstract: While the renin-angiotensin system (RAS) is widely recognized to be involved in atherosclerosis, its potential role in the progression from atherosclerotic lesions to abdominal aortic aneurysm (AAA) is poorly understood. The present study aimed to investigate which components of the RAS may render the atherosclerotic aorta aneurysmatic. The expression of renin, prorenin/renin receptor, angiotensinogen, AT1- and AT2 receptors, cathepsin D, cathepsin G and chymase was examined by immunoblotting and immunohistochemistry in human atherosclerotic, aneurysmatic and healthy aortic tissues obtained from patients undergoing elective repair or at autopsy. AT1- and AT2 receptor mRNA expression was determined using quantitative real-time RT-PCR. All investigated local RAS components were up-regulated in atherosclerotic as compared to healthy tissues. AAA compared to atherosclerosis was characterized by a further increase in the expression of all RAS components except for the AT2 receptor. Cathepsin D was exclusively up-regulated in AAA. Most RAS components co-localized with infiltrating leukocytes or mast cells pointing to their contribution to inflammatory processes. Due to their proteolytic features, some RAS components (cathepsin D and cathepsin G and chymase) may contribute to AAA formation by accessory mechanisms. Taken together, our data suggest that in humans, RAS activation is not just a key-player in the pathogenesis of atherosclerosis, but that a further increasing activation may be involved in the transition from atherosclerosis to AAA. [Copyright &y& Elsevier]

Psoriasis is a common skin disease involving keratinocyte proliferation and altered differentiation, as well as T-cell activation. Here, we show that altered gene transcription in psoriatic skin lesions is highly reproducible between independent data sets. Analysis of gene expression confirmed dysregulation in all expected functional categories, such as IFN signaling and keratinocyte differentiation, and allowed molecular fingerprinting of a previously characterized dendritic cell subset associated with psoriasis tumor necrosis factor alpha (TNF-α)- and inducible nitric oxide synthase (iNOS)-producing CD11bINT DC (Tip-DC). Unexpectedly, a large group of dysregulated transcripts was related to fatty acid signaling and adipocyte differentiation, exhibiting a pattern consistent with the activation of peroxisome proliferator-activated receptorδ (PPARδ). PPARδ itself was strongly induced in psoriasis in vivo. In primary keratinocytes, PPARδ was induced by the transcription factor activator protein 1, in particular by junB, but not by canonical WNT signaling, in contrast to its regulation in colon carcinoma cells. Activation of PPARδ enhanced proliferation of keratinocytes, while this was inhibited by knockdown of PPARδ. Finally, heparin-binding EGF-like growth factor (HB-EGF), known to induce epidermal hyperplasia and itself overexpressed in psoriasis, was identified as a direct target gene of PPARδ. The present data suggest that activation of PPARδ is a major event in psoriasis, contributing to the hyperproliferative phenotype by induction of HB-EGF.Journal of Investigative Dermatology (2008) 128, 110–124; doi:10.1038/sj.jid.5700943; published online 19 July 2007 [ABSTRACT FROM AUTHOR]

Abstract: The gene expression and regulation of nerve growth factor (NGF) in atopic dermatitis (AD) and the human mast cell line (HMC)-1 was investigated at the molecular level. NGF-stimulation of HMC-1 cells resulted in increases in tryptase activity and histamine contents, paralleled by an increase of tryptase and histamine at the transcriptional level. Also, an increased expression of NGF was found in AD lesions, in association with increased systemic NGF plasma levels. Further cutaneous sources for increased NGF levels were keratinocytes and fibroblasts. These findings demonstrate an increased expression of NGF in AD and effects on tryptase and histamine. Mast cells may be major mediators of neurotrophin effects in AD. [Copyright &y& Elsevier]