Dániel Törőcsik

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes.

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in keratinocytes in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal keratinocytes (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. 20-40mJ/cm(2) dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis factor alpha (TNFalpha) and vascular endothelial growth factor (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in keratinocytes after UVB treatment.

Functional ABCG1 expression induces apoptosis in macrophages and other cell types.

The expression of the ATP-binding cassette transporter ABCG1 is greatly increased in macrophages by cholesterol loading via the activation of the nuclear receptor LXR. Several recent studies demonstrated that ABCG1 expression is associated with increased cholesterol efflux from macrophages to high-density lipoprotein, suggesting an atheroprotective role for this protein. Our present study uncovers an as yet not described cellular function of ABCG1. Here we demonstrate that elevated expression of human ABCG1 is associated with apoptotic cell death in macrophages and also in other cell types. We found that overexpression of the wild type protein results in phosphatidyl serine (PS) translocation, caspase 3 activation, and subsequent cell death, whereas neither the inactive mutant variant of ABCG1 (ABCG1K124M) nor the ABCG2 multidrug transporter had such effect. Induction of ABCG1 expression by LXR activation in Thp1 cells and in human monocyte-derived macrophages was accompanied by a significant increase in the number of apoptotic cells. Thyroxin and benzamil, previously identified inhibitors of ABCG1 function, selectively prevented ABCG1-promoted apoptosis in transfected cells as well as in LXR-induced macrophages. Collectively, our results suggest a causative relationship between ABCG1 function and apoptotic cell death, and may offer new insights into the role of ABCG1 in atherogenesis.

Sebocytes differentially express and secrete adipokines.

In addition to producing sebum, sebocytes link lipid metabolism with inflammation at a cellular level and hence, greatly resemble adipocytes. However, so far no analysis was performed to identify and characterize the adipocyte‐associated inflammatory proteins, the members of the adipokine family in sebocytes. Therefore, we determined the expression profile of adipokines [adiponectin, interleukin (IL) 6, resistin, leptin, serpin E1, visfatin, apelin, chemerin, retinol‐binding protein 4 (RBP4) and monocyte chemoattractant protein 1 (MCP1)] in sebaceous glands of healthy and various disease‐affected (acne, rosacea, melanoma and psoriasis) skin samples. Sebaceous glands in all examined samples expressed adiponectin, IL6, resistin, leptin, serpin E1 and visfatin, but not apelin, chemerin, RBP4 and MCP1. Confirming the presence of the detected adipokines in the human SZ95 sebaceous gland cell line we further characterized their expression and secretion patterns under different stimuli mimicking bacterial invasion [by using Toll‐like receptor (TLR)2 and 4 activators], or by 13‐cis retinoic acid (13CRA; also known as isotretinoin), a key anti‐acne agent. With the exception of resistin, the expression of all of the detected adipokines (adiponectin, IL6, leptin, serpin E1 and visfatin) could be further regulated at the level of gene expression, showing a close correlation with the secreted protein levels. Besides providing further evidence on similarities between adipocytes and sebocytes, our results strongly suggest that sebocytes are not simply targets of inflammation but may exhibit initiatory and modulatory roles in the inflammatory processes of the skin through the expression and secretion of adipokines.

Beyond acne: Current aspects of sebaceous gland biology and function

The sebaceous gland is most commonly found in association with a hair follicle. Its traditional function is the holocrine production of sebum, a complex mixture of lipids, cell debris, and other rather poorly characterized substances. Due to the gland’s central role in acne pathogenesis, early research had focused on its lipogenic activity. Less studied aspects of the sebaceous gland, such as stem cell biology, the regulation of cellular differentiation by transcription factors, the significance of specific lipid fractions, the endocrine and specially the neuroendocrine role of the sebaceous gland, and its contribution to the innate immunity, the detoxification of the skin, and skin aging have only recently attracted the attention of researchers from different disciplines. Here, we summarize recent multidisciplinary progress in sebaceous gland research and discuss how sebaceous gland research may stimulate the development of novel therapeutic strategies targeting specific molecular pathways of the pathogenesis of skin diseases.

The prevalence of obesity is increased in patients with late compared with early onset psoriasis

PURPOSE We compared the clinical and epidemiologic characteristics of early and late onset psoriasis with an emphasis on potential differences in the comorbidities associated with each subtype. METHODS An observational, multicenter study was performed, and associations between the age at the time of diagnosis and binary comorbidity outcomes were evaluated using multiple logistic regression analysis adjusted for age and other relevant confounders. RESULTS An increased prevalence of positive family history, psoriatic arthritis, and depression was observed in patients with early onset psoriasis. On the other hand, late onset psoriasis was more frequently associated with obesity and elevated waist circumference compared with early onset form. Elderly psoriatic patients (at the age of 75 years) with late onset psoriasis are at an especially high risk for obesity compared with individuals at the same age with an early onset disease. CONCLUSIONS The increased frequency of psoriasis in the family of early onset patients may suggest that manifestation of psoriasis at younger age is driven by strong genetic influence. However, such a remarkable association of abdominal obesity with late onset psoriasis may suggest that obesity can be one of the acquired factors that may predispose for the development of psoriasis in the elderly.

Leptin promotes a proinflammatory lipid profile and induces inflammatory pathways in human SZ95 sebocytes

Leptin, the adipocyte‐secreted hormone that regulates weight, is known to link lipid metabolism with inflammation in various cell types. However, its role in human sebocytes has not yet been investigated.

Sebaceous Gland-Rich Skin Is Characterized by TSLP Expression and Distinct Immune Surveillance Which Is Disturbed in Rosacea

The microbial community exhibits remarkable diversity on topographically distinct skin regions, which may be accompanied by differences in skin immune characteristics. Our aim was to compare the immune milieu of healthy sebaceous gland-rich (SGR) and sebaceous gland-poor skin areas, and to analyze its changes in an inflammatory disease of SGR skin. For this purpose, immunohistochemical, immunocytochemical, and quantitative real-time PCR analyses of thymic stromal lymphopoietin (TSLP) and other cytokines, phenotypic immune cell markers and transcription factors were carried out in samples from sebaceous gland-poor, SGR skin and from papulopustular rosacea. TSLP mRNA and protein production was also studied in cultured keratinocytes. In SGR skin, higher TSLP expression, dendritic cell appearance without prominent activation, and T cell presence with IL-17/IL-10 cytokine milieu were detected compared with sebaceous gland-poor skin. Linoleic acid, a major sebum component, was found to induce TSLP expression dose-dependently in keratinocytes. In papulopustular rosacea, significantly decreased TSLP level and influx of inflammatory dendritic cells and T cells with IL-17/interferon-γ cytokine milieu were observed. According to our results, SGR skin is characterized by a distinct, noninflammatory immune surveillance, which may explain the preferred localization of inflammatory skin diseases, and can influence future barrier repair therapeutic concepts.

Reduced lipoxygenase and cyclooxygenase mediated signaling in PBMC of atopic dermatitis patients

Lipoxygenases (LOX) and cyclooxygenases (COX) are the main enzymes for poly-unsaturated fatty acid (PUFA) metabolism to highly bioactive prostaglandins, leukotrienes, thromboxanes and protectins. LOX and COX pathways are highly important for the regulation of pro- and anti-inflammatory active metabolite synthesis and metabolism in various inflammatory diseases like atopic diseases (AD). In this study using QRT-PCR, we found that in PBMCs the expression of 5-LOX, 12-LOX, 15-LOX and COX pathways and further enzymatic pathways like various leukotriene-hydoxylases, leukotriene-, prostaglandin-, and thromboxane-synthases as well as various of their membrane based receptors are mainly significantly down-regulated in AD-patients vs. healthy volunteers. In addition, using HPLC MS-MS we determined up to 19 different metabolites originating from eicosapentaenoic acid (EPA), docosapentaenoic acid (DHA) and arachidonic acid (AA) ranging from hydroxylated-PUFA derivatives and further bioactive derivatives like thromboxanes, leukotrienes, prostaglandins and protectins originating from LOX and COX metabolism. In PBMCs from AD-patients LOX and COX pathways were down-regulated. We conclude from this study, that in PBMCs from AD-patients in comparison to healthy volunteers, a systemic down-regulation of LOX- and COX-responses occurs to generally reduce eicosanoid/docosanoid synthesis during the current allergic inflammatory status.

Sebum lipids influence macrophage polarization and activation

As lipids are known to regulate macrophage functions, it is reasonable to suppose that a sebocyte–macrophage axis mediated by sebum lipids may exist.

TSLP expression in the skin is mediated via RARγ-RXR pathways

TSLP is an important trigger and initiator for various atopic diseases mainly atopic dermatitis (AD). Activators of nuclear hormone receptors like bioactive vitamin A and D derivatives are known to induce TSLP up-regulation in the skin. In this study, various combinations of synthetic specific agonists and antagonists of the retinoic acid receptors (RARs), retinoid X receptors (RXRs) and vitamin D receptor (VDR) were topically administered to mice. The aim of the study was to elucidate via which nuclear hormone receptor pathways TSLP is regulated and how this regulation is connected to the development and phenotype of atopic dermatitis. TSLP expression was monitored using QRT-PCR and serum TSLP levels using ELISA. Synthetic agonists of the VDR and RARγ as well as the natural agonist all-trans retinoic acid (ATRA) increased TSLP expression in the skin, while an RXR agonist was not active. Treatments with antagonists of RXRs and RARs in addition to RARα-agonists reduced skin TSLP expression. Strong activation was found after a combination of a VDR and an RXR agonist (ca. 5 times induction) and even stronger by an RARγ and an RXR agonist treatment (ca. 48 times induction). We conclude that besides VDR-mediated signaling mainly RARγ-RXR mediated pathways in the skin are important patho-physiological triggers for increased skin TSLP expression. We conclude that topical synthesized retinoids stimulated by internal or external triggers or topically applied induce TSLP production and are thereby important triggers for atopic dermatitis prevalence.