Mads A. Røpke

Diagnostic microRNA profiling in cutaneous T-cell lymphoma (CTCL)

Cutaneous T-cell lymphomas (CTCLs) are the most frequent primary skin lymphomas. Nevertheless, diagnosis of early disease has proven difficult because of a clinical and histologic resemblance to benign inflammatory skin diseases. To address whether microRNA (miRNA) profiling can discriminate CTCL from benign inflammation, we studied miRNA expression levels in 198 patients with CTCL, peripheral T-cell lymphoma (PTL), and benign skin diseases (psoriasis and dermatitis). Using microarrays, we show that the most induced (miR-326, miR-663b, and miR-711) and repressed (miR-203 and miR-205) miRNAs distinguish CTCL from benign skin diseases with > 90% accuracy in a training set of 90 samples and a test set of 58 blinded samples. These miRNAs also distinguish malignant and benign lesions in an independent set of 50 patients with PTL and skin inflammation and in experimental human xenograft mouse models of psoriasis and CTCL. Quantitative (q)RT-PCR analysis of 103 patients with CTCL and benign skin disorders validates differential expression of 4 of the 5 miRNAs and confirms previous reports on miR-155 in CTCL. A qRT-PCR-based classifier consisting of miR-155, miR-203, and miR-205 distinguishes CTCL from benign disorders with high specificity and sensitivity, and with a classification accuracy of 95%, indicating that miRNAs have a high diagnostic potential in CTCL.

MicroRNA-223 and miR-143 are important systemic biomarkers for disease activity in psoriasis.

BACKGROUND Psoriasis is a systemic inflammatory skin disease. MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that recently have been found in the blood to be relevant as disease biomarkers. OBJECTIVE We aimed to explore miRNAs potential as blood biomarkers for psoriasis. METHODS Using microarray and quantitative real-time PCR we measured the global miRNA expression in whole blood, plasma and peripheral blood mononuclear cells (PBMCs) from patients with psoriasis and healthy controls. RESULTS We identified several deregulated miRNAs in the blood from patients with psoriasis including miR-223 and miR-143 which were found to be significantly upregulated in the PBMCs from patients with psoriasis compared with healthy controls (FCH=1.63, P<0.01; FCH=2.18, P<0.01, respectively). In addition, miR-223 and miR-143 significantly correlated with the PASIscore (r=0.46, P<0.05; r=0.55, P<0.02, respectively). Receiver-operating characteristic analysis (ROC) showed that miR-223 and -143 have the potential to distinguish between psoriasis and healthy controls (miR-223: area under the curve (AUC)=0.80, miR-143: AUC=0.75). Interestingly, after 3-5 weeks of treatment with methotrexate following a significant decrease in psoriasis severity, miR-223 and miR-143 were significantly downregulated in the PBMCs from patients with psoriasis. CONCLUSION We suggest that changes in the miR-223 and miR-143 expressions in PBMCs from patients with psoriasis may serve as novel biomarkers for disease activity in psoriasis; however, further investigations are warranted to clarify their specific roles.

Laser capture microdissection followed by next‐generation sequencing identifies disease‐related microRNAs in psoriatic skin that reflect systemic microRNA changes in psoriasis

Psoriasis is a systemic disease with cutaneous manifestations. MicroRNAs (miRNAs) are small non‐coding RNA molecules that are differentially expressed in psoriatic skin; however, only few cell‐ and region‐specific miRNAs have been identified in psoriatic lesions. We used laser capture microdissection (LCM) and next‐generation sequencing (NGS) to study the specific miRNA expression profiles in the epidermis (Epi) and dermal inflammatory infiltrates (RD) of psoriatic skin (N = 6). We identified 24 deregulated miRNAs in the Epi and 37 deregulated miRNAs in the RD of psoriatic plaque compared with normal psoriatic skin (FCH > 2, FDR < 0.05). Interestingly, 9 of the 37 miRNAs in RD, including miR‐193b and miR‐223, were recently described as deregulated in circulating peripheral blood mononuclear cells (PBMCs) from patients with psoriasis. Using flow cytometry and qRT‐PCR, we found that miR‐193b and miR‐223 were expressed in Th17 cells. In conclusion, we demonstrate that LCM combined with NGS provides a robust approach to explore the global miRNA expression in the epidermal and dermal compartments of psoriatic skin. Furthermore, our results indicate that the altered local miRNA changes seen in the RD are reflected in the circulating immune cells, suggesting that miRNAs may contribute to the pathogenesis of psoriasis.

Validation of a diagnostic microRNA classifier in cutaneous T-cell lymphomas

Troels Marstrand 1 * , Charlotte B. Ahler 1,2 * , Ulrik Ralfkiaer 3,4 , Anders Clemmensen 5 , Katharina L. Kopp 3 , Nina A. Sibbesen 3 , Thorbj ø rn Krejsgaard 3 , Thomas Litman 1 , Mariusz A. Wasik 6 , Charlotte M. Bonefeld 3 , Kirsten Gr ø nb æ k 4 , Lise Mette Rahbek Gjerdum 7 , Robert Gniadecki 8 , Elisabeth Ralfkiaer 7 , Carsten Geisler 3 , Anders Woetmann 3 , Mads A. R ø pke 1 , Christian Glue 2 , Lone Skov 5 & Niels Odum 2

Comparison of microRNA expression using different preservation methods of matched psoriatic skin samples

Abstract:  MicroRNAs are non‐coding RNA molecules modulating gene expression post‐transcriptionally. Formalin‐fixed, paraffin‐embedding (FFPE) is a standard preservation method often used in clinical practices, but induces RNA degradation. Extracting high‐quality RNA from human skin can be challenging as skin contains high levels of RNases. As microRNAs are 19‐23 nucleotides long and lack a poly‐A tail, they may be less prone to RNA degradation than mRNAs. We investigated whether microRNAs in psoriatic (FFPE) samples reliably reflect microRNA expression in samples less prone to RNA degradation such as fresh‐frozen (FS) and Tissue‐Tek‐embedding (OCT). We found a strong correlation of the microRNA expression levels between all preservation methods of matched psoriatic skin samples (rs ranging from 0.91 to 0.95 (P < 0.001)). These observations were further confirmed with qRT‐PCR. Our results demonstrate that microRNA detection in human skin is robust irrespective of preservation method; thus, microRNAs offer an appropriate and flexible approach in clinical practices and for diagnostic purposes in skin disorders.

Comparison of the effects of vitamin D products in a psoriasis plaque test and a murine psoriasis xenograft model

The aim of the present study was to compare the effects of Daivobet® and calcipotriol on clinical score and biomarker responses in a modified version of the Scholtz-Dumas psoriasis plaque assay. Furthermore, it was the aim to compare the effects of calcipotriol and betamethasone in the murine psoriasis xenograft model. Twenty four patients with psoriasis were treated topically once daily for three weeks, whereas the grafted mice were treated for four weeks. Clinical responses were scored twice weekly and biopsies were taken at the end of each study to analyse for skin biomarkers by histology and immunohistochemistry. The results clearly demonstrate effects on both clinical signs and biomarkers. In the patient study the total clinical score was reduced significantly with both Daivobet® and calcipotriol. Both treatments reduced epidermal thickness, Ki-67 and cytokeratin 16 expression. T cell infiltration was significantly reduced by Daivobet® but only marginally by calcipotriol. Both treatments showed strong effects on the epidermal psoriatic phenotype.Results from the xenograft model essentially showed the same results. However differences were observed when investigating subtypes of T cells.The study demonstrates the feasibility of obtaining robust biomarker data in the psoriasis plaque test that correlate well with those obtained in other clinical studies. Furthermore, the biomarker data from the plaque test correlate with biopsy data from the grafted mice.

Calcipotriol and betamethasone dipropionate exert additive inhibitory effects on the cytokine expression of inflammatory dendritic cell–Th17 cell axis in psoriasis

BACKGROUND Psoriasis vulgaris is characterised by epidermal hyper-proliferation and infiltration of immune cells including dendritic cells (DCs) and T cells. The inflammation is driven by a complex interplay between immune and skin cells involving interleukin (IL)-17A, IL-23 and TNF-α as key drivers. The calcipotriol/betamethasone dipropionate two-compound fixed combination product is widely used for topical treatment of psoriasis. However, the mechanism behind its high efficacy has not been elucidated in detail. OBJECTIVE Here, we investigated and compared the immune modulatory effects of betamethasone, calcipotriol and the combination in ex vivo cultures of psoriatic skin and in vitro cultures of primary human cells that recapitulate key cellular activities of psoriatic inflammation. METHOD The immune modulatory effect of the treatments on psoriatic skin and on in vitro differentiated Th1/Th17 cells, Tc1/Tc17 cells, monocyte-derived inflammatory dendritic cells and primary keratinocytes was assessed by a panel of inflammatory and phenotypic related transcription factors and cytokines. The expression was evaluated by both gene and protein analysis. RESULTS Compared to vehicle control or mono-treatments, the effect of calcipotriol/betamethasone combination was significantly better in inhibiting the secretion of IL-17A and TNF-α in psoriatic skin. Additionally, the two components showed additive inhibitory effects on secretion of IL-23 and TNF-α by DCs, of IL-17A and TNF-α by both CD4(+) and CD8(+) T cells and reduced inflammatory responses in Th17-stimulated keratinocytes. Furthermore, calcipotriol was found to enhance IL-10 secretion in psoriatic skin and in human T cells, to induce secretion of type 2 cytokines by T cells and, lastly, to significantly modulate the differentiation of DCs and T cells. CONCLUSIONS In summary, we demonstrate a unique and supplementary immune modulatory effect of calcipotriol/betamethasone combination on TNF-α and IL-23/Th17 immune axis, supporting the superior clinical efficacy of the combination product compared to the respective mono-treatments in psoriasis patients.

Calcipotriol counteracts betamethasone-induced decrease in extracellular matrix components related to skin atrophy

The calcipotriol/betamethasone dipropionate fixed-combination gel is widely used for topical treatment of psoriasis vulgaris. It has been hypothesized that calcipotriol counteracts glucocorticoid-induced skin atrophy which is associated with changes in the extracellular matrix (ECM). To elucidate the combined effects of calcipotriol and betamethasone on key ECM components, a comparative study to the respective mono-treatments was carried out. The effect on collagen I synthesis, matrix metalloproteinase (MMP) secretion, and hyaluronic acid (HA) production was investigated in primary human fibroblast and keratinocyte cultures as well as in a human skin explant model. We show that calcipotriol counteracts betamethasone-induced suppression of collagen I synthesis. Similarly, calcipotriol and betamethasone have opposing effects on MMP expression in both fibroblasts and keratinocytes. Moreover, calcipotriol is able to restore betamethasone-impaired HA synthesis in keratinocytes and prevent betamethasone-induced epidermal thinning in minipigs upon treatment with the calcipotriol/betamethasone gel. In summary, our results show for the first time in primary human skin cultures that calcipotriol reduces early signs of betamethasone-induced skin atrophy by modulation of key ECM components. These results indicate that the calcipotriol component of the fixed-combination gel counteracts the atrophogenic effects of betamethasone on the skin.

Psoriasis drug discovery: methods for evaluation of potential drug candidates

Introduction: Psoriasis is a complex disease with several clinical subtypes, as well as variations in body location and severity. Many patients suffering from psoriasis now benefit from the increased understanding of the pathogenesis of the disease, which in turn drives translational efforts to test new therapeutic concepts in the clinic. However, a multitude of treatment options is currently needed to satisfy patient needs. Areas covered: This review describes the drug discovery platform in relation to psoriasis with special emphasis on how the major disease mechanisms of psoriasis can be studied in experimental in vitro and in vivo settings. The value of using humanized models and experimental clinical studies is highlighted. Expert opinion: The successful development of novel therapies requires a translational approach to develop and implement the best preclinical and experimental clinical models and analytical tools that capture the various biological aspects of the disease. There is a need for more advanced in vitro skin models that contain the relevant cellular constituents as well as a need for careful validation of relevant in vivo models for psoriasis.

The gene expression and immunohistochemical time-course of diphenylcyclopropenone induced contact allergy in healthy humans following repeated epicutaneous challenges

The gene expression time‐course of repeated challenge of contact allergy (CA) remains largely unknown. Therefore, using diphenylcyclopropenone (DPCP) as model allergen in healthy humans we set out to examine: (i) the monotonous and complex gene expression time‐course trajectories following repeated DPCP challenges to find the predominant gene expression pattern, (ii) the time‐course of cell infiltration following repeated DPCP challenges and (iii) the transcriptome of a repeated CA exposure model. We obtained punch biopsies from control and DPCP‐exposed skin from ten DPCP sensitized individuals at 5‐6 monthly elicitation challenges. Biopsies were used for microarray gene expression profiling, histopathology and immunohistochemical staining. Validation of microarray data by qRT‐PCR was performed on 15 selected genes. Early gene expression time points were also validated in an independent data set. An increasing and decreasing trend in gene expression followed by a plateau was predominantly observed during repeated DPCP challenges. Immune responses reached a plateau after two challenges histopathologically, immunohistochemically and in the time‐course gene expression analysis. Transcriptional responses over time revealed a Th1/Th17 polarization as three upstream regulators (IFN‐γ, IL‐1 and IL‐17) activated most of the top upregulated genes. Of the latter genes, 9 of 10 were the same throughout the time course. Excellent correlations between array and PCR data were observed. The transcriptional responses to DPCP over time followed a monotonous pattern. This response pattern confirms and supports the newly reported clinical time‐course observations in de novo‐sensitized individuals showing a plateau response, and thus, there is concordance between clinical response, histopathology, immunohistochemistry and microarray gene expression in volunteers de novo‐sensitized to DPCP.