Skin and systemic inflammation in adults with atopic dermatitis before and after whole-body topical betamethasone 17-valerate 0.1% or tacrolimus 0.1% treatment: A randomized controlled study.

BACKGROUND: Atopic dermatitis (AD) is mainly driven by type 2 inflammation and often treated with topical agents. Studies comparing differences in biomarkers between these treatments are lacking. OBJECTIVES: The aim of this study was to evaluate the effects of topical betamethasone 17-valerate 0.1% and tacrolimus 0.1% ointment on skin barrier function and inflammatory biomarkers in skin and blood in adults with AD. METHODS: In this randomized parallel-group double-blind double-dummy active-comparator study design, 36 adults with AD were treated with either whole-body topical corticosteroid (betamethasone ointment 0.1% plus placebo once daily, n = 18) or calcineurin inhibitor (tacrolimus ointment 0.1% twice daily, n = 18). At baseline, after 2 weeks of daily treatment and after further 4 weeks of twice-weekly maintenance treatment, we evaluated AD severity, levels of natural moisturizing factor (NMF) and cytokines in the skin and blood and characterized circulating T cells. RESULTS: Mean AD severity at baseline corresponded to moderate disease and decreased significantly in both groups. Levels of NMF increased significantly in the tacrolimus group after 2 weeks of treatment (p = 0.002) and tended to increase more than betamethasone at week 6 (p = 0.06). Most skin cytokines decreased with both treatments. However, IL-8, IL-18, IL-22, IP-10, MDC, MMP-9 and TARC were significantly more decreased with betamethasone than tacrolimus after 2 weeks, while after 6 weeks this was only the case for IL-8 and MMP-9. Approximate half of the systemic cytokines decreased significantly with both treatments, but betamethasone decreased MDC significantly more after 2 weeks of treatment. T-cell characterization analyses indicated slight differences in the expression and activation of T cells between groups. CONCLUSION: Topical treatment of AD with betamethasone and tacrolimus ointment effectively reduced disease severity, cutaneous and systemic inflammatory markers. Betamethasone was more effective in decreasing inflammation, but tacrolimus improved skin hydration (NMF levels) more than betamethasone.

Effects of topical corticosteroid versus tacrolimus on insulin sensitivity and bone homeostasis in adults with atopic dermatitis-A randomized controlled study.

INTRODUCTION: Topical corticosteroids (TCS), used to treat atopic dermatitis (AD), have been associated with type 2 diabetes and osteoporosis in epidemiological studies, possibly explained by systemic absorption. OBJECTIVES: We examined whether intensive daily whole-body TCS treatment over 2 weeks followed by twice weekly application for 4 weeks could elicit insulin resistance and increase bone resorption in adults with AD. METHODS: A randomized parallel-group double-blind double-dummy non-corticosteroid-based active comparator study design was completed in Copenhagen, Denmark. Thirty-six non-obese, non-diabetic adults with moderate-to-severe AD were randomized to whole-body treatment with betamethasone 17-valerate 0.1% plus a vehicle once daily or tacrolimus 0.1% twice daily after washout. Insulin sensitivity assessed by the hyperinsulinemic-euglycemic clamp combined with tracer infusions and biomarkers of bone formation (P1NP) and resorption (CTX) were evaluated at baseline, after 2 weeks of daily treatment and after further 4 weeks of twice-weekly maintenance treatment. RESULTS: AD severity improved with both treatments and systemic inflammation was reduced. After 2 weeks, we observed similar increase in peripheral insulin sensitivity with use of betamethasone (n = 18) and tacrolimus (n = 18). Bone resorption biomarker, CTX, was unchanged, while bone formation marker, P1NP, decreased after betamethasone treatment after both 2 and 6 weeks but remained unchanged in the tacrolimus arm. CONCLUSIONS: Whole-body treatment with TCS leads to systemic exposure but appears not to compromise glucose metabolism during short-term use, which may be a result of reduced systemic inflammatory activity. The negative impact on bone formation could be regarded an adverse effect of TCS.

Tralokinumab does not impact vaccine-induced immune responses: Results from a 30-week, randomized, placebo-controlled trial in adults with moderate-to-severe atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease. Interleukin (IL) 13 is a type 2 cytokine that is key to the inflammation underlying AD. Tralokinumab is a first-in-class, fully human, monoclonal antibody that specifically binds with high affinity to IL-13, neutralizing it in AD. Immunomodulatory treatments may impair vaccine-induced immune responses. OBJECTIVE: Assess the immune responses to standard vaccines in adults with moderate-to-severe AD who are undergoing treatment with tralokinumab. METHODS: ECZema TRAlokinumab Trial No. 5 (ECZTRA 5; NCT03562377) was a phase 2, double-blind, randomized, placebo-controlled trial taking place over 30 weeks. Eligible adults were randomized 1:1, with 107 patients receiving tralokinumab 300 mg and 108 patients receiving a placebo every 2 weeks for 16 weeks. All patients received Tdap (tetanus/diphtheria/pertussis) and meningococcal vaccines at week 12. The primary end points were positive antitetanus and antimeningococcal responses between weeks 12 and 16 (noninferiority margin, -25%; responder, >3-fold increase in IgG). RESULTS: The noninferiority of tralokinumab versus placebo for immune response to Tdap (91.9% vs 96.1%) and meningococcal (86.0% vs 84.2%) vaccines was demonstrated at week 16. During treatment, the rates of adverse events were lower for tralokinumab than for the placebo, with most events being mild or moderate. LIMITATIONS: Responses to other vaccines (including influenza) were not examined. CONCLUSIONS: Treatment with tralokinumab 300 mg every 2 weeks did not affect immune responses to Tdap and meningococcal vaccines. Treatment was well tolerated when administered concomitantly with the vaccines and demonstrated a safety profile comparable to phase 3 trials.

Multispectral imaging of hand eczema.

BACKGROUND: Hand eczema is a disease with large variation in clinical presentation and severity. Scoring systems for quantitative severity assessment exist. However, they are observer-dependent. An objective quantitative tool for scoring of hand eczema would improve categorization of hand eczema. OBJECTIVE: To investigate the usefulness of multispectral imaging in assessing severity of hand eczema with respect to extent and the different morphological features. METHODS: Patients with hand eczema (n = 60) and healthy controls (n = 28) were included. The severity of hand eczema was assessed by a dermatologist using the Hand Eczema Severity Index (HECSI) and a global assessment (Physician Global Assessment [PGA]). Multispectral imaging of the hand was performed on all patients and controls using the VideometerLab Instrument. RESULTS: Areas of the morphological elements identified by multispectral imaging were statistically significantly correlated with the PGA scores. Analyzed by Cohen's kappa, a moderate agreement between imaging-based severity assessment and PGA was found. The imaging-based severity assessment was also correlated with HECSI (Spearman rho 0.683, P < .001). Still, the imaging-based algorithm was not capable of differentiating hand eczema patients from controls. CONCLUSIONS: Multispectral imaging allows quantitative measurements of different skin parameters to be performed. In its present form, multispectral imaging cannot replace the clinical assessment of a dermatologist. However, after refinement, this or similar technologies could prove useful.

Anti-inflammatory potency testing of topical corticosteroids and calcineurin inhibitors in human volunteers sensitized to diphenylcyclopropenone.

AIMS: To quantify the anti-inflammatory potency of topical corticosteroids and topical calcineurin inhibitors by measuring the contact allergic response to a diphenylcyclopropenone (DPCP) challenge in de novo sensitized human volunteers. METHODS: Two randomized, double-blind, vehicle-controlled studies were performed encompassing 76 volunteers: 29 in the first and 47 in the second study. Topical drugs were applied pre- and/or post-treatment in block designs. The compounds were tested simultaneously under occluded patch tests covering DPCP-induced dermatitis. Inhibitory responses were assessed by visual scoring and measurements of the oedema thickness with ultrasound. RESULTS: When applied both before and after the DPCP challenge, significant anti-inflammatory effects were seen in descending order for tacrolimus 0.1% ointment, clobetasol propionate ointment, betamethasone valerate ointment and hydrocortisone butyrate ointment, while pimecrolimus cream, hydrocortisone ointment and vehicles had no significant effect. Only tacrolimus ointment (P < 0.01) demonstrated a consistent significant pre-treatment inhibitory effect compared with an untreated DPCP control. CONCLUSIONS: This human testing method in which the inflammation of experimentally induced allergic patch test reactions is quantified by objective measurement allows an analysis of the anti-inflammatory potency of not only topical corticosteroids, but also of drugs that have no effect on vasoconstriction. The method allowed comparison of the potencies of four topical corticosteroids and two calcineurin inhibitors.

Effects of glucocorticoids on stratum corneum lipids and function in human skin-A detailed lipidomic analysis.

BACKGROUND: Topical glucocorticoids (GCs) are known to induce atrophy of human skin including thinning of epidermal and dermal compartments by influencing keratinocyte proliferation and synthesis of extracellular matrix proteins. GCs are also known to reduce skin barrier integrity but little is known about the changes in lipid composition in human skin following topical administration of GCs. OBJECTIVE: This study investigated the effects of GCs on stratum corneum (SC) function and lipid profile of human skin in vivo. METHOD: Over a period of 4 weeks, 16 healthy volunteers were treated on the forearms once daily with topical clobetasol proprionate (CP), betamethasone diproprionate (BDP) or vehicle. One day after last application (Day 29) SC lipids were collected by tape stripping and analysed by a high sensitivity liquid chromatography-mass spectrometry method. Gene expression was analysed in skin biopsies. The full skin, epidermal and SC thickness were assessed by ultrasound, optical coherence tomography and confocal microscopy, respectively, and barrier integrity was assessed by measuring transepidermal water loss (TEWL). RESULTS: Compared to vehicle controls, GCs induced significant alterations in SC lipid profiles. CP caused a reduction in 98 lipids of 226 analysed while BDP treatment only resulted in a significant change of 29 lipids. Most pronounced changes occurred among long chain, ester-linked, ceramide classes while other ceramide classes were much less affected. Almost the complete profile of triacylglycerols (TGs) was significantly decreased by CP while more modest changes were observed in free fatty acids. Topical GCs reduced the thickness of skin layers and increased TEWL. GC treatment also induced changes in expression of genes coding for extracellular markers and enzymes involved in lipid synthesis. CONCLUSIONS: This study shows a reduction in specific SC lipid classes following topical GC treatment of human skin and contributes to the characterisation of the barrier disruption in human skin induced by topical steroids.

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.

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.

Objective Quantification of Immune Cell Infiltrates and Epidermal Proliferation in Psoriatic Skin: A Comparison of Digital Image Analysis and Manual Counting.

Digital pathology and image analysis have developed extensively during the last couple of years. Especially the advance in whole-slide scanning, software, and computer processing makes it possible to apply these methods in tissue-based research. Today this task is dominated by tedious manual assessments by pathologists with the interobserver and intraobserver variation this includes. Automated quantitative assessment of immunohistochemical staining has the potential to objectively extract numerical measures from cell and tissue structures, and allows efficient high throughput analysis in clinical research. Published data of manual cell counts in psoriatic skin samples were in this study reevaluated using the digital image analysis (DIA) software. Whole slides immunohistochemically stained for CD3, CD4, CD8, CD45R0, and Ki-67 were scanned and quantitatively evaluated using simple threshold analysis. Regression analysis with R values in the range of 0.85 to 0.95 indicates a good correlation between the manual count of cell numbers and the staining density obtained by automated DIA. Moreover, we show that the automated image analysis is reliable over a broad range of thresholds and that it is robust to differences in staining intensities and hence useful for high throughput analysis. DIA is a viable technical approach for automated cell quantification. Its output highly correlates to the conventional manual cell counting and hence allows for increasing the throughput and reducing the analysis time significantly.

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.

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.

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.

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

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 (r(s) 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.

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.

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.