ERAP1 and ERAP2 Gene Variants as Potential Clinical Biomarkers of Anti-IL-17A Response in Psoriasis Vulgaris.

BACKGROUND: Interleukin-17A (IL-17A) is a proinflammatory cytokine, playing an essential role in the development of psoriasis. Although treatment with anti-IL-17A monoclonal antibodies has demonstrated high efficacy in psoriasis patients, not all patients respond equally well, highlighting the need for biomarkers to predict treatment response. Specific single nucleotide polymorphisms (SNPs) in the endoplasmatic reticulum aminopeptidase (ERAP) 1 and 2 genes have been associated with psoriasis and other immune-mediated diseases. OBJECTIVES: We aimed to investigate the association between the ERAP1 and ERAP2 genotypes and response to secukinumab treatment in psoriasis patients. METHODS: A total of 75 patients with plaque psoriasis were included. All patients were genotyped for the ERAP1 rs27524, rs27044, rs30187, rs2287987, and rs26653 SNPs, the ERAP2 rs2248374 SNP, and human leukocyte antigen-C*06:02 (HLA-C*06:02) status. RESULTS: Our results demonstrated that individuals with specific ERAP1 and ERAP2 genotypes had a considerably lower response rate to secukinumab treatment. Patients with the ERAP2 rs2248374 G/G genotype had a more than 6-fold increased risk of treatment failure compared with patients with the rs2248374 A/G or -A/A genotypes. Stratifying for HLA-C*06:02 status, the ERAP2 G/G genotype pointed towards an increased risk of treatment failure among HLA-C*06:02-positive patients, although this was not statistically significant. CONCLUSION: Taken together, this unique study breaks new ground by identifying distinct ERAP1 and ERAP2 gene variants that may serve as potential biomarkers for predicting the treatment response to secukinumab in psoriasis patients. Notable, out data extends existing knowledge by linking specific ERAP1 and ERAP2 gene variants to treatment outcome.

Method for high-plex analysis of immune cells in human skin using the GeoMx system.

Specific T cell populations in the skin have been demonstrated as important disease drivers in several dermatoses. Due to the unique skin architecture, these cells are not grouped together in structures but dispersedly spread out throughout the epidermis. Following tissue disruption and isolation, only about 10% of skin T cells are recovered and any in vitro expansion may alter their bona fide phenotype. The Nanostring GeoMx system was developed to address cellular phenotype and protein expression in a tissue spatial context. To do so, regions of interest (ROI) must exceed a certain area threshold (usually 100 µm in diameter) to generate a sufficient signal-to-noise ratio. Here, we present an approach that allows for the pooling of numerous smaller ROIs within the skin, enabling T cell and melanocyte phenotyping. Skin samples from healthy individuals and vitiligo patients were analysed using the GeoMx system and several immune profiling panels. A sufficient signal-to-noise ratio was achieved by pooling smaller ROIs and analysing them as a single group. While this prevents spatial analysis, this method allows for detailed analysis of cells as a population in the context of their physiological environment, making it possible to investigate in situ phenotype of rare cells in different tissue compartments.

Efficacy and Safety of the Heat Shock Protein 90 Inhibitor RGRN-305 in Hidradenitis Suppurativa: A Parallel-Design Double-Blind Trial.

Importance: Hidradenitis suppurativa is a painful immune-mediated disorder with limited treatment options; hence, a need exists for new treatments. Objective: To evaluate the feasibility of heat shock protein 90 inhibition by RGRN-305 as a novel mechanism of action in treating moderate to severe hidradenitis suppurativa. Design, Setting, and Participants: This was a parallel-design, double-blind, proof-of-concept, placebo-controlled randomized clinical trial conducted between September 22, 2021, and August 29, 2022, at the Department of Dermatology, Aarhus University Hospital in Denmark. The study included a 1- to 30-day screening period, a 16-week treatment period, and a 4-week follow-up period. Eligibility criteria included age 18 years or older and moderate to severe hidradenitis suppurativa with 6 or more inflammatory nodules or abscesses in at least 2 distinct anatomic regions. Of 19 patients screened, 15 patients were enrolled in the study. Intention-to-treat analysis was performed. Interventions: Patients were randomly assigned (2:1) to receive oral RGRN-305, 250-mg tablet, or matching placebo once daily for 16 weeks. Main Outcomes and Measures: The primary efficacy end point was the percentage of patients achieving Hidradenitis Suppurativa Clinical Response 50 (HiSCR-50) at week 16. Secondary efficacy end points included HiSCR-75 or HiSCR-90, Hidradenitis Suppurativa Physician's Global Assessment, Dermatology Life Quality Index scores, and a pain numeric rating scale. Safety was assessed by adverse events, physical examinations, clinical laboratory measurements, and electrocardiograms. Results: A total of 15 patients were enrolled, completed the study, and were included in all analyses (10 [67%] female; median age, 29 [IQR, 23-41] years). The primary end point HiSCR-50 at week 16 was achieved by a higher percentage in the RGRN-305 group (60% [6 of 10]) than in the placebo group (20% [1 of 5]). Improvements were also observed across all secondary end points at week 16, including higher rates of the harder-to-reach HiSCR levels; 50% (5 of 10) achieved HiSCR-75 and 30% (3 of 10) achieved HiSCR-90, whereas none of the placebo-treated patients achieved HiSCR-75 or HiSCR-90. RGRN-305 was well tolerated, with no deaths or serious adverse events, and treatment-emergent adverse events were similarly frequent between the RGRN-305 and placebo groups. Conclusions and Relevance: The findings of this trial suggest that heat shock protein 90 inhibition by RGRN-305 offers a novel mechanism of action in treating hidradenitis suppurativa, warranting further evaluation in larger trials. Trial Registration: ClinicalTrials.gov Identifier: NCT05286567.

Contemporary Advances in Computer-Assisted Bone Histomorphometry and Identification of Bone Cells in Culture.

Static and dynamic bone histomorphometry and identification of bone cells in culture are labor-intensive and highly repetitive tasks. Several computer-assisted methods have been proposed to ease these tasks and to take advantage of the increased computational power available today. The present review aimed to provide an overview of contemporary methods utilizing specialized computer software to perform bone histomorphometry or identification of bone cells in culture. In addition, a brief historical perspective on bone histomorphometry is included. We identified ten publications using five different computer-assisted approaches (1) ImageJ and BoneJ; (2) Histomorph: OsteoidHisto, CalceinHisto, and TrapHisto; (3) Fiji/ImageJ2 and Trainable Weka Segmentation (TWS); (4) Visiopharm and artificial intelligence (AI); and (5) Osteoclast identification using deep learning with Single Shot Detection (SSD) architecture, Darknet and You Only Look Once (YOLO), or watershed algorithm (OC_Finder). The review also highlighted a substantial need for more validation studies that evaluate the accuracy of the new computational methods to the manual and conventional analyses of histological bone specimens and cells in culture using microscopy. However, a substantial evolution has occurred during the last decade to identify and separate bone cells and structures of interest. Most early studies have used simple image segmentation to separate structures of interest, whereas the most recent studies have utilized AI and deep learning. AI has been proposed to substantially decrease the amount of time needed for analyses and enable unbiased assessments. Despite the clear advantages of highly sophisticated computational methods, the limited nature of existing validation studies, particularly those that assess the accuracy of the third-generation methods compared to the second-generation methods, appears to be an important reason that these techniques have failed to gain wide acceptance.

Quantification of Immunohistochemically Stained Cells in Skin Biopsies.

Immunohistochemical quantification of inflammatory cells in skin biopsies is a valuable tool for diagnosing skin diseases and assessing treatment response. The quantification of individual cells in biopsies is time-consuming, tedious, and difficult. In this study, we presented and compared two methods for the quantification of CD8+ T cells in skin biopsies from patients with psoriasis using both commercial software (Adobe Photoshop) and open-source software (Qupath). In addition, we provided a detailed, step-by-step description of both methods. The methods are scalable by replacing the CD8 antibody with other antibodies to target different cells. Moreover, we investigated the correlation between quantifying CD8+ cells normalized to area or epidermal length and cell classifications, compared cell classifications in QuPath with threshold classifications in Photoshop, and analyzed the impact of data normalization to epidermal length or area on inflammatory cell densities in skin biopsies from patients with psoriasis. We found a satisfactory correlation between normalizing data to epidermal length and area for psoriasis skin. However, when non-lesional and lesional skin samples were compared, a significant underestimation of inflammatory cell density was found when data were normalized to area instead of epidermal length. Finally, Bland-Altman plots comparing Qupath and Photoshop to quantify inflammatory cell density demonstrated a good agreement between the two methods.

Climatotherapy at the Dead Sea for psoriasis is a highly effective anti-inflammatory treatment in the short term: An immunohistochemical study.

Climatotherapy is a well-described treatment of psoriasis. Dead Sea climatotherapy (DSC) in Israel consists of intensive sun and Dead Sea bathing and is very effective in improving clinical and patient-reported outcomes. However, the effect of DSC has not been widely studied. We aimed to investigate the effect of DSC on psoriasis skin using quantitative immunohistochemistry techniques and analysis of blood samples. Skin punch biopsies from 18 psoriasis patients from a previous cohort study were used. Biopsies were obtained from non-lesional skin and from a psoriasis target lesion at baseline. A biopsy was acquired from the target lesion after DSC. Among patients who achieved complete visual clearance, a biopsy was also obtained at relapse. Blood samples were obtained at the same time points. We performed haematoxylin and eosin staining and quantitative immunohistochemical analysis of CD3, CD4, CD8, CD11c, CD103, CD163, CD207, forkhead box P3, Ki67 and myeloperoxidase. We performed blood tests of cholesterol, c-reactive protein, glucose, haemoglobin A1c and triglycerides. All skin biomarkers except for CD207 were decreased after DSC. At relapse, none of the biomarkers were significantly different from the baseline lesional measurements. Total CD207 staining correlated with psoriasis area and severity index at baseline while CD163 staining correlated with psoriasis area and severity index at EOT. No changes were observed in selected blood tests during the study. Consistent with clinical results, DSC is highly effective in the short term almost normalising all investigated biomarkers. However, at relapse, biomarkers were upregulated to the baseline level.

Hypobaric hypoxia deteriorates bone mass and strength in mice.

Mountaineers at high altitude are at increased risk of acute mountain sickness as well as high altitude pulmonary and cerebral edema. A densitometric study in mountaineers has suggested that expeditions at high altitude decrease bone mineral density. Surprisingly, the in vivo skeletal effects of hypobaric hypoxia are largely unknown, and have not been studied using advanced contemporary methods to assess bone microstructure. Eighty-four 22-week-old female mice were divided into seven groups with 12 mice in each group: 1. Baseline; 2. Normobaric, 4 weeks; 3. Hypobaric hypoxia, 4 weeks; 4. Normobaric, 8 weeks; 5. Hypobaric hypoxia, 8 weeks; 6. Normobaric, 12 weeks; and 7. Hypobaric hypoxia, 12 weeks. Hypobaric hypoxia mice were housed in hypobaric chambers at an ambient pressure of 500 mbar (5500 m altitude), while normobaric mice were housed at sea level atmospheric pressure for 4, 8, or 12 weeks, respectively. Hypobaric hypoxia had a profound impact on femoral cortical bone and L4 trabecular bone, while the effect on femoral trabecular bone was less pronounced. Hypobaric hypoxia reduced the bone strength of the femoral mid-diaphysis and L4 at all time-points. At femoral cortical bone, hypobaric hypoxia reduced bone formation through fewer mineralizing surfaces and lower bone formation rate after 2 weeks. In addition, bone strength decreased, and C-terminal telopeptide of type I collagen (CTX-I) increased independently of the duration of exposure to simulated high altitude. At L4, hypobaric hypoxia resulted in a substantial reduction in bone volume fraction, trabecular thickness, and trabecular number after 4 weeks of exposure. Hypobaric hypoxia reduced bone strength and femoral bone mass, while femoral trabecular bone was much less affected, indicating the skeletal response to hypobaric hypoxia differ between cortical and trabecular bone. These findings provide initial preclinical support for future clinical studies in mountaineers to assess bone status and bone strength after exposure to prolonged high altitude exposure.

Artificial intelligence-assisted identification and quantification of osteoclasts.

Quantification of osteoclasts to assess bone resorption is a time-consuming and tedious process. Since the inception of bone histomorphometry and manual counting of osteoclasts using bright-field microscopy, several approaches have been proposed to accelerate the counting process using both free and commercially available software. However, most of the present alternatives depend on manual or semi-automatic color segmentation and do not take advantage of artificial intelligence (AI). The present study directly compare estimates of osteoclast-covered surfaces (Oc.S/BS) obtained by the conventional manual method using a bright-field microscope to that obtained by a new AI-assisted method. We present a detailed step-by-step guide for the AI-based method. Tibiae from Wistar rats were either enzymatically stained for TRAP or immunostained for cathepsin K to identify osteoclasts. We found that estimation of Oc.S/BS by the new AI-assisted method was considerably less time-consuming, while still providing similar results to the conventional manual method. In addition, the retrainable AI-module used in the present study allows for fully automated overnight batch processing of multiple annotated sections.•Bone histomorphometry•AI-assisted osteoclast identification•TRAP and cathepsin K.

Tissue-Resident Memory T Cells in Skin Diseases: A Systematic Review.

In health, the non-recirculating nature and long-term persistence of tissue-resident memory T cells (TRMs) in tissues protects against invading pathogens. In disease, pathogenic TRMs contribute to the recurring traits of many skin diseases. We aimed to conduct a systematic literature review on the current understanding of the role of TRMs in skin diseases and identify gaps as well as future research paths. EMBASE, PubMed, SCOPUS, Web of Science, Clinicaltrials.gov and WHO Trials Registry were searched systematically for relevant studies from their inception to October 2020. Included studies were reviewed independently by two authors. This study was conducted in accordance with the PRISMA-S guidelines. This protocol was registered with the PROSPERO database (ref: CRD42020206416). We identified 96 studies meeting the inclusion criteria. TRMs have mostly been investigated in murine skin and in relation to infectious skin diseases. Pathogenic TRMs have been characterized in various skin diseases including psoriasis, vitiligo and cutaneous T-cell lymphoma. Studies are needed to discover biomarkers that may delineate TRMs poised for pathogenic activity in skin diseases and establish to which extent TRMs are contingent on the local skin microenvironment. Additionally, future studies may investigate the effects of current treatments on the persistence of pathogenic TRMs in human skin.

Effects of bendroflumethiazide on bone mineral density; results from the BONATHIAD randomized double-blind placebo-controlled cohort study.

PURPOSE: Thiazide diuretics (TD) may play a role in preventing osteoporosis. The objective was to investigate the effects of bendroflumethiazide in combination with bisphosphonates on bone mineral density, selected blood parameters, blood pressure, pulse, and muscle function. METHODS: Double-blinded, randomized, placebo-controlled interventional study in postmenopausal osteoporotic women over the age of 50 years consisting of four arms: 1) 24 weeks with bendroflumethiazide +24 weeks of washout, 2) 24 weeks with placebo +24 weeks of washout, 3) 48 weeks with bendroflumethiazide, or 4) 48 weeks with placebo. At inclusion, participants were on oral bisphosphonates. Intervention consisted of either bendroflumethiazide or placebo. Dual energy X-ray absorptiometry (DXA), vertebral fracture assessment (VFA), quantitative CT (QCT) and selected blood parameters were acquired at baseline and at 48 weeks and Timed-Up-and-Go, handgrip strength, blood pressure, pulse and balance additionally at 24 weeks. RESULTS: 139 postmenopausal Caucasian women over 50 years were randomized (mean age 64.7 years (SEM 0.6, range 51-79)). 109 (78%) completed the study. No difference in the effect of bendroflumethiazide on DXA, VFA, QCT, biochemistry or muscle function were found between the treatment arms. CONCLUSION: Bendroflumethiazide for 24- or 48 weeks in combination with bisphosphonates does not improve bone mineral density, selected blood parameters or muscle function compared to placebo combined with bisphosphonates. Studies with longer treatment periods and more patients are needed to further characterize the effects of bendroflumethiazide on bone and subpopulations that might benefit from the treatment.

MicroRNA-106b Regulates Expression of the Tumour Suppressors p21 and TXNIP and Promotes Tumour Cell Proliferation in Mycosis Fungoides.

A prognostic 3-miRNA classifier for early-stage mycosis fungoides has been developed recently, with miR-106b providing the strongest prognostic power. The aim of this study was to investigate the molecular function of miR-106b in mycosis fungoides disease progression. The cellular localization of miR-106b in mycosis fungoides skin biopsies was determined by in situ hybridization. The regulatory role of miR-106b was assessed by transient miR-106b inhibitor/mimic transfection of 2 mycosis fungoides derived cell lines, followed by quantitative real-time PCR (RT-qPCR), western blotting and a proliferation assay. MiR-106b was found to be expressed by dermal T-lymphocytes in mycosis fungoides skin lesions, and miR-106b expression increased with advancing mycosis fungoides stage. Transfection of miR-106b in 2 mycosis fungoides derived cell lines showed that miR-106b represses the tumour suppressors cyclin-dependent kinase inhibitor 1 (p21) and thioredoxin-interacting protein (TXNIP) and promotes mycosis fungoides tumour cell proliferation. In conclusion, these results substantiate that miR-106b has both a functional and prognostic role in progression of mycosis fungoides.

Effect of Dead Sea Climatotherapy on Psoriasis; A Prospective Cohort Study.

Background: Dead Sea climatotherapy (DSC) is a treatment option for psoriasis in Denmark. However, the response to DSC has not been particularly well studied. Aim: We sought to determine effectiveness and response duration of DSC on psoriasis-related outcome parameters. Methods: Eighteen patients participated in a 4-week treatment program in Ein Gedi in Israel. Treatment, consisting of sun exposure and bathing, was individualized. Results: DSC was associated with a mean 13.0-point reduction (88%) in Psoriasis Area and Severity Index and a mean reduction of 2.3 (76.7%) on the 5-point Investigator's Global Assessment Scale. Furthermore, patients' quality of life improved measured by the Dermatology Quality of Life Index and EuroQol 5D index values. The mean time from treatment end to reappearance of visible skin symptoms was 93.8 days (SD: 62.5, range: 31-219 days). Conclusions: Our results confirm that DSC has an immediate effect on skin manifestations and improves quality of life, but long-term disease control is not observed.