A genome-wide meta-analysis of palmoplantar pustulosis implicates TH2 responses and cigarette smoking in disease pathogenesis.

BACKGROUND: Palmoplantar pustulosis (PPP) is an inflammatory skin disorder that mostly affects smokers and manifests with painful pustular eruptions on the palms and soles. Although the disease can present with concurrent plaque psoriasis, TNF and IL-17/IL-23 inhibitors show limited efficacy. There is therefore a pressing need to uncover PPP disease drivers and therapeutic targets. OBJECTIVES: We sought to identify genetic determinants of PPP and investigate whether cigarette smoking contributes to disease pathogenesis. METHODS: We performed a genome-wide association meta-analysis of 3 North-European cohorts (n = 1,456 PPP cases and 402,050 controls). We then used the scGWAS program to investigate the cell-type specificity of the association signals. We also undertook genetic correlation analyses to examine the similarities between PPP and other immune-mediated diseases. Finally, we applied Mendelian randomization to analyze the causal relationship between cigarette smoking and PPP. RESULTS: We found that PPP is not associated with the main genetic determinants of plaque psoriasis. Conversely, we identified genome-wide significant associations with the FCGR3A/FCGR3B and CCHCR1 loci. We also observed 13 suggestive (P < 5 × 10-6) susceptibility regions, including the IL4/IL13 interval. Accordingly, we demonstrated a significant genetic correlation between PPP and TH2-mediated diseases such as atopic dermatitis and ulcerative colitis. We also found that genes mapping to PPP-associated intervals were preferentially expressed in dendritic cells and often implicated in T-cell activation pathways. Finally, we undertook a Mendelian randomization analysis, which supported a causal role of cigarette smoking in PPP. CONCLUSIONS: The first genome-wide association study of PPP points to a pathogenic role for deregulated TH2 responses and cigarette smoking.

Loss-of-Function Myeloperoxidase Mutations Are Associated with Increased Neutrophil Counts and Pustular Skin Disease.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031-2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031-2A>C;c.2031-2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031-2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031-2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10-6 and p = 3.6 × 10-5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10-10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

Pustular psoriasis: Molecular pathways and effects of spesolimab in generalized pustular psoriasis.

BACKGROUND: The IL-36 pathway plays a key role in the pathogenesis of generalized pustular psoriasis (GPP). In a proof-of-concept clinical trial, treatment with spesolimab, an anti-IL-36 receptor antibody, resulted in rapid skin and pustular clearance in patients presenting with GPP flares. OBJECTIVE: We sought to compare the molecular profiles of lesional and nonlesional skin from patients with GPP or palmoplantar pustulosis (PPP) with skin from healthy volunteers, and to investigate the molecular changes after spesolimab treatment in the skin and blood of patients with GPP flares. METHODS: Pre- and post-treatment skin and blood samples were collected from patients with GPP who participated in a single-arm, phase I study (n = 7). Skin biopsies from patients with PPP (n = 8) and healthy volunteers (n = 16) were obtained for comparison at baseline. Biomarkers were assessed by RNA-sequencing, histopathology, and immunohistochemistry. RESULTS: In GPP and PPP lesions, 1287 transcripts were commonly upregulated or downregulated. Selected transcripts from the IL-36 signaling pathway were upregulated in untreated GPP and PPP lesions. In patients with GPP, IL-36 pathway-related signatures, TH1/TH17 and innate inflammation signaling, neutrophilic mediators, and keratinocyte-driven inflammation pathways were downregulated by spesolimab as early as week 1. Spesolimab also decreased related serum biomarkers and cell populations in the skin lesions from patients with GPP, including CD3+ T, CD11c+, and IL-36γ+ cells and lipocalin-2-expressing cells. CONCLUSIONS: In patients with GPP, spesolimab showed rapid modulation of commonly dysregulated molecular pathways in GPP and PPP, which may be associated with improved clinical outcomes.

Single-cell analysis implicates TH17-to-TH2 cell plasticity in the pathogenesis of palmoplantar pustulosis.

BACKGROUND: Palmoplantar pustulosis (PPP) is a severe inflammatory skin disorder characterized by eruptions of painful, neutrophil-filled pustules on the palms and soles. Although PPP has a profound effect on quality of life, it remains poorly understood and notoriously difficult to treat. OBJECTIVE: We sought to investigate the immune pathways that underlie the pathogenesis of PPP. METHODS: We applied bulk and single-cell RNA sequencing (RNA-Seq) methods to the analysis of skin biopsy samples and peripheral blood mononuclear cells. We validated our results by flow cytometry and immune fluorescence microscopy RESULTS: Bulk RNA-Seq of patient skin detected an unexpected signature of T-cell activation, with a significant overexpression of several TH2 genes typically upregulated in atopic dermatitis. To further explore these findings, we carried out single-cell RNA-Seq in peripheral blood mononuclear cells of healthy and affected individuals. Memory CD4+ T cells of PPP patients were skewed toward a TH17 phenotype, a phenomenon that was particularly significant among cutaneous lymphocyte-associated antigen-positive skin-homing cells. We also identified a subset of memory CD4+ T cells that expressed both TH17 (KLRB1/CD161) and TH2 (GATA3) markers, with pseudotime analysis suggesting that the population was the result of TH17 to TH2 plasticity. Interestingly, the GATA3+/CD161+ cells were overrepresented among the peripheral blood mononuclear cells of affected individuals, both in the single-cell RNA-Seq data set and in independent flow cytometry experiments. Dual-positive cells were also detected in patient skin by immune fluorescence microscopy. CONCLUSIONS: PPP is associated with complex T-cell activation patterns and may explain why biologic drugs that target individual T helper cell populations have shown limited therapeutic efficacy.

Cigarette Smoke Specifically Affects Small Airway Epithelial Cell Populations and Triggers the Expansion of Inflammatory and Squamous Differentiation Associated Basal Cells.

Smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and causes remodeling of the small airways. However, the exact smoke-induced effects on the different types of small airway epithelial cells (SAECs) are poorly understood. Here, using air-liquid interface (ALI) cultures, single-cell RNA-sequencing reveals previously unrecognized transcriptional heterogeneity within the small airway epithelium and cell type-specific effects upon acute and chronic cigarette smoke exposure. Smoke triggers detoxification and inflammatory responses and aberrantly activates and alters basal cell differentiation. This results in an increase of inflammatory basal-to-secretory cell intermediates and, particularly after chronic smoke exposure, a massive expansion of a rare inflammatory and squamous metaplasia associated KRT6A+ basal cell state and an altered secretory cell landscape. ALI cultures originating from healthy non-smokers and COPD smokers show similar responses to cigarette smoke exposure, although an increased pro-inflammatory profile is conserved in the latter. Taken together, the in vitro models provide high-resolution insights into the smoke-induced remodeling of the small airways resembling the pathological processes in COPD airways. The data may also help to better understand other lung diseases including COVID-19, as the data reflect the smoke-dependent variable induction of SARS-CoV-2 entry factors across SAEC populations.

Inhibiting Interleukin 36 Receptor Signaling Reduces Fibrosis in Mice With Chronic Intestinal Inflammation.

BACKGROUND & AIMS: Intestinal fibrosis is a long-term complication in inflammatory bowel diseases (IBD) that frequently results in functional damage, bowel obstruction, and surgery. Interleukin (IL) 36 is a group of cytokines in the IL1 family with inflammatory effects. We studied the expression of IL36 and its receptor, interleukin 1 receptor like 2 (IL1RL2 or IL36R) in the development of intestinal fibrosis in human tissues and mice. METHODS: We obtained intestinal tissues from 92 patients with Crohn's disease (CD), 48 patients with ulcerative colitis, and 26 patients without inflammatory bowel diseases (control individuals). Tissues were analyzed by histology to detect fibrosis and by immunohistochemistry to determine the distribution of fibroblasts and levels of IL36R ligands. Human and mouse fibroblasts were incubated with IL36 or control medium, and transcriptome-wide RNA sequences were analyzed. Mice were given neutralizing antibodies against IL36R, and we studied intestinal tissues from Il1rl2-/- mice; colitis and fibrosis were induced in mice by repetitive administration of DSS or TNBS. Bone marrow cells were transplanted from Il1rl2-/- to irradiated wild-type mice and intestinal tissues were analyzed. Antibodies against IL36R were applied to mice with established chronic colitis and fibrosis and intestinal tissues were studied. RESULTS: Mucosal and submucosal tissue from patients with CD or ulcerative colitis had higher levels of collagens, including type VI collagen, compared with tissue from control individuals. In tissues from patients with fibrostenotic CD, significantly higher levels of IL36A were noted, which correlated with high numbers of activated fibroblasts that expressed α-smooth muscle actin. IL36R activation of mouse and human fibroblasts resulted in expression of genes that regulate fibrosis and tissue remodeling, as well as expression of collagen type VI. Il1rl2-/- mice and mice given injections of an antibody against IL36R developed less severe colitis and fibrosis after administration of DSS or TNBS, but bone marrow cells from Il1rl2-/- mice did not prevent induction of colitis and fibrosis. Injection of antibodies against IL36R significantly reduced established fibrosis in mice with chronic intestinal inflammation. CONCLUSION: We found higher levels of IL36A in fibrotic intestinal tissues from patients with IBD compared with control individuals. IL36 induced expression of genes that regulate fibrogenesis in fibroblasts. Inhibition or knockout of the IL36R gene in mice reduces chronic colitis and intestinal fibrosis. Agents designed to block IL36R signaling could be developed for prevention and treatment of intestinal fibrosis in patients with IBD.

Global skin gene expression analysis of early diffuse cutaneous systemic sclerosis shows a prominent innate and adaptive inflammatory profile.

OBJECTIVES: Determine global skin transcriptome patterns of early diffuse systemic sclerosis (SSc) and how they differ from later disease. METHODS: Skin biopsy RNA from 48 patients in the Prospective Registry for Early Systemic Sclerosis (PRESS) cohort (mean disease duration 1.3 years) and 33 matched healthy controls was examined by next-generation RNA sequencing. Data were analysed for cell type-specific signatures and compared with similarly obtained data from 55 previously biopsied patients in Genetics versus Environment in Scleroderma Outcomes Study cohort with longer disease duration (mean 7.4 years) and their matched controls. Correlations with histological features and clinical course were also evaluated. RESULTS: SSc patients in PRESS had a high prevalence of M2 (96%) and M1 (94%) macrophage and CD8 T cell (65%), CD4 T cell (60%) and B cell (69%) signatures. Immunohistochemical staining of immune cell markers correlated with the gene expression-based immune cell signatures. The prevalence of immune cell signatures in early diffuse SSc patients was higher than in patients with longer disease duration. In the multivariable model, adaptive immune cell signatures were significantly associated with shorter disease duration, while fibroblast and macrophage cell type signatures were associated with higher modified Rodnan Skin Score (mRSS). Immune cell signatures also correlated with skin thickness progression rate prior to biopsy, but did not predict subsequent mRSS progression. CONCLUSIONS: Skin in early diffuse SSc has prominent innate and adaptive immune cell signatures. As a prominently affected end organ, these signatures reflect the preceding rate of disease progression. These findings could have implications in understanding SSc pathogenesis and clinical trial design.

Psoriatic skin molecular and histopathologic profiles after treatment with risankizumab versus ustekinumab.

BACKGROUND: IL-23 contributes to the activation, maintenance, and proliferation of TH17 cells and plays a major role in psoriasis pathophysiology. IL-23p19 inhibition with risankizumab resulted in superior clinical responses in patients with psoriasis compared with ustekinumab (dual IL-12/IL-23 inhibitor), but comparative molecular effects have not been established. OBJECTIVE: We investigated the similarities and differences in molecular and histopathologic profiles in skin lesions from patients with psoriasis receiving risankizumab versus ustekinumab at an early time point. METHODS: Lesional skin biopsy samples from 81 patients with moderate-to-severe plaque psoriasis participating in 2 different studies (a phase I risankizumab study and a phase II study of risankizumab vs ustekinumab) were analyzed by using histopathology, immunohistochemistry, and RNA sequencing. RESULTS: Risankizumab induced a rapid decrease in levels of proteins and transcriptomic biomarkers associated with the IL-23 pathway, which were maintained through 8 weeks. At week 4, risankizumab decreased histopathologic expression of biomarkers, including K16, Ki67, CD3, lipocalin-2, CD11c, dendritic cell lysosome-associated membrane glycoprotein, ß-defensin 2, and S100A7; global histopathologic scoring revealed that 54% and 69% of patients treated with 90 or 180 mg of risankizumab, respectively, were graded as experiencing "excellent improvement" versus 29% of patients treated with ustekinumab. At week 4, there was a common decrease in expression of 2645 genes expressed in lesional skin between patients receiving risankizumab and ustekinumab and a significant decrease in 2682 genes unique to risankizumab treatment. Risankizumab more strongly downregulated expression of genes associated with keratinocytes, epidermal cells, and monocytes, versus ustekinumab. CONCLUSION: Risankizumab demonstrated more pronounced changes in the molecular and histopathologic profile of psoriatic skin lesions compared with ustekinumab at week 4.

Exosomal miRNAs as Potential Biomarkers to Monitor Phosphodiesterase 5 Inhibitor Induced Anti-Fibrotic Effects on CCl4 Treated Rats.

MicroRNAs (miRNAs) are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and play an important role in the pathogenesis of non-alcoholic fatty liver disease. Here, we investigated the phosphodiesterase 5 (PDE5) inhibitor induced effects on hepatic and plasma exosomal miRNA expression in CCl4-treated rats. In the present study, hepatic miRNA profiling was conducted using the Nanostring nCounter technology and mRNA profiling using RNA sequencing from PDE5 treated rats in the model of CCl4-induced liver fibrosis. To evaluate if the PDE5 inhibitor affected differentially expressed miRNAs in the liver can be detected in plasma exosomes, qRT-PCR specific assays were used. In livers from CCl4-treated rats, the expression of 22 miRNAs was significantly increased (> 1.5-fold, adj. p < 0.05), whereas the expression of 16 miRNAs was significantly decreased (> 1.5-fold, adj. p < 0.05). The majority of the deregulated miRNA species are implicated in fibrotic and inflammatory processes. The PDE5 inhibitor suppressed the induction of pro-fibrotic miRNAs, such as miR-99b miR-100 and miR-199a-5p, and restored levels of anti-fibrotic miR-122 and miR-192 in the liver. In plasma exosomes, we observed elevated levels of miR-99b, miR-100 and miR-142-3p after treatment with the PDE5-inhibitor compared to CCl4/Vehicle-treated. Our study demonstrated for the first time that during the development of hepatic fibrosis in the preclinical model of CCl4-induced liver fibrosis, defined aspects of miRNA regulated liver pathogenesis are influenced by PDE5 treatment. In conclusion, miRNA profiling of plasma exosomes might be used as a biomarker for NASH progression and monitoring of treatment effects.

Effects of BI 655064, an antagonistic anti-CD40 antibody, on clinical and biomarker variables in patients with active rheumatoid arthritis: a randomised, double-blind, placebo-controlled, phase IIa study.

OBJECTIVE: To evaluate the safety, efficacy and therapeutic mechanism of BI 655064, an antagonistic anti-CD40 monoclonal antibody, in patients with rheumatoid arthritis (RA) and an inadequate response to methotrexate (MTX-IR). METHODS: In total, 67 patients were randomised to receive weekly subcutaneous doses of 120 mg BI 655064 (n=44) or placebo (n=23) for 12 weeks. The primary endpoint was the proportion of patients who achieved 20% improvement in American College of Rheumatology criteria (ACR20) at week 12. Safety was assessed in patients who received at least one dose of study drug. RESULTS: At week 12, the primary endpoint was not met, with 68.2% of patients treated with BI 655064 achieving an ACR20 vs 45.5% with placebo (p=0.064); using Bayesian analysis, the posterior probability of seeing a difference greater than 35% was 42.9%. BI 655064 was associated with greater changes in CD40-CD40L pathway-related markers, including reductions in inflammatory and bone resorption markers (interleukin-6, matrix metalloproteinase-3, receptor activator of nuclear factor-κB ligand), concentration of autoantibodies (immunoglobulin [Ig]G rheumatoid factor [RF], IgM RF, IgA RF) and CD95+ activated B-cell subsets. No serious adverse events (AEs) related to BI 655064 treatment or thromboembolic events occurred; reported AEs were mainly of mild intensity. CONCLUSION: Although blockade of the CD40-CD40L pathway with BI 655064 in MTX-IR patients with RA resulted in marked changes in clinical and biological parameters, including reductions in activated B-cells, autoantibody production and inflammatory and bone resorption markers, with a favourable safety profile, clinical efficacy was not demonstrated in this small phase IIa study. TRIAL REGISTRATION NUMBER: NCT01751776.

Monocyte-derived alveolar macrophages are key drivers of smoke-induced lung inflammation and tissue remodeling.

Smoking is a leading risk factor of chronic obstructive pulmonary disease (COPD), that is characterized by chronic lung inflammation, tissue remodeling and emphysema. Although inflammation is critical to COPD pathogenesis, the cellular and molecular basis underlying smoking-induced lung inflammation and pathology remains unclear. Using murine smoke models and single-cell RNA-sequencing, we show that smoking establishes a self-amplifying inflammatory loop characterized by an influx of molecularly heterogeneous neutrophil subsets and excessive recruitment of monocyte-derived alveolar macrophages (MoAM). In contrast to tissue-resident AM, MoAM are absent in homeostasis and characterized by a pro-inflammatory gene signature. Moreover, MoAM represent 46% of AM in emphysematous mice and express markers causally linked to emphysema. We also demonstrate the presence of pro-inflammatory and tissue remodeling associated MoAM orthologs in humans that are significantly increased in emphysematous COPD patients. Inhibition of the IRAK4 kinase depletes a rare inflammatory neutrophil subset, diminishes MoAM recruitment, and alleviates inflammation in the lung of cigarette smoke-exposed mice. This study extends our understanding of the molecular signaling circuits and cellular dynamics in smoking-induced lung inflammation and pathology, highlights the functional consequence of monocyte and neutrophil recruitment, identifies MoAM as key drivers of the inflammatory process, and supports their contribution to pathological tissue remodeling.

Interleukin 36 receptor-inducible matrix metalloproteinase 13 mediates intestinal fibrosis.

Background: Fibrostenotic disease is a common complication in Crohn's disease (CD) patients hallmarked by transmural extracellular matrix (ECM) accumulation in the intestinal wall. The prevention and medical therapy of fibrostenotic CD is an unmet high clinical need. Although targeting IL36R signaling is a promising therapy option, downstream mediators of IL36 during inflammation and fibrosis have been incompletely understood. Candidate molecules include matrix metalloproteinases which mediate ECM turnover and are thereby potential targets for anti-fibrotic treatment. Here, we have focused on understanding the role of MMP13 during intestinal fibrosis. Methods: We performed bulk RNA sequencing of paired colon biopsies taken from non-stenotic and stenotic areas of patients with CD. Corresponding tissue samples from healthy controls and CD patients with stenosis were used for immunofluorescent (IF) staining. MMP13 gene expression was analyzed in cDNA of intestinal biopsies from healthy controls and in subpopulations of patients with CD in the IBDome cohort. In addition, gene regulation on RNA and protein level was studied in colon tissue and primary intestinal fibroblasts from mice upon IL36R activation or blockade. Finally, in vivo studies were performed with MMP13 deficient mice and littermate controls in an experimental model of intestinal fibrosis. Ex vivo tissue analysis included Masson's Trichrome and Sirius Red staining as well as evaluation of immune cells, fibroblasts and collagen VI by IF analysis. Results: Bulk RNA sequencing revealed high upregulation of MMP13 in colon biopsies from stenotic areas, as compared to non-stenotic regions of patients with CD. IF analysis confirmed higher levels of MMP13 in stenotic tissue sections of CD patients and demonstrated αSMA+ and Pdpn+ fibroblasts as a major source. Mechanistic experiments demonstrated that MMP13 expression was regulated by IL36R signaling. Finally, MMP13 deficient mice, as compared to littermate controls, developed less fibrosis in the chronic DSS model and showed reduced numbers of αSMA+ fibroblasts. These findings are consistent with a model suggesting a molecular axis involving IL36R activation in gut resident fibroblasts and MMP13 expression during the pathogenesis of intestinal fibrosis. Conclusion: Targeting IL36R-inducible MMP13 could evolve as a promising approach to interfere with the development and progression of intestinal fibrosis.

Into the unknown: expression profiling without genome sequence information in CHO by next generation sequencing.

The arrival of next-generation sequencing (NGS) technologies has led to novel opportunities for expression profiling and genome analysis by utilizing vast amounts of short read sequence data. Here, we demonstrate that expression profiling in organisms lacking any genome or transcriptome sequence information is feasible by combining Illumina's mRNA-seq technology with a novel bioinformatics pipeline that integrates assembled and annotated Chinese hamster ovary (CHO) sequences with information derived from related organisms. We applied this pipeline to the analysis of CHO cells which were chosen as a model system owing to its relevance in the production of therapeutic proteins. Specifically, we analysed CHO cells undergoing butyrate treatment which is known to affect cell cycle regulation and to increase the specific productivity of recombinant proteins. By this means, we identified sequences for >13,000 CHO genes which added sequence information of approximately 5000 novel genes to the CHO model. More than 6000 transcript sequences are predicted to be complete, as they covered >95% of the corresponding mouse orthologs. Detailed analysis of selected biological functions such as DNA replication and cell cycle control, demonstrated the potential of NGS expression profiling in organisms without extended genome sequence to improve both data quantity and quality.

Alveolar macrophages in early stage COPD show functional deviations with properties of impaired immune activation.

Despite its high prevalence, the cellular and molecular mechanisms of chronic obstructive pulmonary disease (COPD) are far from being understood. Here, we determine disease-related changes in cellular and molecular compositions within the alveolar space and peripheral blood of a cohort of COPD patients and controls. Myeloid cells were the largest cellular compartment in the alveolar space with invading monocytes and proliferating macrophages elevated in COPD. Modeling cell-to-cell communication, signaling pathway usage, and transcription factor binding predicts TGF-ß1 to be a major upstream regulator of transcriptional changes in alveolar macrophages of COPD patients. Functionally, macrophages in COPD showed reduced antigen presentation capacity, accumulation of cholesteryl ester, reduced cellular chemotaxis, and mitochondrial dysfunction, reminiscent of impaired immune activation.

Pilot study of an occupational healthcare program to assess the SARS-CoV-2 infection and immune status of employees in a large pharmaceutical company.

OBJECTIVE: To safeguard key workers involved in development and production of medicines and ensure business continuity, we developed an occupational healthcare program, performed by our company's occupational healthcare services, to assess the infection and immune status for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This pilot program, conducted at our company facilities, evaluated the suitability of diagnostic tools in our setting for program upscaling. METHODS: We used different marketed in vitro diagnostics (including tests for antibodies against spike protein subunits S1 and S2 and nucleocapsid [N] protein) combined with medical history, symptoms and likelihood of infection. We evaluated the testing strategy over four visits in 141 employees (known positive COVID-19 history, n = 20; unknown status, n = 121) between April and June 2020 at four company locations in Germany. Digital self-monitoring over the pilot program duration was also included. RESULTS: No incident infections were detected. Based on immune status, medical history and likelihood of infection, 10 participants (8.3%) with previously unknown history of COVID-19 were identified to have been infected before entering the program. These participants, who recalled no or mild symptoms in the preceding months, were primarily identified using an assay that detected both S1 and S2 immunoglobulin (Ig) G. The frequency of positive lateral flow assay (LFA) results (IgM or IgG directed against the N-protein) in this cohort was lower compared with participants with a known history of COVID-19 (0‒10.8% vs. 33.8‒75.7%, respectively). CONCLUSIONS: Data from this pilot program suggest that LFA for antibodies may not always reliably detect current, recent or past infections; consequently, these have not been included in our upscaled occupational healthcare program. Regular testing strategies for viral RNA and antibodies directed against different SARS-CoV-2 proteins, combined with hygiene rules and a comprehensive baseline assessment, are recommended to ensure avoidance of infections at workplace as reliably as possible.

Spesolimab, an Anti-Interleukin-36 Receptor Antibody, in Patients with Palmoplantar Pustulosis: Results of a Phase IIa, Multicenter, Double-Blind, Randomized, Placebo-Controlled Pilot Study.

INTRODUCTION: Palmoplantar pustulosis (PPP) is a chronic, inflammatory skin disease, with high disease burden, that is often refractory to treatment. There is a high unmet clinical need for the treatment of patients with PPP. The objectives of this study were to evaluate the safety and efficacy of spesolimab, a novel anti-interleukin-36 receptor antibody, in patients with PPP. METHODS: This was a phase IIa, multicenter, double-blind, randomized, placebo-controlled pilot study comparing 900 mg spesolimab (n = 19), 300 mg spesolimab (n = 19), and placebo (n = 21) administered intravenously every 4 weeks until week 12 in patients with PPP. The primary efficacy endpoint was the achievement of Palmoplantar Pustulosis Area and Severity Index 50 (PPP ASI50) at week 16, defined as achieving an ≥ 50% decrease from baseline PPP ASI. RESULTS: At week 16, 31.6% of patients in both spesolimab dose groups achieved PPP ASI50 versus 23.8% receiving placebo (risk difference 0.078; 95% confidence interval -0.190, 0.338). Thus, the primary endpoint was not met. Spesolimab was well tolerated with no clinically relevant treatment-emergent safety signals observed. CONCLUSIONS: PPP severity declined over time in all treatment groups after the start of treatment, with a faster decline in the spesolimab arms than in the placebo arm, indicating a potential treatment effect for spesolimab. Limitations to the study included a small sample size and lower overall disease severity than expected at baseline. It is possible that the primary efficacy endpoint may have coincided with natural disease resolution in some patients. Further effects of the efficacy of spesolimab in PPP are being explored in a phase IIb trial.

Comparison of normalization methods for Illumina BeadChip HumanHT-12 v3.

BACKGROUND: Normalization of microarrays is a standard practice to account for and minimize effects which are not due to the controlled factors in an experiment. There is an overwhelming number of different methods that can be applied, none of which is ideally suited for all experimental designs. Thus, it is important to identify a normalization method appropriate for the experimental setup under consideration that is neither too negligent nor too stringent. Major aim is to derive optimal results from the underlying experiment. Comparisons of different normalization methods have already been conducted, none of which, to our knowledge, comparing more than a handful of methods. RESULTS: In the present study, 25 different ways of pre-processing Illumina Sentrix BeadChip array data are compared. Among others, methods provided by the BeadStudio software are taken into account. Looking at different statistical measures, we point out the ideal versus the actual observations. Additionally, we compare qRT-PCR measurements of transcripts from different ranges of expression intensities to the respective normalized values of the microarray data. Taking together all different kinds of measures, the ideal method for our dataset is identified. CONCLUSIONS: Pre-processing of microarray gene expression experiments has been shown to influence further downstream analysis to a great extent and thus has to be carefully chosen based on the design of the experiment. This study provides a recommendation for deciding which normalization method is best suited for a particular experimental setup.

Characterization of iCell cardiomyocytes using single-cell RNA-sequencing methods.

INTRODUCTION: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are being evaluated for their use in pharmacological and toxicological testing, particularly for electrophysiological side effects. However, little is known about the composition of the commercially available iCell cardiomyocyte (Fuijifilm Cellular Dynamics) cultures and the transcriptomic phenotype of individual cells. METHODS: We characterized iCell cardiomyocytes (assumed to be a mixture of nodal-, atrial-, and ventricular-like cardiomyocytes together with potential residual non-myocytes) using bulk RNA-sequencing, followed by investigation of cellular heterogeneity using two different single-cell RNA-sequencing platforms. RESULTS: Bulk RNA-sequencing identified key cardiac markers (TNNT2, MYL7) as well as fibroblast associated genes (P4HB, VIM), and cardiac ion channels in the iCell cardiomyocyte culture. High-resolution single cell RNA-sequencing demonstrated that both, cardiac and fibroblast-related genes were co-expressed throughout the cell population. This approach resolved two cell clusters within iCell cardiomyocytes. Interestingly, these clusters could not be associated with known cardiac subtypes. However, transcripts of ion channels potentially useful as functional markers for cardiac subtypes were below the detection limits of the single-cell approaches used. Instead, one cluster (10.8% of the cells) is defined by co-expression of cardiac and cell cycle-related genes (e.g. TOP2A). Incorporation of bromodeoxyuridine further confirmed the capability of iCell cardiomyocytes to enter cell cycle. DISCUSSION: The co-expression of cardiac related genes with cell cycle or fibroblast related genes may be interpreted either as aberrant or as an immature feature. However, this excludes the presence of a non-cardiomyocyte sub-population and indicates that some cardiomyocytes themselves enter cell cycle.