Additional Analysis of the APPLE (Atherosclerosis Prevention in Paediatric Lupus Erythematosus) Trial Identifies Novel Determinants of Patient Heterogeneity and a Distinct Lipid Metabolomic Signature Associated with Atherosclerosis Progression

Background: Juvenile-onset systemic lupus erythematosus (JSLE) is associated with chronic inflammation and increased risk of atherosclerosis. The APPLE trial was a randomised, placebo-controlled trial of atorvastatin for atherosclerosis progression in JSLE, using carotid intima-media thickness (CIMT) measurements as primary outcome. Methods: Unsupervised clustering analysis was used to stratify JSLE patients by their baseline CIMT and identify patterns of CIMT progression over 36 months. An additional in-depth metabolomic analysis was performed to identify lipidomic signatures predictive of CIMT progression. Correlation and univariate regression analyses explored associations between patient and disease characteristics and serum biomarkers. Machine learning techniques and ROC analyses were used to identify and validate a serum metabolomic signature of high CIMT progression. Findings: Baseline CIMT measurements stratified JSLE patients into three groups with distinct CIMT progression trajectories irrespective of the treatment allocation. Two distinct CIMT progression rates (high vs. low), characterised by higher total and low-density lipoprotein (LDL) cholesterol levels (P=0.001 and P=0.002, respectively) were found in the placebo group, while patients treated with atorvastatin had three distinct CIMT trajectories (high, intermediate and low progression), not associated with any relevant biomarkers. A robust metabolomic signature predictive of high CIMT progression in the placebo arm was identified (AUC = 80.7%). Interpretation: This complementary analysis of the APPLE trial provides new evidence for the significant heterogeneity of subclinical atherosclerosis in JSLE and its distinct progression trajectories irrespective of treatment allocation. Clinical trial patient stratification using the newly identified metabolomic signature predictive of increased natural atherosclerosis progression rate may improve results. Despite being effective in lowering serum lipids, atorvastatin did not prevent the CIMT progression in many at risk JSLE patients, highlighting the need for personalised therapies to address various molecular mechanism driving atherosclerosis in JSLE

Protein interaction, monocyte toxicity and immunogenic properties of cerium oxide crystals with 5% or 14% gadolinium, cobalt oxide and iron oxide nanoparticles - an interdisciplinary approach

Metal oxide nanoparticles are widely used in both consumer products and medical applications, but the knowledge regarding exposure-related health effects is limited. However, it is challenging to investigate nanoparticle interaction processes with biological systems. The overall aim of this project was to improve the possibility to predict exposure-related health effects of metal oxide nanoparticles through interdisciplinary collaboration by combining workflows from the pharmaceutical industry, nanomaterial sciences, and occupational medicine. Specific aims were to investigate nanoparticle-protein interactions and possible adverse immune reactions. Four different metal oxide nanoparticles; CeOx nanocrystals with 5% or 14% Gd, Co3O4, and Fe2O3, were characterized by dynamic light scattering and high-resolution transmission electron microscopy. Nanoparticle-binding proteins were identified and screened for HLA-binding peptides in silico. Monocyte interaction with nanoparticle-protein complexes was assessed in vitro. Herein, for the first time, immunogenic properties of nanoparticle-binding proteins have been characterized. The present study indicates that especially Co3O4-protein complexes can induce both danger signals, verified by the production of inflammatory cytokines and simultaneously bind autologous proteins, which can be presented as immunogenic epitopes by MHC class II. The clinical relevance of these findings should be further evaluated to investigate the role of metal oxide nanoparticles in the development of autoimmune disease. The general workflow identified experimental difficulties, such as nanoparticle aggregate formation and a lack of protein-free buffers suitable for particle characterization, protein analyses, as well as for cell studies. This confirms the importance of future interdisciplinary collaborations.Funding Agencies|Region Ostergotland ALF [LIO-606891]; AFA insurances [150246]; Swedish Government Strategic Research Area in Materials Science on Functional Materials at Linkoping University [2009-00971]; Knut and Alice Wallenberg FoundationKnut &amp; Alice Wallenberg Foundation [2012.0083 CTS, 18:399]; Center in Nano Science and Nano technology at LiTH (CeNano) at Linkoping University</p

Significantly improved prediction of subcellular localization by integrating text and protein sequence data

Computational prediction of protein subcellular localization is a challenging problem. Several approaches have been presented during the past few years; some attempt to cover a wide variety of localizations, while others focus on a small number of localizations and on specific organisms. We present a comprehensive system, integrating protein sequence-derived data and text-based information. It is tested on three large data sets, previously used by leading prediction methods. The results demonstrate that our system performs significantly better than previously reported results, for a wide range of eukaryotic subcellular localizations. 1

Monocyte NOTCH2 expression predicts IFN-beta immunogenicity in multiple sclerosis patients

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-beta is an established treatment for MS; however, up to 30% of IFN-beta-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14(+) monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-beta. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-beta administration. Reduced monocyte NOTCH2 expression in nADA(+) MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA(+) patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-beta administration

Long-term SARS-CoV-2-specific and cross-reactive cellular immune responses correlate with humoral responses, disease severity, and symptomatology

Background: Cellular immune memory responses post coronavirus disease 2019 (COVID-19) have been difficult to assess due to the risks of contaminating the immune response readout with memory responses stemming from previous exposure to endemic coronaviruses. The work herein presents a large-scale long-term follow-up study investigating the correlation between symptomology and cellular immune responses four to five months post seroconversion based on a unique severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific peptide pool that contains no overlapping peptides with endemic human coronaviruses. Methods: Peptide stimulated memory T cell responses were assessed with dual interferon-gamma (IFN gamma) and interleukin (IL)-2 Fluorospot. Serological analyses were performed using a multiplex antigen bead array. Results: Our work demonstrates that long-term SARS-CoV-2-specific memory T cell responses feature dual IFN gamma and IL-2 responses, whereas cross-reactive memory T cell responses primarily generate IFN gamma in response to SARS-CoV-2 peptide stimulation. T cell responses correlated to long-term humoral immune responses. Disease severity as well as specific COVID-19 symptoms correlated with the magnitude of the SARS-CoV-2-specific memory T cell response four to five months post seroconversion. Conclusion: Using a large cohort and a SARS-CoV-2-specific peptide pool we were able to substantiate that initial disease severity and symptoms correlate with the magnitude of the SARS-CoV-2-specific memory T cell responses

Detection and kinetics of persistent neutralizing anti-interferon-beta antibodies in patients with multiple sclerosis. Results from the ABIRISK prospective cohort study

Two validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-beta) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-beta-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-beta ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA