Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus.

BACKGROUND: Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described. OBJECTIVE: We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes. METHODS: Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE. RESULTS: Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. CONCLUSION: Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

Assessing Pediatric Rheumatology Fellow Competence in the Milestone Era: Past, Present, and Future.

Starting in 2015, pediatric rheumatology fellowship training programs were required by the Accreditation Council for Graduate Medical Education to assess fellows' academic performance within 21 subcompetencies falling under six competency domains. Each subcompetency had four or five milestone levels describing developmental progression of knowledge and skill acquisition. Milestones were standardized across all pediatric subspecialties. As part of the Milestones 2.0 revision project, the Accreditation Council for Graduate Medical Education convened a workgroup in 2022 to write pediatric rheumatology-specific milestones. Using adult rheumatology's Milestones 2.0 as a starting point, the workgroup revised the patient care and medical knowledge subcompetencies and milestones to reflect requirements and nuances of pediatric rheumatology care. Milestones within four remaining competency domains (professionalism, interpersonal and communication skills, practice-based learning and improvement, and systems-based practice) were standardized across all pediatric subspecialties, and therefore not revised. The workgroup created a supplemental guide with explanations of the intent of each subcompetency, 25 in total, and examples for each milestone level. The new milestones are an important step forward for competency-based medical education in pediatric rheumatology. However, challenges remain. Milestone level assignment is meant to be informed by results of multiple assessment methods. The lack of pediatric rheumatology-specific assessment tools typically result in clinical competency committees determining trainee milestone levels without such collated results as the foundation of their assessments. Although further advances in pediatric rheumatology fellowship competency-based medical education are needed, Milestones 2.0 importantly establishes the first pediatric-specific rheumatology Milestones to assess fellow performance during training and help measure readiness for independent practice.

Synthetic oligonucleotide antigens modified with locked nucleic acids detect disease specific antibodies.

New techniques to detect and quantify antibodies to nucleic acids would provide a significant advance over current methods, which often lack specificity. We investigate the potential of novel antigens containing locked nucleic acids (LNAs) as targets for antibodies. Particularly, employing molecular dynamics we predict optimal nucleotide composition for targeting DNA-binding antibodies. As a proof of concept, we address a problem of detecting anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We test the best oligonucleotide binders in surface plasmon resonance studies to analyze binding and kinetic aspects of interactions between antigens and target DNA. These DNA and LNA/DNA sequences showed improved binding in enzyme-linked immunosorbent assay using human samples of pediatric lupus patients. Our results suggest that the novel method is a promising tool to create antigens for research and point-of-care monitoring of anti-DNA antibodies.

Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases.

Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid cardiolipin, and as the last step, coupling of the product with azide-containing linker and copper-catalyzed click chemistry with ß2-glycoprotein I and prothrombin. To prove utility of the product antigens, we used enzyme-linked immunosorbent assay and three cohorts of samples obtained from patients in Denmark (n = 34) and the USA (n = 27 and n = 14). Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity and specificity. Unlike previously used antigens the obtained autoantibody titers strongly correlate with high disease activity and in particular, with arthritis, renal involvement, anti-Smith antibodies and high lymphocyte count. Importantly, chemical composition of antigens has a strong influence on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.

Multiplex giant magnetoresistive biosensor microarrays identify interferon-associated autoantibodies in systemic lupus erythematosus.

High titer, class-switched autoantibodies are a hallmark of systemic lupus erythematosus (SLE). Dysregulation of the interferon (IFN) pathway is observed in individuals with active SLE, although the association of specific autoantibodies with chemokine score, a combined measurement of three IFN-regulated chemokines, is not known. To identify autoantibodies associated with chemokine score, we developed giant magnetoresistive (GMR) biosensor microarrays, which allow the parallel measurement of multiple serum antibodies to autoantigens and peptides. We used the microarrays to analyze serum samples from SLE patients and found individuals with high chemokine scores had significantly greater reactivity to 13 autoantigens than individuals with low chemokine scores. Our findings demonstrate that multiple autoantibodies, including antibodies to U1-70K and modified histone H2B tails, are associated with IFN dysregulation in SLE. Further, they show the microarrays are capable of identifying autoantibodies associated with relevant clinical manifestations of SLE, with potential for use as biomarkers in clinical practice.

A sea anemone's environment affects discharge of its isolated nematocysts.

Nematocysts were isolated from individuals of Calliactis tricolor maintained under different feeding schedules or in different salinities in an attempt to determine how these culture conditions influence the discharge of isolated nematocysts. In addition, the discharge frequencies of nematocysts isolated from two different populations of sea anemones found in two different environments were also compared. Undischarged acontial nematocysts were isolated by extrusion into 1 M sodium citrate and were then treated with 5 mM EGTA to initiate discharge. Nematocysts isolated from anemones maintained under three different feeding schedules showed significantly different responses to the test solution. Nematocysts isolated from anemones maintained in two different salinities did not differ significantly in discharge frequency. Nematocysts isolated from individuals from two separate populations of C. tricolor responded significantly differently to 5 mM EGTA and to deionized water, and these responses also depended upon the isolation solution used. Environmental conditions are known to have an impact on the physiological state of most organisms, but this is the first study providing evidence that the environment or feeding state of an anemone affects discharge of isolated nematocysts. Inherent differences in ionic and osmotic characteristics among nematocysts could explain some of the ambiguities when comparing past studies of isolated nematocyst discharge.