Myositis-related autoantibody profile and clinical characteristics stratified by anti-cytosolic 5'-nucleotidase 1A status in connective tissue diseases.

INTRODUCTION/AIMS: Cytosolic 5'-nucleotidase 1A (cN-1A) autoantibodies have been recognized as myositis-related autoantibodies. However, their correlations with clinical characteristics and other myositis-specific and myositis-associated autoantibodies (MSAs/MAAs) are still unclear. We aimed to establish the prevalence and clinical and laboratory associations of cN-1A autoantibodies in a cohort of patients with connective tissue diseases. METHODS: A total of 567 participants (182 idiopathic inflammatory myopathies [IIM], 164 systemic lupus erythematosus [SLE], 121 systemic sclerosis [SSc], and 100 blood donors [BD]) were tested for the presence of cN-1A autoantibodies and other myositis-specific and myositis-associated autoantibodies (MSAs/MAAs). Clinical and laboratory characteristics were compared between anti-cN-1A positive and negative patients with sporadic inclusion body myositis (sIBM) and between anti-cN-1A positive and negative patients with non-IBM IIM. RESULTS: In the sIBM cohort, 30 patients (46.9%) were anti-cN-1A positive vs. 18 (15.2%) in the non-IBM IIM cohort, 17 (10%) were anti-cN-1A positive in the SLE cohort and none in the SSc or the BD cohorts. Anti-cN-1A positivity had an overall sensitivity of 46.9% and a specificity of 93.2% for sIBM. Dysphagia was more frequent in the anti-cN-1A positive vs. negative sIBM patients (p = .04). In the non-IBM IIM group, being anti-cN-1A antibody positive was associated with the diagnosis polymyositis (p = .04) and overlap-myositis (p = .04) and less disease damage evaluated by physician global damage score (p < .001). DISCUSSION: cN-1A autoantibodies were predominantly found in IIM patients and was associated with dysphagia in sIBM patients. Notably, anti-cN-1A appears to identify a distinct phenotype of anti-cN-1A positive non-IBM IIM patients with a milder disease course.

Elevated Antibody Titers to Epstein-Barr Virus and Cytomegalovirus in Patients with Drug-Induced Lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which has been associated with Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) infection. Drug-induced lupus (DIL) is a lupus-like disease caused by the intake of therapeutic drugs, which has been estimated to cause approximately 10-15% of lupus-like cases. Although SLE and DIL share common clinical symptoms, there are some fundamental differences between DIL and SLE onset. Moreover, it remains to be examined whether environmental factors, such as EBV and CMV infections, may contribute to the development of DIL. This study focused on examining the possible association between DIL and EBV and CMV infections, by examining IgG titers to EBV and CMV antigens in serum samples by enzyme-linked immunosorbent assays. Antibody titers to EBV early antigen-diffuse and CMV pp52 were found to be significantly elevated in both SLE and DIL patients compared to healthy controls, although no correlation was found for antibodies to the two virus antigens in the respective disease groups. Moreover, total IgG titers were reduced in SLE and DIL serum samples, which may reflect a general lymphocytopenia, which commonly is associated with SLE. The current findings support that EBV and CMV infections may contribute to the development of DIL and that onset of both diseases are related.

A cluster of type II interferon-regulated genes associates with disease activity in patients with systemic lupus erythematosus.

Upregulation of interferon-regulated genes (IRGs), denoted IFN signature, in peripheral blood has been used as an indirect measure of IFN pathway activation in patients with systemic lupus erythematosus (SLE). However, it has not been determined, which IFN signatures that optimally reflect clinical disease activity. In this study, we determined an IFN signature based on the expression of 128 IRGs in whole blood from 34 SLE patients in a cross-sectional (CS) study, 11 with active lupus nephritis followed longitudinally (LS) and 15 healthy controls. Blood samples were collected in PAXgene tubes and RNA was extracted and purified using a PAXgene blood RNA kit (Qiagen). Gene expression was measured using the NanoString nCounter Gene Expression platform. The CS SLE patients with higher disease activity displayed thrice as many upregulated IRGs (n = 46) as the rest. These IRGs clustered in three groups, consisting of IRGs known to be predominantly stimulated by type I (gene cluster K1) and type II (gene clusters K2 and 3) IFNs. SLEDAI-2K scores associated with the K2 and K3 gene scores (ß = 0.372 and ß = 0.419, both p < 0.015) but not with K1. In the longitudinal study, the mean SLEDAI-2K score decreased after an average follow-up of 360 days (ß = -2.08, P = 5.09 × 10-12). The mean K1, K2 and K3 gene scores did not change over time, however longitudinal changes in SLEDAI-2K and K3 scores were associated (ß = 0.814, p = 0.007). This study validates the presence of type I IRG subsets that do not associate with disease activity in SLE patients. The novel finding in this study is the association between a type II IRG subset and disease activity. Both findings may have significant implications for choosing IRGs defining clinically relevant IFN signatures.

Galectin-3-binding protein is a novel predictor of venous thromboembolism in systemic lupus erythematosus.

OBJECTIVES: Venous (VTE) and arterial (AT) thrombosis in systemic lupus erythematosus (SLE) are poorly explained and difficult to predict. Leptin and tumour necrosis factor-like weak inducer of apoptosis (TWEAK) have been linked to subclinical atherosclerosis and galectin-3-binding protein (G3BP) to type I interferon activation and a pro-thrombotic environment. Thus, we explore serum G3BP, interferon gamma-induced protein 10 (IP-10), soluble CD163 (sCD163), TWEAK and leptin as predictors of VTE and AT, damage accrual, and all-cause mortality during follow-up in a Swedish SLE cohort. METHODS: Baseline data were available from 162 SLE patients. VTE (deep vein thrombosis and/or pulmonary embolism), AT (myocardial infarction and/or stroke), damage accrual, and survival data were the main study outcomes and available at follow-up (median of five years). Baseline serum G3BP, IP-10, sCD163, TWEAK and leptin were measured and analysed by univariable and multivariable methods for association to the study outcomes. RESULTS: During the follow-up, 10 (6%) VTE and 13 (8%) AT events occurred. The SLICC/ACR Damage Index increased in 78 (48%) patients, and 19 (12%) patients died. In the univariable regression analysis G3BP levels were significantly associated with an increased risk of VTE (hazard ratio (HR) 1.11, 95% confidence interval (CI): 1.01-1.22, p=0.03). This persisted in the adjusted multivariable analyses (HR 1.18, 95% CI: 1.05-1.33, p=0.007). The other biomarkers were not associated with AT/VTE, damage accrual, or all-cause mortality. CONCLUSIONS: Our study identifies serum G3BP as a novel predictor of VTE in SLE. Further studies are needed to understand the role of G3BP in VTE and translate this into clinical practice.

Stimulation of Mononuclear Cells Through Toll-Like Receptor 9 Induces Release of Microvesicles Expressing Double-Stranded DNA and Galectin 3-Binding Protein in an Interferon-α-Dependent Manner.

Background: Microvesicles (MVs) expressing the type 1 interferon (IFN)-inducible protein galectin-3 binding protein (G3BP) may play a pathogenic role in systemic lupus erythematosus (SLE). Co-expression of double-stranded DNA (dsDNA) on such MVs may render them immunogenic and targets for anti-dsDNA antibodies. Little is known about the mechanisms underlying generation of this MV population. In this study, we investigated how Toll-like receptors (TLRs), IFN-α, and T cells are involved in this process in healthy subjects. Methods: Peripheral blood mononuclear cells (PBMCs) isolated from 12 healthy donors were stimulated in-vitro for 24 h with a series of TLR-agonists or the T cell activating antibody OKT3 or were subjected to apoptosis by incubation with staurosporine. MVs in the supernatants were subsequently isolated by differential centrifugation and were quantified and characterized with respect to expression of G3BP and dsDNA by flow cytometry. Results: Stimulation of PBMCs with the TLR9-agonist and strong IFN-α inducer ODN2395 significantly increased the release of MVs expressing G3BP. The production of MVs with this phenotype was markedly enhanced by co-stimulation of T cells. Furthermore, dependency on IFN-α in the generation of G3BP-expressing MVs was indicated by a marked reduction following addition of the IFN-α inhibitor IFN alpha-IFNAR-IN-1 hydrochloride. Conclusion: Release of G3BP-expressing MVs from healthy donor PBMCs is induced by stimulation of TLR9 in an IFN-α-dependent manner and is enhanced by co-stimulation of T cells.

Clinical and Serological Associations with the Development of Incident Proteinuria in Danish Patients with Systemic Lupus Erythematosus.

OBJECTIVE: In a longitudinal cohort study, we investigated whether clinical and serological manifestations at the time of classification of systemic lupus erythematosus (SLE) were predictive of subsequent development of incident proteinuria as a biomarker of incident lupus nephritis. METHODS: Patients fulfilling SLE classification criteria but having no proteinuria prior to or at the time of classification were included. Data on SLE manifestations, vital status, criteria-related autoantibodies, and SLE-associated medications were collected during clinical visits and supplemented by chart review. HR were calculated by Cox regression analyses. RESULTS: Out of 850 patients with SLE, 604 had not developed proteinuria at the time of SLE classification. Of these 604 patients, 184 (30%) developed incident proteinuria following SLE classification. The patients had a median followup of 11 years and 7 months. Younger age and history of psychosis at the time of classification were associated with development of incident proteinuria, just as were lymphopenia (HR 1.49, 95% CI 1.08-2.06), anti-dsDNA (HR 1.38, 95% CI 1.01-1.87), and a high number of autoantibodies (HR 1.26, 95% CI 1.06-1.48). CONCLUSION: The risk of incident proteinuria after onset of SLE was increased by the presence of lymphopenia, anti-dsDNA antibodies, psychosis, younger age, and a high number of autoantibodies at onset.

Antibodies to a strain-specific citrullinated Epstein-Barr virus peptide diagnoses rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Anti-citrullinated protein antibodies (ACPA) are crucial for the serological diagnosis of RA, where Epstein-Barr virus (EBV) has been suggested to be an environmental agent in triggering the onset of the disease. This study aimed to analyse antibody reactivity to citrullinated EBV nuclear antigen-2 (EBNA-2) peptides from three different EBV strains (B95-8, GD1 and AG876) using streptavidin capture enzyme-linked immunosorbent assay. One peptide, only found in a single strain (AG876), obtained a sensitivity and specificity of 77% and 95%, respectively and showed high sequence similarity to the filaggrin peptide originally used for ACPA detection. Comparison of antibody reactivity to commercial assays found that the citrullinated peptide was as effective in detecting ACPA as highly sensitive and specific commercial assays. The data presented demonstrate that the citrullinated EBNA-2 peptide indeed is recognised specifically by RA sera and that the single peptide is able to compete with assays containing multiple peptides. Furthermore, it could be hypothesized that RA may be caused by (a) specific strain(s) of EBV.

The use of synthetic peptides for detection of anti-citrullinated protein antibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology. A characteristic feature of RA is the presence of anti-citrullinated protein antibodies (ACPA). Since ACPAs are highly specific for RA and are often present before the onset of RA symptoms, they have become valuable diagnostic and prognostic. As a result, several assays for detection of ACPAs exist, which vary in sensitivity and specificity. In this study, we analyzed the reactivity of RA sera to selected peptides by solid-phase immunoassays in order to develop an ACPA assay with improved sensitivity and specificity. ACPA levels were determined with respect to sensitivity and specificity in 332 serum samples using the newly developed peptide panel, which was compared to the commercial assays CCPlus (Eurodiagnostica) and CCP3.1 (Inova Diagnostics). A primary panel (peptides 814, 33062 and 33156) was identified, which obtained a sensitivity of 71%, while the complete peptide panel reacted with 79% of RA sera screened. Total specificities of 89% and 80% were obtained for the primary peptide panel and the complete peptide panel. Sensitivities for the commercial assays ranged between 71% and 76% and specificities between 88% and 90%. These findings indicate that the generated peptide panel is optimal for ACPA detection and able to compete with commercial available assays. Collectively, this study may contribute to characterize autoimmunity towards citrullinated proteins and to the development of new and improved diagnostic assays for detection of ACPA and determination of RA.

Endothelial dysfunction is associated with activation of the type I interferon system and platelets in patients with systemic lupus erythematosus.

OBJECTIVES: Endothelial dysfunction may be connected to cardiovascular disease (CVD) in systemic lupus erythematosus (SLE). Type I interferons (IFNs) are central in SLE pathogenesis and are suggested to induce both endothelial dysfunction and platelet activation. In this study, we investigated the interplay between endothelial dysfunction, platelets and type I IFN in SLE. METHODS: We enrolled 148 patients with SLE and 79 sex-matched and age-matched healthy controls (HCs). Type I IFN activity was assessed with a reporter cell assay and platelet activation by flow cytometry. Endothelial dysfunction was assessed using surrogate markers of endothelial activation, soluble vascular cell adhesion molecule-1 (sVCAM-1) and endothelial microparticles (EMPs), and finger plethysmograph to determine Reactive Hyperaemia Index (RHI). RESULTS: In patients with SLE, type I IFN activity was associated with endothelial activation, measured by high sVCAM-1 (OR 1.68, p<0.01) and elevated EMPs (OR 1.40, p=0.03). Patients with SLE with high type I IFN activity had lower RHI than HCs (OR 2.61, p=0.04), indicating endothelial dysfunction.Deposition of complement factors on platelets, a measure of platelet activation, was seen in patients with endothelial dysfunction. High levels of sVCAM-1 were associated with increased deposition of C4d (OR 4.57, p<0.01) and C1q (OR 4.10, p=0.04) on platelets. High levels of EMPs were associated with C4d deposition on platelets (OR 3.64, p=0.03). CONCLUSIONS: Endothelial dysfunction was associated with activation of platelets and the type I IFN system. We suggest that an interplay between the type I IFN system, injured endothelium and activated platelets may contribute to development of CVD in SLE.

Distinct proteome pathology of circulating microparticles in systemic lupus erythematosus.

BACKGROUND: The pathogenesis of systemic lupus erythematosus (SLE) is poorly understood but has been linked to defective clearance of subcellular particulate material from the circulation. This study investigates the origin, formation, and specificity of circulating microparticles (MPs) in patients with SLE based on comprehensive MP proteome profiling using patients with systemic sclerosis (SSc) and healthy donors (HC) as controls. METHODS: We purified MPs from platelet-poor plasma using differential centrifugation of samples from SLE (n = 45), SSc (n = 38), and two sets of HC (n = 35, n = 25). MP proteins were identified and quantitated after trypsin digestion by liquid chromatography-tandem mass spectrometry. The abundance of specific proteins was compared between the groups using univariate statistics and false discovery rate correction for multiple comparisons. Specific proteins and protein ratios were explored for diagnostic and disease activity information using receiver-operating characteristic curves and by analysis of correlations of protein abundance with disease activity scores. RESULTS: We identify and quantitate more than 1000 MP proteins and show that a subpopulation of SLE-MPs (which we propose to call luposomes) are highly specific for SLE, i.e. not found in MP preparations from HC or patients with another autoimmune, systemic disease, SSc. In SLE-MPs platelet proteins and mitochondrial proteins are significantly diminished, cytoskeletal proteins deranged, and glycolytic enzymes and apoptotic proteins significantly increased. CONCLUSIONS: Normal MPs are efficiently removed in SLE, but aberrant MPs, derived from non-lymphoid leukocytes, are less efficiently removed and abundantly produced leading to an altered MP proteome in SLE. The data suggest that an abnormal generation of MPs may partake in the pathology of SLE and that new diagnostic, monitoring, and treatment strategies targeting these processes may be advantageous.

Complexes of DNA with fluorescent dyes are effective reagents for detection of autoimmune antibodies.

To date, there are multiple assays developed that detect and quantify antibodies in biofluids. Nevertheless, there is still a lack of simple approaches that specifically detect autoimmune antibodies to double-stranded DNA. Herein we investigate the potential of novel nucleic acid complexes as targets for these antibodies. This is done in a simple, rapid and specific immunofluorescence assay. Specifically, employing 3D nanostructures (DNA origami), we present a new approach in the detection and study of human antibodies to DNA. We demonstrate the detection of anti-DNA antibodies that are characteristic of systemic lupus erythematosus, a chronic autoimmune disease with multiple manifestations. We tested the most potent non-covalent pairs of DNA and fluorescent dyes. Several complexes showed specific recognition of autoimmune antibodies in human samples of lupus patients using a simple one-step immunofluorescence method. This makes the novel assay developed herein a promising tool for research and point-of-care monitoring of anti-DNA antibodies. Using this method, we for the first time experimentally confirm that the disease-specific autoimmune antibodies are sensitive to the 3D structure of nucleic acids and not only to the nucleotide sequence, as was previously thought.

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.

Application of synthetic peptides for detection of anti-citrullinated peptide antibodies.

Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides and analyze their potential as substrates for ACPA detection by streptavidin capture enzyme-linked immunosorbent assay. Using systematically overlapping peptides, containing a 10 amino acid overlap, labelled with biotin C-terminally or N-terminally, sera from 160 individuals (RA sera (n=60), healthy controls (n=40), systemic lupus erythematosus (n=20), Sjögren's syndrome (n=40)) were screened for antibody reactivity. Antibodies to a panel of five citrullinated EBNA-1 peptides were found in 67% of RA sera, exclusively of the IgG isotype, while 53% of the patient sera reacted with a single peptide, ARGGSRERARGRGRG-Cit-GEKR, accounting for more than half of the ACPA reactivity alone. Moreover, these antibodies were detected in 10% of CCP2-negative RA sera. In addition, 47% of the RA sera reacted with two or three citrullinated EBNA-1 peptides from the selected peptide panel. Furthermore, a negative correlation between the biotin attachment site and the location of citrulline in the peptides was found, i.e. the closer the citrulline was located to biotin, the lower the antibody reactivity. Our data suggest that citrullinated EBNA-1 peptides may be considered a substrate for the detection of ACPAs and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies.

Detection of antibodies to the 20s proteasome by ELISA.

The presence of antibodies against the 20S proteasome has been correlated with diseases like multiple sclerosis (MS) and systemic lupus erythematosus (SLE) but no definite association has been established. In order to investigate this further, we optimized an ELISA for proteasome antibodies and applied it to test a total of 324 serum and plasma samples from MS patients, SLE patients, and healthy controls. Our results yield a functional and reliable assay but no correlation between the amount of proteasome antibodies present and the development of MS or SLE could be established.

Elevated platelet expression of CD36 may contribute to increased risk of thrombo-embolism in active inflammatory bowel disease.

CONTEXT: Inflammatory bowel disease (IBD) induces increased risk of thrombo-embolism. CD36 is involved in platelet activation, glucose metabolism and inflammation. OBJECTIVE: The relationship between CD36 expression on platelets and monocytes, plasma sCD36, and CD36-positive platelet-derived microparticles (PDMPs) and inflammation in both active IBD and after one week of anti-tumour necrosis alpha antibody (anti-TNF) treatment was investigated. MATERIAL AND METHODS: Patients with exacerbation of Crohn's disease (n = 8) or ulcerative colitis (n = 5) and 13 healthy controls were enrolled. Seven patients underwent anti-TNF treatment for one week. Platelet, monocyte, and PDMP-CD36 were measured by flow-cytometry. RESULTS: Platelet CD36 expression was 34% higher in patients, and correlated with insulin resistance and fasting glucose. sCD36 was 37% lower and restored after anti-TNF treatment. CONCLUSION: Elevated platelet CD36 expression may contribute to increased risk of thrombo-embolism in active IBD. This may not entirely be attributed to inflammation and secondary insulin resistance may play a role.

Circulating microparticles in systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease presenting with a wide array of clinical manifestations and an elusive pathogenesis. A characteristic feature in SLE is the occurrence of autoantibodies against chromatin, double-stranded DNA, and RNA-binding ribonucleoproteins. Observations of defective clearance of dying cells in SLE combined with the generation and exposure of nuclear autoantigens during apoptosis have led to the hypothesis that improperly cleared apoptotic debris constitutes a source of autoantigens capable of triggering autoimmune disease. In blood, circulating, heterogeneous subcellular microparticles (MPs) are released from cells and platelets constitutively and upon cellular activation or apoptosis. Such MPs may reflect the state of their parental cells and tissues, and could serve as markers of pathology. Particular in SLE MPs may serve as carriers of autoantigens and constituents of immune complexes (ICs). The purposes of this PhD thesis were to develop and apply qualitative and quantitative methods to characterize circulating MPs with respect to numbers, cellular origins and composition in a large cohort of well-characterized SLE patients compared to healthy and disease controls and to explore associations with clinical, biochemical and serological parameters. The PhD thesis consists of a review and three papers. In the first paper we show that SLE patients have significantly decreased numbers of annexin V binding MPs and MPs from platelets, leukocytes and endothelial cells using flow cytometry. Two morphologically distinguishable populations of annexin V non-binding MPs were increased in the SLE patients. The annexin V non-binding MPs of most likely cellular origin were associated with the presence of lupus nephritis, markers of increased disease activity and levels of endothelial cell-derived MPs. In the second paper we present the development of a proteomic method to characterize the protein composition of purified MPs using high-resolution mass spectrometry and establish a set of proteins which may serve as normalizers for MP protein quantitation enabling comparison across samples and studies. We identify a core proteome of more than 330 proteins in MPs from healthy individuals. The method enables an unbiased, comprehensive coverage of all proteins present in MPs irrespectively of the availability and utility of immunological reagents. In the third paper we use the established flow cytometry and mass spectrometry platforms to show that SLE-MPs carry more surface-bound IgG, IgM and C1q indicating that SLE-MPs could be antigenic targets and constituents of ICs. Additionally, the numbers of MPs carrying IgG are also increased in SLE. The load of IgG on SLE-MPs was associated with markers of complement activation, indicators of disease activity in SLE. In conclusion, using both antibody-dependent and independent methods we demonstrate that SLE-MPs deviate distinctly from controls and may serve as precursors of ICs associated with complement activation and disease activity. This supports the hypothesis of MPs being directly involved in or reflecting tissue-specific or systemic inflammation in addition to carrying accessible antigens. Accordingly, further characterization of the proteome and functional properties of SLE-MPs seem highly warranted in future studies.