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Objective To review: 1) degree of conformity to the American College of Rheumatology neuropsychological battery (ACR-NB) among studies that used a NB, 2) review definitions of cognitive impairment (CI) from studies that used a NB, and 3) characterize measurement tools used to assess CI in systemic lupus erythematosus (SLE). Methods The literature search was conducted in Ovid Medline, Embase, and PsycINFO for articles on CI in adult SLE patients. We reviewed studies that used a NB and compared their tests to the ACR-NB to assess the degree of conformity. Definitions of CI from studies that used a NB were reviewed when sufficient information was available. We reviewed and categorized CI measurement tools into four broad categories: NB, screening, incomplete/mixed batteries, and computerized batteries. Results Of 8727 references, 118 were selected for detailed review and 97 were included in the final analysis. Of 43 studies that used a NB, none of the studies used the ACR-NB exactly as published. Many studies supplemented with other tests. Overall, there was inconsistent use of ACR-NB tests. Definitions for CI varied, with cut-offs ranging from 1 to 3 standard deviations below normative values on domains/tests varying in type and number. The most frequently used measurement tool for assessing CI in SLE was a NB. Use of screening tests and computerized batteries have also increased over the last decade. Conclusion The assessment and definition of CI in SLE remains heterogeneous. A consensus meeting to address existing inconsistencies should be considered to harmonize the field of CI in SLE.

Objectives Type I interferons (IFNs) play an important role in the pathophysiology of systemic lupus erythematosus (SLE). While cross-sectional data suggest an association between IFN-induced gene expression and SLE disease activity, interest in this as a biomarker of flare has been tempered by a lack of fluctuation with disease activity in the majority of patients. This led us to question whether IFN-induced gene expression might instead be a biomarker of overall disease severity, with patients with high levels spending more time in an active disease state. Methods Levels of five interferon-responsive genes were measured in the whole peripheral blood at baseline visit for 137 SLE patients subsequently followed for 5 years. Log transformed values were summed to yield a composite IFN5 score, and the correlation with various disease outcomes examined. Receiver operator characteristic analyses were performed for outcomes of interest. Kaplan-Meier curves were generated to compare the proportion of flare-free patients with high and low IFN5 scores over time. Results The baseline IFN5 score was positively correlated with the adjusted mean SLE disease activity index-2000, number of flares, adjusted mean prednisone dose, and number of new immunosuppressive medications over the subsequent 5 years. Optimal cut-offs for the IFN5 score were determined using Youden’s index and predicted more severe outcomes with 57–67% accuracy. A high baseline IFN5 level was associated with a significantly increased risk of subsequent flare. Conclusions Measurement of the type I IFN signature is a useful tool for predicting the subsequent disease activity course.

Objectives To systematically review and synthesize literature on 1) the overall prevalence of depression and anxiety in SLE patients in identified studies, and 2) the pooled prevalence per metrics of depression and anxiety in adult SLE patients. Methods This review used (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) PRISMA guidelines and in-depth searches in four databases (1954-2016; Ovid-based Medline, Embase, PsycINFO and CINAHL) to identify articles on the prevalence of depression and/or anxiety in adult SLE patients. Included studies were critically appraised and analyzed. The prevalence of depression and anxiety was studied for all included studies, and whenever possible, pooled prevalence (PP) was determined for more commonly used metrics. Statistical and publication bias was assessed using funnel plots. Result A total of 3103 references were identified, 226 were selected for detailed review and 72 were included in the final analysis. Overall Prevalence The depression PP, obtained from 69 studies representing 23,386 SLE patients, was 35.0% (95% CI: 29.9%-40.3%). The anxiety PP, obtained from 38 studies representing 4439 SLE patients, was 25.8% (95% CI: 19.2%-32.9%). Prevalence per metrics used The more commonly used instruments included the Centre for Epidemiological Studies - Depression (CES-D), Beck Depression Inventory (BDI), Beck Anxiety Inventory (BAI), Hospital Anxiety and Depression Scales (HADS-A/D), and Hamilton Rating Scales for Depression/Anxiety (HAM-D/A)]. The CES-D was utilized in 13 studies including 1856 SLE patients; depression PP was 41.5% (95% CI: 35.1%-48.1%). The BDI was utilized in 14 studies including 1355 SLE patients and the BAI in 3 studies including 489 patients; depression PP was 39.9% (95% CI: 31.1%-49.1) and anxiety PP was 38.4% (95% CI: 34.2%-42.8%). The HADS-D was utilized in 14 studies including 1238 SLE patients and the HADS-A in 12 studies including 1099 patients respectively; it's depression PP was 24.4% (95% CI: 19.1%-30.1%) and anxiety PP was 38.3% (95% CI: 29.1%-47.9%). The HAM-D was utilized in 4 studies including 267 SLE patients and the HAM-A in 4 studies including 213 patients respectively; its depression PP was 40.0% (95% CI: 23.0%-59.0%) and anxiety PP was 39.0% (95% CI: 32.0%-45.0%). Conclusion There was high variability in the prevalence of depression and anxiety, ranging from 8.7%-78.6% and 1.1%-71.4%, respectively. This could be attributed to the lack of consistency in the metrics used and its definition for depression and anxiety in SLE. Studies that used a specific metric, such as the CES-D, BDI or HAM-D, yielded similar depression prevalence. The HADS-D had the lowest prevalence. All metrics of anxiety yielded similar anxiety prevalence.

Objective We investigated the autoantibody (autoAb) profiles in ANA+ individuals lacking systemic autoimmune rheumatic disease (SARD) and early SARD patients to determine the key differences between these groups and identify factors that are associated with an increased risk of symptomatic progression within the next 2 years in ANA+ individuals. Methods Using custom antigen (Ag) microarrays, 144 IgM and IgG autoAbs were surveyed in 84 asymptomatic and 123 symptomatic (48 UCTD and 75 SARD patients) ANA+ individuals. AutoAbs were compared in ANA+ individuals lacking a SARD diagnosis with ≥2 years follow-up (n = 52), including all those who demonstrated progression (n = 14) during this period, with changes over time assessed in a representative subset. Results We show that ANA+ individuals have autoAb to many self-Ags that are not being captured by current screening techniques and very high levels of these autoAbs are predominantly restricted to early SARD patients, with SLE patients displaying reactivity to many more autoAgs than the other groups. In general, the symptoms that developed in progressors mirrored those seen in SARD patients with similar patterns of autoAbs. Only anti-Ro52 Abs were found to predict progression (positive predictive value 46%, negative predictive value 89%). Surprisingly, over 2 years of follow-up the levels of autoAbs remained remarkably stable regardless of whether individuals progressed or not. Conclusion Our findings strongly argue that development of assays with an expanded set of auto-Ags and enhanced dynamic range would improve the diagnostic and prognostic ability of autoAb testing.

Objectives To examine i) the relationship between neuropsychological performance and depression and anxiety over time, and ii) the overlap between classification of cognitive dysfunction, anxiety, and depression in SLE. Methods 301 patients with SLE were included. Cognition was measured using a modified version of the ACR neuropsychological battery; cognitive dysfunction was defined as z-scores ≤-1.5 on ≥2 domains. Depression and anxiety were measured using the Beck Depression Inventory-II and the Beck Anxiety Inventory, respectively. All measures were assessed at baseline, 6, and 12 months. Their relationships were analyzed using Multiple Factor Analysis (MFA). Results Anxiety and depression and neuropsychological performance were stable across time. Factor analysis identified two dimensions explaining 42.2% of the variance in neuropsychological performance. The first dimension (33.1% of the variance) included primarily complex cognitive tests measuring executive function; verbal, visual, and working memory; and complex processing speed. The second dimension (9.1% of the variance) included primarily measures of simple information processing speed or motor dexterity. Anxiety and depression scores were consistently related to the first cognitive dimension. There was substantial overlap in participants classified with cognitive dysfunction and anxiety and depression. Conclusions Depression and anxiety symptoms in SLE patients are related to a cognitive dimension incorporating memory, executive function and complex processing speed in a stable manner across one year. Many patients with cognitive dysfunction exhibit clinically significant anxiety and depression. Further research should examine whether cognition improves when anxiety and depression are treated and mechanistic links between anxiety and depression and cognitive dysfunction in SLE.

Chapter 1 provides an overview of the five core domains for the appropriate assessment of patients with systemic lupus erythematosus (SLE) for research and clinical settings: disease activity, chronic damage resulting from lupus activity or its treatment, health-related quality of life (HRQoL), adverse events, and economic impact. Emerging concepts in SLE such as frailty are also introduced along with deep insights on selected existing concepts, in particular cognition, depression, and anxiety as well as fatigue. Having explored what needs to be measured in SLE, this chapter then outlines the decision-making process by which candidate instruments are identified and determined to be a match for the target construct (e.g., disease activity).

ObjectiveDispersion, or variability in an individual’s performance across multiple tasks at a single assessment visit, has been associated with cognitive dysfunction (CD) in many neurodegenerative and neurodevelopmental disorders. We aimed to compute a dispersion score using neuropsychological battery (NB) tests and determine its association with CD in patients with SLE.MethodsCD was defined as a z-score of ≤−1.5 on ≥2 domains of the NB. To compute a type of dispersion score known as the intraindividual SD (ISD), the SD of age-adjusted and sex-adjusted z-scores was calculated for each visit in each patient. To estimate the association between ISD and cognitive status (CD and non-CD), we used multilevel logistic regression, adjusting for clinically important covariates.ResultsA total of 301 adult patients with SLE completed the NB at baseline, 187 of whom were reassessed at 6 months and 189 at 12 months. CD was observed in 35.2% of patients at baseline, 27.8% at 6 months and 28.0% at 12 months. Prior to covariate adjustment, the mean ISD for non-CD was 1.10±0.31 compared with 1.50±0.70 for CD. After adjusting for ethnicity, education, employment, socioeconomic status and anxiety/depression, there was a statistically significant association between ISD and CD (OR for one-unit increase in ISD: 13.56, 95% CI 4.80 to 38.31; OR for 1/10th-unit increase in ISD: 1.30, 95% CI 1.17 to 1.44). Findings were valid across multiple sensitivity analyses.ConclusionThis is the first study to show that patients with SLE who were classified as having CD by the NB had more variability across the NB tests (ie, higher ISD score) compared with those who were not classified as having CD.

OBJECTIVE Screening for cognitive impairment in systemic lupus erythematosus (SLE) conventionally relies on the American College of Rheumatology (ACR) neuropsychologic battery (NB), which is not universally available. To develop a more accessible screening approach, we assessed validity of the Automated Neuropsychological Assessment Metrics (ANAM). Using the ACR NB as the gold standard for cognitive impairment classification, the objectives were 1) to measure overall discriminative validity of the ANAM for cognitive impairment versus no cognitive impairment, 2) to identify ANAM subtests and scores that best differentiate patients with cognitive impairment from those with no cognitive impairment, and 3) to derive ANAM composite indices and cutoffs. METHODS A total of 211 consecutive adult patients, female and male, with SLE were administered the ANAM and ACR NB. 1) For overall discriminative validity of the ANAM, we compared patients with cognitive impairment versus those with no cognitive impairment on 4 scores. 2) Six ANAM models using different scores were developed, and the most discriminatory subtests were selected using logistic regression analyses. The area under the receiver operating characteristic curve (AUC) was calculated to establish ANAM validity against the ACR NB. 3) ANAM composite indices and cutoffs were derived for the best models, and sensitivities and specificities were calculated. RESULTS Patients with no cognitive impairment performed better on most ANAM subtests, supporting ANAM's discriminative validity. Cognitive impairment could be accurately identified by selected ANAM subtests with top models, demonstrating excellent AUCs of 81% and 84%. Derived composite indices and cutoffs demonstrated sensitivity of 78-80% and specificity of 70%. CONCLUSION This study provides support for ANAM's discriminative validity for cognitive impairment and utility for cognitive screening in adult SLE. Derived composite indices and cutoffs enhance clinical applicability.

Background Fatigue is a common symptom of systemic autoimmune rheumatic disease (SARD). Patients with SARD have a protracted pre-clinical phase during which progressive immunologic derangements occur culminating in disease. In this study, we sought to determine when fatigue develops and whether its presence correlates with inflammatory factors or predicts disease progression. Methods Anti-nuclear antibody (ANA)-negative healthy controls (HCs) and ANA-positive participants with no criteria, at least one clinical criteria (undifferentiated connective tissue disease, UCTD), or meeting SARD classification criteria were recruited. Fatigue was assessed using a modified version of the FACIT-F questionnaire and the presence of fibromyalgia determined using a questionnaire based on the modified 2010 ACR criteria. Peripheral blood expression of five IFN-induced genes was quantified by NanoString and the levels of IL-1β, IL-6, or TNF-α by ELISA. Results Fatigue was as prevalent and severe in individuals lacking SARD criteria as it was in UCTD and SARD. Overall, ~ 1/3 of ANA+ subjects met fibromyalgia criteria, with no differences between sub-groups. Although fatigue was more severe in these individuals, those lacking fibromyalgia remained significantly more fatigued than ANA− HC. However, even in these subjects, fatigue correlated with the widespread pain index and symptom severity scores on the fibromyalgia questionnaire. Fatigue was not associated with elevated cytokine levels in any of the ANA+ sub-groups and did not predict imminent disease progression. Conclusions Fatigue is common in ANA+ individuals lacking sufficient criteria for a SARD diagnosis, correlates with fibromyalgia-related symptoms, and is not associated with inflammation or predictive of disease progression.

Background Lupus nephritis (LN) is one of the most common and severe manifestations of systemic lupus erythematosus (SLE). We tested the association of SLE-risk loci with LN risk in childhood- (cSLE) and adult-onset SLE (aSLE). Methods Two Toronto-based tertiary care SLE cohorts included cSLE (diagnosed Results Of 1237 participants, 572 had cSLE (41% with LN) and 665 had aSLE (30% with LN). Increasing non-HLA GRS was significantly associated with increased LN risk (OR=1.26; 95% CI: 1.09, 1.46, p=0.0006) as was increasing HLA GRS in Europeans (OR=1.55; 95% CI: 1.07, 2.25; p=0.03). There was a trend for stronger associations between both GRSs and LN risk in Europeans with cSLE compared with aSLE. Conclusions We observed an association between known SLE-risk loci and LN risk in children and adults with SLE, with the strongest effect observed among Europeans with cSLE. Future directions will include incorporating SLE-risk SNPs specific to non-European ancestral groups and validating findings in an independent cohort. Funding Source(s): Dr. Hiraki: Canadian Institute of Health Research (CIHR) Project Scheme grant

Objective.Case ascertainment through self-report is a convenient but often inaccurate method to collect information. The purposes of this study were to develop, assess the sensibility, and validate a tool to identify cases of systemic autoimmune rheumatic diseases (SARD) in the outpatient setting.Methods.The SARD tool was administered to subjects sampled from specialty clinics. Determinants of sensibility — comprehensibility, feasibility, validity, and acceptability — were evaluated using a numeric rating scale from 1–7. Comprehensibility was evaluated using the Flesch Reading Ease and the Flesch-Kincaid Grade Level. Self-reported diagnoses were validated against medical records using Cohen’s κ statistic.Results.There were 141 participants [systemic lupus erythematosus (SLE), systemic sclerosis (SSc), rheumatoid arthritis, Sjögren syndrome (SS), inflammatory myositis (polymyositis/dermatomyositis; PM/DM), and controls] who completed the questionnaire. The Flesch Reading Ease score was 77.1 and the Flesch-Kincaid Grade Level was 4.4. Respondents endorsed (mean ± SD) comprehensibility (6.12 ± 0.92), feasibility (5.94 ± 0.81), validity (5.35 ± 1.10), and acceptability (3.10 ± 2.03). The SARD tool had a sensitivity of 0.91 (95% CI 0.88–0.94) and a specificity of 0.99 (95% CI 0.96–1.00). The agreement between the SARD tool and medical record was κ = 0.82 (95% CI 0.77–0.88). Subgroup analysis by SARD found κ coefficients for SLE to be κ = 0.88 (95% CI 0.79–0.97), SSc κ = 1.0 (95% CI 1.0–1.0), PM/DM κ = 0.72 (95% CI 0.49–0.95), and SS κ = 0.85 (95% CI 0.71–0.99). The screening questions had sensitivity ranging from 0.96 to 1.0 and specificity ranging from 0.88 to 1.0.Conclusion.This SARD case ascertainment tool has demonstrable sensibility and validity. The use of both screening and confirmatory questions confers added accuracy.

ObjectiveLN is one of the most common and severe manifestations of SLE. Our aim was to test the association of SLE risk loci with LN risk in childhood-onset SLE (cSLE) and adult-onset SLE (aSLE).MethodsTwo Toronto-based tertiary care SLE cohorts included cSLE (diagnosed ResultsOf 1237 participants, 572 had cSLE (41% with LN) and 665 had aSLE (30% with LN). Increasing non-HLA GRS was significantly associated with increased LN risk [odds ratio (OR) = 1.26; 95% CI 1.09, 1.46; P = 0.0006], as was increasing HLA GRS in Europeans (OR = 1.55; 95% CI 1.07, 2.25; P = 0.03). There was a trend for stronger associations between both GRSs and LN risk in Europeans with cSLE compared with aSLE. When restricting cases to proliferative LN, the magnitude of these associations increased for both the non-HLA (OR = 1.30; 95% CI 1.10, 1.52; P = 0.002) and HLA GRS (OR = 1.99; 95% CI 1.29, 3.08; P = 0.002).ConclusionWe observed an association between known SLE risk loci and LN risk in children and adults with SLE, with the strongest effect observed among Europeans with cSLE. Future studies will include SLE-risk single nucleotide polymorphisms specific to non-European ancestral groups and validate findings in an independent cohort.

Background Diagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA+ individuals to determine whether they share any of the cellular immunologic features seen in SARD. Methods Healthy ANA− controls and ANA+ (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString. Results A number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA+ subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA+ subjects and subjects with UCTD clustered with patients with early SARD, rather than ANA− healthy controls. Conclusions ANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD. Electronic supplementary material The online version of this article (10.1186/s13075-018-1752-3) contains supplementary material, which is available to authorized users.

Background The diagnosis of Systemic Autoimmune Rheumatic Diseases (SARD), including Systemic Lupus Erythematosus (SLE), relies on the presence of ANAs, many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals most of whom will not develop SARD. A number of cellular immune changes are seen in SARD, and thus could constitute potential biomarkers/treatment targets for SARD, however it is not known at what point in disease progression these develop. Methods Healthy ANA- controls (n=32) and ANA+ (≥1:160 by immunofluorescence) participants with no (asymptomatic ANA+, n=61), at least one (UCTD, n=35), or meeting SARD classification criteria (n=59) were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry. Results Consistent with previous reports, SARD patients had increased proportions of activated B cells (CD86+ or CD95+) and in the SLE patient subset there were increased proportions of plasma cells/plasmablasts, as compared to ANA- controls. SARD patients also had reduced proportions of iNKT and IFN-γ producing cells, as well as, increased proportions of memory Tfh (CD4+CXCR5hiPD1hi) and T regulatory (Treg, CD4+FOXP3+HELIOS+) cells, especially in the SLE and Sjogren’s Disease patient subsets. In asymptomatic ANA+ individuals and UCTD patients, similar increases in the proportion of activated B cells, Tfh, and Treg cells, and decreases in the proportion of iNKT and IFN-γ producing cells were seen to those in SARD. In asymptomatic ANA+ individuals and SARD patients, the extent of serologic changes (number of specific ANAs detected by Bioplex® 2200 ANA screening system) positively correlated with activation in the switched memory B cell compartment and the proportion of Tfh cells, with the later being an independent predictor of serologic status in a multivariate analysis. However, significantly elevated levels of Tfh cells could still be seen in asymptomatic ANA+ individuals who lacked specific ANAs. Consistent with a role for Tfh cell in ANA production there was a strong correlation between the proportion of Tfh and plasma cells in asymptomatic ANA+ individuals. In preliminary studies, the majority of Tfh cells in asymptomatic ANA+ and UCTD patients were Tfh2 cells, with a trend to increased proportions of Tfh2 cells and decreased proportions Tfh17 cells as compared to active SLE patients. Conclusions Tfh cells appear to play an important role in the development of a positive ANA and in the epitope spreading that may accompany disease progression, and therefore constitute a promising target for treatment of early disease.

Background Cognitive impairment (CI) is a common neurobehavioural manifestation of systemic lupus erythematosus (SLE). In our recent systematic review, the prevalence of CI was 38% (95% CI 33% to 43%) with a wide variation (15%–79%), which may be due to differences in CI definitions and selection of neuropsychological tests across studies. We aim to report the prevalence of CI in a large cohort using a comprehensive battery (CB) of tests in which we operationalized the classification of CI. Methods Consecutive consenting SLE patients, aged 18–65 years, who attended a single center (Jul 2016-Feb 2018) were recruited. Patients were administered a CB that evaluates the major cognitive domains: Manual motor speed and dexterity, simple attention and processing speed, visual-spatial construction, verbal fluency, learning and memory (visuospatial and memory), executive functioning (untimed and timed). Patient scores were compared to a normative sample of age- and gender-matched healthy controls to obtain z-scores. CI was operationalized on the CB as a z-score of ≤−1.5 (as compared to controls) on ≥2 domains or z≤−2.0 on ≥1 domain. Descriptive statistics were used. Results Of the 199 patients (89% female), the mean age at SLE diagnosis was 28.3±10.4 and disease duration at enrolment was 14.3±10.2 years. The prevalence of CI was 37.7% (z≤−1.5 in ≥2 domains) and 49.8% (z≤−2.0 in ≥1 domains). Prevalence of patients with domain z-scores of ≤−1.5 and ≤−2.0 varied from 3.0%–46.2% and 0.5%–25.1% respectively (figure 1). The most affected domain was learning and memory (visuospatial and memory) in 92 (46.2%) patients based on z≤−1.5 on ≥2 subtests and 50 (25.1%) patients based on z≤−2.0 in ≥1 subtest. Conclusion Prevalence of CI using our CB ranged between 37.7%–49.8% (z≤−1.5 in≥2 domains and z≤−2.0 in≥1 domains respectively), which was higher than the pooled prevalence from previous reports of 38%. These differences in CI prevalence across studies could be attributed to different factors including the heterogeneity in patients’ demographics/comorbidities, sample size, the use of different metrics to determine CI, and the lack of a standardized definition of CI. Further studies are required to identify the best definition for CI and its metrics. Acknowledgment This project is funded by the Arthritis Society and the Physicians’ Services Incorporated Foundation.

Objective.Serologically active clinically quiescent (SACQ) patients with systemic lupus erythematosus (SLE) are clinically quiescent despite serologic activity. Since studies suggest that antichromatin antibodies are more sensitive than anti-dsDNA antibodies in detecting active SLE, and that immunoglobulin (Ig) G, in particular complement-fixing subclasses, may be more pathogenic than IgM, we investigated the levels of anti-dsDNA and antichromatin isotypes in SACQ patients as compared to non-SACQ patients with SLE.Methods.Levels of IgM, IgA, IgG, and IgG1–4 antichromatin and anti-dsDNA were measured by ELISA. SACQ was defined as ≥ 2 years with the SLE Disease Activity Index 2000 (SLEDAI-2K) at 2 or 4 from serologic activity, during which patients could be taking antimalarials, but not corticosteroids or immunosuppressives. Unselected non-SACQ patients with SLE were used as comparators. SACQ patient serum samples were further stratified based on subsequent development of flare, defined as clinical SLEDAI-2K ≥ 1 and/or treatment initiation. Nonparametric statistics were used, and generalized estimating equations were applied to account for multiple samples in the same patient.Results.SACQ patients’ complement-fixing antichromatin and anti–dsDNA IgG subclasses were significantly higher than those of non-SACQ patients. When the sample drawn latest in a SACQ period was analyzed, there was no difference between antichromatin or anti-dsDNA isotype or IgG subclass levels between patients who flared and those who remained SACQ, nor were consistent trends seen when samples were examined during SACQ and flare in the same patient.Conclusion.The SACQ phenotype does not arise from a lack of pathogenic anti-dsDNA and/or antichromatin autoantibodies. Neither increases in antichromatin nor anti-dsDNA isotype or IgG subclass levels were predictive of or coincident with flare in SACQ patients.

Both a lack of biomarkers and relatively ineffective treatments constitute impediments to management of lupus nephritis (LN). Here we used gene expression microarrays to contrast the transcriptomic profiles of active SLE patients with and without LN to identify potential biomarkers for this condition. RNA isolated from whole peripheral blood of active SLE patients was used for transcriptomic profiling and the data analyzed by linear modeling, with corrections for multiple testing. Results were validated in a second cohort of SLE patients, using NanoString technology. The majority of genes demonstrating altered transcript abundance between patients with and without LN were neutrophil-related. Findings in the validation cohort confirmed this observation and showed that levels of RNA abundance in renal remission were similar to active patients without LN. In secondary analyses, RNA abundance correlated with disease activity, hematuria and proteinuria, but not renal biopsy changes. As abundance levels of the individual transcripts correlated strongly with each other, a composite neutrophil score was generated by summing all levels before examining additional correlations. There was a modest correlation between the neutrophil score and the blood neutrophil count, which was largely driven by the dose of glucocorticosteroids and not the proportion of low density and/or activated neutrophils. Analysis of longitudinal data revealed no correlation between baseline neutrophil score or changes over the first year of follow-up with subsequent renal flare or treatment outcomes, respectively. The findings argue that although the neutrophil score is associated with LN, its clinical utility as a biomarker may be limited.

Objective.The occurrence of monoclonal gammopathy of undetermined significance (MGUS) is common in chronic immune mediated disorders. This increased monoclonal antibody production could result from chronic stimulation of lymphocytes, with the immunoglobulin G (IgG) subtype accounting for the majority of cases in psoriatic arthritis (PsA). We aimed to identify IgG subclass profiles in patients with PsA and to determine association with specific disease characteristics.Methods.Serum samples from 221 patients with PsA from a single cohort were analyzed for their serum IgG subclass levels. All patients fulfilled the ClASsification for Psoriatic ARthritis (CASPAR) criteria and were followed at 6-month to 12-month intervals according to a standard protocol. MGUS was defined as the occurrence of a discrete band in the gammaglobulin region on at least 2 separate serum protein electrophoresis tests performed 6 months apart. Patients with high abnormal IgG subclass levels were compared to patients with normal levels using descriptive tests.Results.Elevations of IgG1-4 were common in PsA, with ∼20%–49% of patients having elevations of each subclass, IgG2 being the most common subclass abnormality. However, no clinical-serological correlation was found in the group with abnormal IgG2 levels. Of the 38 patients with MGUS, elevations in IgG1 were most common. Patients with an abnormal IgG1 subclass level were more likely to have a discrete band in the gammaglobulin region, higher prevalence of MGUS, and abnormal erythrocyte sedimentation rate or C-reactive protein levels.Conclusion.Determination of the IgG subclass concentration in PsA did not seem to add any significant value in identifying specific disease manifestations. However, this study provides insight into the pathological process leading to MGUS in PsA.

Objective. The aim of this study was to determine whether anti-nucleosome antibodies function as activity-specific biomarkers in SLE. Methods. Fifty-one patients were recruited and followed prospectively with periodic clinical and biochemical assessments over a 14-month period. Disease activity was determined by the SLEDAI-2K. Anti-nucleosome antibody levels were measured by an ELISA and its utility as an activity-specific biomarker as compared with that of anti-dsDNA antibodies and C3 was assessed both at baseline and in longitudinal analysis. Results. Anti-nucleosome antibodies were significantly elevated in SLE patients vs controls and showed a moderate positive correlation with disease activity. The utility of anti-nucleosome antibodies in identifying patients with active disease in a cross-sectional analysis was comparable to that of anti-dsDNA antibodies and C3. Analysis of variance demonstrated that the level of anti-nucleosome antibodies and C3 varied significantly with changes in disease activity over time. Changes in clinical state were not mirrored by changes in anti-dsDNA antibodies. In time-dependent analysis, anti-nucleosome antibodies showed a better fit over time than anti-dsDNA antibodies and C3. In pairwise comparisons, C3 and anti-nucleosome antibodies outperformed other models, including the conventional pairing of C3 and anti-dsDNA antibodies, however, no biomarker alone or as a group accurately predicted impending remissions or exacerbations. Conclusion. Anti-nucleosome antibodies demonstrate greater fidelity as a biomarker for changes in SLE disease activity than traditional biomarkers, supporting the routine monitoring of this antibody in clinical practice.

The International Society of Nephrology/Renal Pathology Society (ISN/RPS) lupus nephritis (LN) classification is under reconsideration, given challenges with inter-rater reliability and resultant inconsistent relationship with treatment response. Integration of molecular classifiers into histologic evaluation can improve diagnostic precision and identify therapeutic targets. This study described the relationship between histological and molecular phenotypes and clinical responses in LN. Renal compartmental mRNA abundance was measured in 54 biopsy specimens from LN patients and correlated to ISN/RPS classification and individual histologic lesions. A subset of transcripts was also evaluated in sequential biopsies of a separate longitudinal cohort of 36 patients with paired samples obtained at the time of flare and at follow up. Unsupervised clustering based on mRNA abundance did not demonstrate a relationship with the (ISN/RPS) classification, nor did univariate statistical analysis. Exploratory analyses suggested a correlation with individual histologic lesions. Glomerular FN1 (fibronectin), SPP1 (secreted phosphoprotein 1), and LGALS3 (galectin 3) abundance correlated with disease activity and changed following treatment. Exploratory analyses suggested relationships between specific transcripts and individual histologic lesions, with the important representation of interferon-regulated genes. Our findings suggested that the current LN classification could be refined by the inclusion of molecular descriptors. Combining molecular and pathologic kidney biopsy phenotypes may hold promise to better classify disease and identify actionable treatment targets and merits further exploration in larger cohorts.

The objective of this study was to determine the clinical significance of renal vascular lesions in lupus nephritis.Renal vascular lesions defined as thrombotic microangiopathy, lupus vasculopathy, uncomplicated vascular immune deposits, and arterial sclerosis were evaluated in relation to renal and vascular morbidity and overall mortality.Biopsies from 161 patients revealed thrombotic microangiopathy (13), lupus vasculopathy (5), and arterial sclerosis (93). No renal vascular lesions were found in 24.8% of patients. At the time of biopsy, arterial sclerosis or lupus vasculopathy patients were older (arterial sclerosis=37.9±13.0 and lupus vasculopathy=44.4±8.9 versus controls=33.1±8.9 years, P0.05), and the mean arterial pressure was higher in all groups compared with controls. Nephritis subtype, activity indices, and proteinuria were similar between groups, estimated GFR was lower in arterial sclerosis (70.5±33.3 versus 84.5±26.6 ml/min per 1.73 m(2), P=0.03), and chronicity index (thrombotic microangiopathy=3.5, lupus vasculopathy=4.5, and arterial sclerosis=2.5) was higher in all renal vascular lesions subgroups versus controls (1.0, P0.05). In 133 patients with similar follow-up, the association between renal vascular lesions and vascular events was significant (Fisher exact test, P=0.002) and remained so after multivariate analysis (exact conditional scores test, P=0.04), where the difference between arterial sclerosis and uncomplicated vascular immune deposits was most noticeable (odds ratio [95% confidence interval]=8.35[0.98, 83.12], P=0.05). The associations between renal vascular lesions, renal outcomes, and death were not significant, likely because of insufficient power.Renal vascular lesions are common in SLE patients with nephritis and may be associated with arterial vascular events.

Objective.Associations between the use of micronutrient supplements (MS) and disease activity, quality of life (QOL), and healthcare resource utilization were studied in a Canadian population of patients with systemic lupus erythematosus (SLE).Methods.QOL was assessed by the Medical Outcomes Study 36-item Short Form. Healthcare resource utilization and disease activity/damage were determined.Results.Of the 259 subjects studied, 53% were MS users and 34% used only calcium/vitamin D. MS users had a higher Systemic Lupus International Collaborating Clinics score and utilized more healthcare resources. Disease activity and QOL were similar between MS users and nonusers.Conclusion.MS are frequently used by patients with SLE and are not associated with concomitant benefit on QOL. MS users utilized more healthcare resources.

Objective:To study the longitudinal expression of interferon (IFN)-inducible genes in systemic lupus erythematosus (SLE) and determine their suitability as disease biomarkers.Methods:RNA was isolated from the peripheral blood of 94 patients with SLE and 11 controls and reverse transcribed into cDNA. The expression levels of five IFN-responsive genes (LY6E, OAS1, IFIT1, ISG15 and MX1) were determined by quantitative PCR, normalised to GAPDH and summed to generate a global IFN score. Patients were followed longitudinally for a period of 3–12 months, and the association between disease activity, as measured by the SLE disease activity index (SLEDAI-2K), and other clinical and laboratory variables was examined.Results:The expression of all five IFN-responsive genes was significantly higher in patients with SLE than in controls. The expression of LY6E, OAS1, IFIT1 and the global IFN score was associated with high disease activity. The global IFN score was also associated with active renal disease, a decreased C3, and the presence of anti-dsDNA or anti-RNA binding protein antibodies at a single point in time. However, there was a poor correlation between changes in this score and changes in disease activity, C3 or anti-dsDNA antibody levels in patients followed longitudinally. In most patients the levels of IFN-induced gene expression remained relatively stable over 3–12 months despite marked changes in disease activity. Nevertheless, in patients with low/moderate disease activity, those with high IFN scores had a more recent history of sustained high disease activity.Conclusion:The findings indicate that IFN-induced gene expression has limited clinical utility as a biomarker of acute changes in disease activity.

Background Patients with systemic autoimmune rheumatic diseases (SARD) often have a prolonged pre-clinical phase during which they are anti-nuclear antibody (ANA)+ but lack clinical symptoms. It has been proposed that progression from asymptomatic autoimmunity to clinical disease is accompanied by immunologic changes that could be used as predictors of disease development. Previous studies indicate that a number of B cell phenotypic changes are seen in SARD patients including changes in the proportions of various naive and memory B cell subsets, increased B cell activation and elevated levels of plasmablasts/plasma cells. Objectives To determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis have B cell phenotypic changes similar to those seen in SARD. Methods Healthy controls (HC) and ANA+ individuals who: 1) lacked clinical symptoms of SARD (ANS); 2) had at least one clinical symptom of SARD (UCTD); or 3) had a recently diagnosed steroid and immunosuppressive naive SARD (SLE, SS, SSc, MCTD, DM) were recruited. PBMCs were isolated over a Ficoll gradient, stained with various combinations of fluorescently labeled antibodies and analyzed by flow cytometry. Anti-nuclear antibodies were measured through the hospital laboratory. Whole blood IFN signature and BAFF RNA levels were measured by NanoString. Results B cell phenotypes were examined for 32 HC, 38 ANS, 28 UCTD, and 59 early SARD (24 SS, 26 SSc, 6 SLE, 2 MCTD, 1 DM) patients. Patients with early SARD had a number of changes in their naive and memory B cell subsets, as previously reported for patients with established disease, including: increased proportions of mature naive B cells (SSc); increased proportions of T1T2 cells (SLE and SS); and trends to decreased proportions of switched memory B cells (CD27+IgD–) in all SARD. Similar decreases in the proportion of switched memory B cells were seen in ANS and UCTD patients, and as seen for the SARD patients, these cells were activated with elevated levels of CD86 as compared to HC. Significantly increased activation of the CD27–IgD– memory compartment was also seen in ANS, UCTD, SLE and SjD patients. Although significantly increased proportions of plasmablasts and/or CD138+ plasma cells were seen in all SARD patients (except those with SSc), these were not seen in ANS and UCTD patients. Nevertheless, in pre-SARD individuals (ANS + UCTD) there was a significant positive correlation between the size of these cell subsets, as well as the proportion of T1T2 cells, and ANA titer and the number of different anti-nuclear antibodies specificities. As observed for early SARD patients, there was a trend to increased BAFF levels as compared to HC in pre-SARD individuals, which achieved statistical significance in UCTD patients. However, there was no association between the levels of BAFF and any of the B cell phenotypes, whereas the IFN signature was positively associated with the proportion of T1T2 cells. Conclusions B cell phenotypic abnormalities precede the onset of clinical disease in ANA+ individuals and have a pattern suggesting ongoing activation through T-B collaboration. Disclosure of Interest None declared

Background The relapsing and remitting nature of lupus nephritis (LN) poses a challenge to clinicians who must balance the risk of long term kidney damage with the side effects of treatment. Management of this condition would be greatly aided by the identification of biomarkers that accurately reflect and predict treatment responses. We previously identified and validated a panel of urinary biomarkers that are specifically elevated in SLE patients with active LN as compared to active patients without LN or LN patients in remission. Objectives To determine whether changes in the levels of these urinary biomarkers predict treatment responses. Methods 21 SLE patients with biopsy-proven LN were followed longitudinally for a minimum of 2 years after treatment. Levels of 15 urinary biomarkers including Clusterin, Cystatin C, NGAL, PF4, vWF,sVCAM-1, GM-CSF, GRO, IL-15, IL-6, MCP-1, Adiponectin, PAI-1, MMP-7, and TIMP-1, were measured by Luminex. Patients were classified as having a complete response (n=12), partial response (n=4), or treatment (Tx) failure (n=5) at 2 years following initiation of treatment, based upon previously established criteria. Urinary biomarker levels were considered abnormal if they were >2 SD above the mean for 24 healthy controls. Data were analyzed using non-parametric statistics. Results At 3–6 months following treatment, the changes in biomarker levels from the first visit were not significantly different between complete responders and partial responders or Tx failures. However, at 1 year (11–16 months) following treatment, 5 urinary biomarkers, sVCAM-1, Adiponectin, IL-15, vWF, and MCP-1, demonstrated significantly different changes from baseline in complete responders as compared to Tx failures, with the majority of responders demonstrating improvement and the majority of Tx failures demonstrating worsening. As urinary biomarker levels not corrected for urinary osmolality correlated best with clinical outcomes, subsequent analyses were done with the uncorrected values. Normalization of urinary Adiponectin by 11–16 months was the best predictor of a complete response, with 11 of 12 patients who normalized being complete responders and 1 a partial responder. Similar but slightly less discriminative results were obtained for the other 4 urinary biomarkers. Conversely, the presence of an abnormal urinary vWF at 11–16 months was the strongest predictor of an adverse outcome with 4 of 6 patients with abnormal levels being Tx failures. Notably, all patients that were Tx failures that had normal levels of urinary biomarkers at 11–16 months subsequently developed abnormal levels, whereas patients who were complete responders eventually normalized the majority of these 5 biomarkers. Partial responders demonstrated normalization with delayed kinetics. Conclusions Measurement of urinary biomarkers can provide valuable insight into treatment responses in lupus nephritis. Disclosure of Interest None declared

Advances in immunotherapy have improved survival of patients with systemic lupus erythematosus who now face an increasing burden of chronic diseases including that of the kidney. As systemic inflammation is also thought to contribute directly to the progression of chronic kidney disease (CKD), we assessed this risk in patients with lupus, with and without a diagnosis of nephritis, and also identified modifiable risk factors. Accordingly, we enrolled 631 patients (predominantly Caucasian), of whom 504 were diagnosed with lupus within the first year and followed them an average of 11 years. Despite the presence of a chronic inflammatory disease, the rate of decline in renal function of 238 patients without nephritis was similar to that described for non-lupus patient cohorts. Progressive loss of kidney function developed exclusively in patients with lupus nephritis who had persistent proteinuria and dyslipidemia, although only six required dialysis or transplantation. The mortality rate was 16% with half of the deaths attributable to sepsis or cancer. Thus, despite the presence of a systemic inflammatory disease, the risk of progressive CKD in this lupus cohort was relatively low in the absence of nephritis. Hence, as in idiopathic glomerular disease, persistent proteinuria and dyslipidemia (modifiable risks) are the major factors for CKD progression in lupus patients with renal involvement.

Objective.To examine the relationship between interferon-α (IFN-α) and dysregulation of B cell activation factor (BAFF) and specific B cell phenotypes in systemic lupus erythematosus (SLE).Methods.Four-color flow cytometry was used to examine the peripheral B cell populations in patients with SLE. RNA was isolated from the peripheral blood of 87 patients and BAFF expression was determined by quantitative polymerase chain reaction (PCR) and normalized to GAPDH. The expression levels of 5 IFN-responsive genes (LY6E, OAS1, IFIT1, ISG15, and MX1) were determined by quantitative PCR and totaled to generate a global IFN score. The correlations were examined between peripheral B cell populations (including transitional, pregerminal, plasmablasts, and memory) and the expression of BAFF and the global IFN score.Results.Examination of the peripheral B cell populations in SLE demonstrated a relative expansion of the transitional B cell and plasmablast compartment and a reduction in the memory B cell population. Expressions of BAFF and global IFN score were elevated in patients with SLE compared to healthy controls. A strong positive correlation was noted between BAFF expression and the relative proportion of late transitional (T2) B cells. The proportions of more mature B cell phenotypes did not correlate with BAFF expression. The global IFN score was strongly associated with the level of BAFF expression and moderately correlated with the proportion of late transitional B cells.Conclusion.The findings suggest that elevated BAFF expression supports expansion of the T2 B cell compartment and contributes to a breach in tolerance in patients with SLE.

Objective.To compare the healthcare cost and loss of productivity in patients with systemic lupus erythematosus (SLE) with (LN) and without lupus nephritis (lupus nephritis-negative, LNN).Method.Patients were classified into those with active (ALN and ALNN) and inactive disease (ILN and ILNN). Patients reported on visits to healthcare professionals and use of diagnostic tests, medications, assistive devices, alternative treatments, hospital emergency visits, surgical procedures, and hospitalizations as well as loss of productivity in the 4 weeks preceding enrollment.Results.Enrollment was 141 patients, 79 with LN and 62 LNN. Patients with LN were more likely to visit rheumatologists and nephrologists, undergo diagnostic tests, and had higher costs for medications than patients who were LNN. The annual healthcare cost averaged $CAN 12,597 ± 9946 for patients with LN and $10,585 ± 13,149 for patients who were LNN, a difference of $2012 (95% CI –$2075, $6100). Patients with ALN had more diagnostic tests and surgical procedures, contributing to a significantly higher annual direct cost ($14,224 ± 10,265) compared to patients with ILN ($9142 ± 8419) and a difference of $5082 (95% CI $591, $9573). The healthcare cost was not different between patients with ALNN and patients with ILNN. In patients with LN and patients who were LNN, < 50% were employed and on average missed 6.5–9 days of work per month. The loss of productivity was significantly higher for caregivers of patients with LN than caregivers of patients who were LNN.Conclusion.Healthcare cost and loss of productivity were similar between patients with LN and patients who were LNN; the loss of productivity for caregivers is higher for patients with LN; and the healthcare cost is greater in ALN than in ILN.

Objectives. We examined occupational and non-occupational exposures in relation to risk of SLE in a case–control study conducted through the Canadian Network for Improved Outcomes in SLE (CaNIOS). Methods. SLE cases (n = 258) were recruited from 11 rheumatology centres across Canada. Controls (without SLE, n = 263) were randomly selected from phone number listings and matched to cases by age, sex and area of residence. Data were collected using a structured telephone interview. Results. An association was seen with outdoor work in the 12 months preceding diagnosis [odds ratio (OR) 2.0; 95% CI 1.1, 3.8]; effect modification by sun reaction was suggested, with the strongest effect among people who reported reacting to midday sun with a blistering sunburn or a rash (OR 7.9; 95% CI 0.97, 64.7). Relatively strong but imprecise associations were seen with work as an artist working with paints, dyes or developing film (OR 3.9; 95% CI 1.3, 12.3) and work that included applying nail polish or nail applications (OR 10.2; 95% CI 1.3, 81.5). Patients were more likely than controls to report participation in pottery or ceramics work as a leisure activity, with an increased risk among individuals with a total frequency of at least 26 days (OR 2.1; 95% CI 1.1, 3.9). Analyses of potential respirable silica exposures suggested an exposure–response gradient (OR 1.0, 1.4. and 2.1 for zero, one and two or more sources of exposure, respectively; trend test P

Objective.To investigate the expression of tumor necrosis factor receptors (TNFR1 and TNFR2) and adapter proteins (TRADD, RIP, and TRAF2) in peripheral blood mononuclear cell (PBMC) subsets from patients with systemic lupus erythematosus (SLE).Methods.PBMC were isolated from 45 SLE patients and 25 controls, and stained with labeled antibodies that enabled identification of various T cell, B cell, and monocyte subpopulations. Expression of TNF-related signaling molecules was measured by staining with labeled antibodies either directly or following fixation and permeabilization. Apoptosis was quantified using an anti-active caspase 3 antibody. RNA expression of TNF-related signaling molecules was assessed by quantitative RT-PCR and serum levels of TNF-α by ELISA.Results.SLE patients had increased levels of TNFR1, TNFR2, and TRAF2, together with decreased levels of RIP, on various B, CD4+ T, and CD8+ T cell subsets as compared to controls. This altered expression was seen in both naive and memory subpopulations, and reflected altered staining of the whole population rather than a subset of cells that were activated. The levels of these molecules were not significantly correlated with serum TNF-α levels or their RNA expression in whole peripheral blood. TNFR1 and TNFR2 expression was negatively correlated with disease activity. There was no association between the proportion of apoptotic cells in any of the subpopulations and serum TNF-α levels or expression of TNF-related signaling molecules.Conclusion.Patients with SLE had altered expression of TNF-related signaling molecules, suggesting that there may be an imbalance in TNF-α signaling favoring cellular activation as opposed to proapoptotic pathways.

Objective.Studies of human inflammatory arthritis would be significantly aided by the development of better animal models. Our hypothesis is that it is possible to develop humanized arthritis models through novel techniques of hematopoietic stem and progenitor cell (HSPC) delivery.Methods.Bone marrow was obtained from patients with osteoarthritis who were undergoing total hip replacement. HSPC were enriched by negative selection and injected into the femur of irradiated anti-CD122 treated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Human cell engraftment was analyzed by flow cytometry. Arthritis was induced by an intraarticular injection of Chlamydia trachomatis and injected knee joints were examined 5 days later by histology and immunohistochemistry.Results.Human bone marrow HSPC successfully engrafted NOD/SCID mice, with some mice showing up to 90% engrafted human cells. Human B lymphoid and myeloid cells were detected in the bone marrow and spleen 6 weeks following transfer of HSPC, and engrafted recipient mice remained healthy up to 12 weeks postinjection. Chlamydia-injected mice that had been repopulated with HSPC had synovial inflammation, consisting of human neutrophils and macrophages.Conclusion.Bone marrow-derived human HSPC engraft NOD/SCID mice and traffic appropriately to an inflammatory stimulus in the joint, thus offering the potential for direct studies on the immunopathogenesis and treatment of human arthritis.

Background The systemic autoimmune rheumatic diseases (SARD; Systemic Lupus Erythematosus, Rheumatoid Arthritis, Sjogren9s Disease, Systemic Sclerosis) are proposed to have a prolonged period of pre-clinical autoimmunity culminating in clinical disease. Evidence from disease-specific studies (e.g., SLE, RA) suggests that the pre-clinical phase is marked by the accrual of immunological abnormalities such as pathogenic auto-antibodies (auto-Ab). Little is known about the cellular derangements that accompany the transition from benign to pathological autoimmunity. Abnormalities in T cell subsets including invariant NKT (iNKT) and T follicular helper (T FH ) cells are implicated in the development of systemic autoimmunity. Both a decrease in iNKT cells and an increase in T FH cells are found in patients with established SARD. Objectives To determine if T cell abnormalities associated with SARD are present in pre-clinical autoimmunity. Methods Patients (n=87) who were ANA + (titer ≥1:160) and healthy controls (HC, n=37) were recruited. Patients were stratified into clinical subsets: (1) no defining SARD symptoms (n=24); (2) undifferentiated connective tissue disease (UCTD, n=17), one or more SARD defining symptom; and (3) early SARD (n=46), fulfilling ACR criteria for a SARD diagnosis. Patients were immunosuppressive and steroid naive. PBMCs were isolated over a Ficoll gradient, stained with combinations of fluorescently labeled antibodies and analyzed by flow cytometry. ANA titer and auto-Ab profile were determined. Results A statistically significant decrease in the proportion of iNKT cells (CD3 + Vα24Jα18 TCR + ) was found for all ANA + patients relative to HC (p=0.0001). Similar results were found for each patient subset when compared to HC; ANA + asymptomatic (p=0.001), UCTD (p=0.02) and SARD (p=0.001), with no differences amongst groups. The proportion of T FH (CD4 + CXCR5 high PD-1 high ) cells was significantly elevated (p=0.01) in patients versus HC. While the proportion of T FH cells was similar between asymptomatic ANA + patients and HC, there was a significant expansion of T FH in UCTD as compared to both groups (p=0.02 and p=0.04, respectively). SARD patients had a non-significant trend to increased proportions of T FH as compared to UCTD. Patients (n=50) with higher ANA titers (≥1:640) had a significant increase (p=001) in T FH when compared to individuals (n=13) with lower ANA levels (1:160). A positive correlation (p FH and the number of auto-Abs within the patient population was found. No significant differences were noted in the iNKT or T FH cell proportions between SARD patients stratified by specific disease diagnosis. Conclusions A decrease in the iNKT cell subset is present at the earliest phase of pre-clinical autoimmunity (asymptomatic ANA + ) suggesting that loss of this T cell subset contributes to a breach of tolerance to nuclear antigens. In contrast, increases in the T FH compartment appear to parallel the onset of clinical symptoms and accrual of auto-Abs. These results suggest that an incremental development of T cell abnormalities marks the progression towards clinically significant autoimmunity. Disclosure of Interest None declared

Background Patients with systemic autoimmune rheumatic diseases (SARD) often have a prolonged pre-clinical phase during which they are anti-nuclear antibody (ANA)+ but lack clinical symptoms. It has been proposed that progression from asymptomatic autoimmunity to clinical disease is accompanied by immunologic changes that could be used as predictors of disease development. Elevated levels of interferon (IFN)-induced gene expression, termed the IFN signature, are found in several SARD conditions, and IFNs appear to play an important role in disease pathogenesis. Objectives To determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis share the IFN-signature. Methods ANA+ individuals who: 1) lacked clinical symptoms of SARD (ANS); 2) had a least one clinical symptom of SARD (Undifferentiated Connective Tissue Disease, UCTD); or 3) had a recently diagnosed SARD (Systemic Lupus Erythematosus, SLE; Sjogren9s Disease, SjD; Scleroderma, SSc; Mixed Connective Tissue Disease, MCTD; Dermatomyositis, DM) were recruited from clinics at UHN/MSH hospitals. None of the patients were on corticosteroids or DMARDs with the exception of hydroxychloroquine. Healthy controls (HC) were also recruited. RNA was prepared from blood archived in Tempus tubes. Expression of 5 IFN-induced genes was quantified by Nanostring, normalized to expression of housekeeping genes, and summed to generate an IFN5 score. ANAs and levels of specific autoantibodies were measured by the hospital laboratory. Results To date we have measured the IFN signature on 95 individuals (21 HC, 21 ANS, 16 UCTD, 22 SjD, 7 SSc, 6 SLE, 1 MCTD, 1 DM). There was a trend to higher mean IFN score in all groups as compared to HC (mean ± SD: HC 7,071±6,321; ANS 27,245±36,037; UCTD 27,624±24,827; SSc 34,940±40,940; SjD 61,877±33,404; SLE 62,769±50,233; MCTD/DM 97,716±24,973), which achieved statistical significance for ANS, UCTD, SjD, and SLE (corrected p=0.044, 0.003, Conclusions An IFN signature is seen in a subset of ANA+ individuals prior to a confirmed diagnosis of SARD and appears to correlate with the type and number of specific ANAs rather than onset of clinical disease. Disclosure of Interest None declared

Background Nephritis is a major cause of morbidity and mortality in SLE. One of impediments to optimal management of this condition is a lack of biomarkers that forecast development of renal disease or that can be used to determine the response to therapy. Objectives In this study we used gene expression microarrays to contrast the gene expression profile in active lupus patients with and without nephritis to identify potential biomarkers and immune mechanisms associated with nephritis. Methods Affymetrix Human Gene 2.0ST arrays were used to assay gene expression in total RNA isolated from blood archived in PAXgene tubes for 38 active SLE patients with ≥4 ACR SLE classification criteria (25 biopsy proven nephritis, 13 active without nephritis) and 17 healthy controls. Data were analyzed with linear modeling, with corrections for multiple testing. Results were validated in a largely independent cohort of 22 healthy controls and 170 SLE patients (129 active (89 with and 40 without nephritis), 41 nephritis in remission), using Nanostring technology. Results Comparison of gene expression between healthy controls and all SLE patients revealed 27 genes that were increased >1 log 2 fold-change in SLE, the majority of which (25) were IFN-induced. When active SLE patients with and without nephritis were compared, there were 25 probes that demonstrated a >1 log 2 fold-change with a false discovery rate of q Conclusions Patients with lupus nephritis have higher levels of LDG gene expression than those without nephritis. Given the high rate of spontaneous NETosis in this cell population, the findings are consistent with the concept that high levels of NETosing neutrophils promote renal disease in SLE. Disclosure of Interest J. Wither Grant/research support from: CIHR and Eli Lilly and Company, S. Prokopec: None declared, B. Noamani: None declared, D. Bonilla: None declared, Z. Touma: None declared, H. Reich: None declared, J. Scholey: None declared, P. Fortin Grant/research support from: CIHR and Eli Lilly and Company, P. Boutros: None declared, C. Landolt-Marticorena: None declared

Background Management of lupus nephritis (LN) would be greatly aided by the discovery of biomarkers that accurately reflect changes in disease activity. Here, we used a proteomics approach to identify potential urinary biomarkers associated with LN. Methods Urine was obtained from 60 LN patients with paired renal biopsies, 25 active non-LN SLE patients, and 24 healthy controls. Using Luminex, 128 analytes were quantified and normalized to urinary creatinine levels. Data were analyzed by linear modeling and non-parametric statistics, with corrections for multiple comparisons. A second cohort of 33 active LN, 16 active non-LN, and 30 remission LN SLE patients was used to validate the results. Results Forty-four analytes were identified that were significantly increased in active LN as compared to active non-LN. This included a number of unique proteins (e.g., TIMP-1, PAI-1, PF4, vWF, and IL-15) as well as known candidate LN biomarkers (e.g., adiponectin, sVCAM-1, and IL-6), that differed markedly (>4-fold) between active LN and non-LN, all of which were confirmed in the validation cohort and normalized in remission LN patients. These proteins demonstrated an enhanced ability to discriminate between active LN and non-LN patients over several previously reported biomarkers. Ten proteins were found to significantly correlate with the activity score on renal biopsy, eight of which strongly discriminated between active proliferative and non-proliferative/chronic renal lesions. Conclusions A number of promising urinary biomarkers that correlate with the presence of active renal disease and/or renal biopsy changes were identified and appear to outperform many of the existing proposed biomarkers. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1120-0) contains supplementary material, which is available to authorized users.

Background Elevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody–positive (ANA+) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA+ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question. Methods Healthy ANA− control subjects and ANA+ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score. Results The mean IFN5 scores were increased in all ANA+ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA+ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA+ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA+ subset, this score also correlated with the ANA titre, whereas in the other ANA+ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores. Conclusions An IFN signature is seen in a significant proportion of ANA+ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.