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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.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

BackgroundThe diagnosis of cognitive impairment (CI) is often delayed requiring use of a comprehensive battery (CB). The Automated Neuropsychological Assessment Metrics (ANAM) is a computerised tool that can be used to screen for CI.ObjectivesTo determine the ability of ANAM (v4) GNS Battery to predict CI in patients with systemic lupus erythematosus (SLE).MethodsSLE patients (n=98), aged 18–65 years, attending a single centre between July 2016-April 2017 were recruited. Participants were administered the ANAM and CB on the same day. ANAM throughput scores were used to provide an estimate of cognitive efficiency. Patient scores on the ANAM and CB were compared to a normative sample of age and gender-matched healthy controls. The CB evaluates the following major cognitive domains: manual motor speed and dexterity, simple attention and processing speed, visual-spatial construction, verbal fluency, learning and memory (visuospatial and memory), and executive functioning (untimed and timed). ANAM evaluates the following major cognitive domains: attention and processing speed, memory, visual-spatial processing, executive functioning, abstract language function and fine motor processing. CI was operationalized on the CB and ANAM as a z-score of ≤−1.5 on≥2 domains or a z-score ≤−2.0 on≥1 domains, or either.The performance of ANAM was compared against the CB using different CI definitions. Descriptive analysis was used to determine prevalence, sensitivity (Sn), specificity (Sp), Positive Predictive Value (PPV) and Negative Predictive Value (NPV).ResultsOf the 98 patients (90.8% female), the mean age at SLE diagnosis was 28.5±10.2 and disease duration at enrolment was 15.5±10.0 years. Prevalence of CI using CB ranged between 40.0%–44.8% (z≤−1.5 in≥2 domains and z≤−2.0 in≥1 domains, respectively) and 55.2% for either. Prevalence of CI using the ANAM ranged between 30.8%–39.3%% (z≤−1.5 in≥2 domains and z≤−2.0 in≥1 domains, respectively) and 43.0% for either.ANAM Sn/Sp was 52/73% and PPV/NPV was 70/55% [based on z≤−1.5 in≥2 domains or z≤−2.0 in≥1 domains for ANAM and CB (corresponding for A+B and E+F in table 1)].Abstract SAT0418 – Table 1Performance of ANAM against the CB Definitions of CIComprehensive Battery (CB) z≤−1.5 in≥2 domainsz≤−2.0 in≥1 domains E Sn/SpPPV/NNVF Sn/SpPPV/NNVE+F Sn/SpPPV/NNV ANAMz≤−1.5 in≥2 domainsA45/81%62/68%37/76%55/61%37/80%69/51%z≤−2.0 in≥1 domainsB55/76%61/71%49/72%58/65%50/80%75/57%A+B55/69%55/69%51/67%55/64%52/73%70/55%Sn sensitivity; Sp specificity; PPV Positive Predictive Value/NPV Negative Predictive ValueConclusionsANAM is a promising tool for the assessment of CI in SLE. Future studies are required to determine if the sensitivity of the ANAM can be improved against the current CB.Disclosure of InterestNone declared

BackgroundPatients 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 naïve and memory B cell subsets, increased B cell activation and elevated levels of plasmablasts/plasma cells.ObjectivesTo determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis have B cell phenotypic changes similar to those seen in SARD.MethodsHealthy 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 naïve 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.ResultsB 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 naïve and memory B cell subsets, as previously reported for patients with established disease, including: increased proportions of mature naïve 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.ConclusionsB 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 InterestNone declared

Systemic lupus erythematosus is a complex autoimmune disorder characterized by the production of pathogenic anti-nuclear antibodies. Previous work from our laboratory has shown that the introgression of a New Zealand Black-derived chromosome 4 interval onto a lupus-prone background suppresses the disease. Interestingly, the same genetic interval promoted the expansion of both Natural Killer T- and CD5 + B cells in suppressed mice. In this study, we show that ablation of NKT cells with a CD1d knockout had no impact on either the suppression of lupus or the expansion of CD5 + B cells. On the other hand, suppressed mice had an expanded population of IL-10-producing B cells that predominantly localized to the CD5 + CD1d low compartment. The expansion of CD5 + B cells negatively correlated with the frequency of pro-inflammatory IL-17 A-producing T-cells and kidney damage. Adoptive transfer with a single injection of total B cells with an enriched CD5 + compartment reduced the frequency of memory/activated, IFNγ-producing, and IL-17 A-producing CD4 T-cells but did not significantly reduce autoantibody levels. Taken together, these data suggest that the expansion of CD5 + IL-10-producing B cells and not NKT cells protects against lupus in these mice, by limiting the expansion of pro-inflammatory IL-17 A- and IFNγ-producing CD4 T-cells.

BackgroundThe 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.ObjectivesTo determine whether changes in the levels of these urinary biomarkers predict treatment responses.Methods21 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.ResultsAt 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.ConclusionsMeasurement of urinary biomarkers can provide valuable insight into treatment responses in lupus nephritis.Disclosure of InterestNone declared

Systemic lupus erythematosus (SLE) is a complex disease trait of unknown aetiology. Genome-wide linkage studies in human SLE identified several linkage regions, including one at 1q23, which contains multiple susceptibility genes, including the members of the signalling lymphocyte activation molecule (SLAM) locus. In mice there is a syntenic linkage region, Sle1. The SLAM genes are functionally related cell-surface receptors, which regulate signal transduction of cells in the immune system. Family-based association study in UK and Canadian SLE families identified variants in the promoter and coding region of SLAMF7 and LY9 contributing to SLE disease susceptibility. The strongest association was from rs509749, in exon 8 of LY9 ( P =0.00209). rs509749 encodes a Val/Met nonsynonymous change in amino acid 602 in the cytoplasmic domain of LY9. In the parents and affected individuals from the Canadian SLE families, the risk allele of rs509049 skews the T-cell population by increasing the number of CD8+ memory T cells, while decreasing the proportion of CD4+ naïve T cells and activated T cells. Since rs509749 lies within the consensus binding site for SAP/SH2D1a, which influences downstream signalling events from LY9, the mechanism for increased CD8+ memory T cells may include differential binding SAP/SH2D1a to the cytoplasmic domain of LY9.

BackgroundPatients 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.ObjectivesTo determine whether ANA+ individuals who lack sufficient symptoms for a SARD diagnosis share the IFN-signature.MethodsANA+ 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; Sjogren's 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.ResultsTo 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, <0.0001, 0.0075, respectively). Using a cutoff of 2 SD above the mean of HC as indicative of an elevated IFN5 score; 8/21 ANS, 8/16 UCTD, 3/7 SSc, 18/22 SjD, 5/6 SLE, and 2/2 MCTD/DM participants had elevated IFN levels. Marked elevations of the IFN5 score were seen in a subset of ANS and UCTD participants, which could not be attributed to recent infection. Although there was a significant correlation between the ANA titer (p=0.002) and IFN5 score for all ANA+ individuals, this was not seen in the ANS or UCTD subsets of this population. However the IFN5 score was positively correlated with the number of different ANA specificities present in the UCTD subset (p=0.048) and all ANA+ individuals (p<0.0001). Within the ANS subset, there was a strong correlation between the presence of anti-Ro/La antibodies with 6/8 IFN5 high as compared to 1/13 IFN low individuals being antibody positive (p=0.003).ConclusionsAn 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 InterestNone declared

BackgroundThe systemic autoimmune rheumatic diseases (SARD; Systemic Lupus Erythematosus, Rheumatoid Arthritis, Sjogren's 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 (TFH) cells are implicated in the development of systemic autoimmunity. Both a decrease in iNKT cells and an increase in TFH cells are found in patients with established SARD.ObjectivesTo determine if T cell abnormalities associated with SARD are present in pre-clinical autoimmunity.MethodsPatients (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 naïve. 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.ResultsA 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 TFH (CD4+CXCR5highPD-1high) cells was significantly elevated (p=0.01) in patients versus HC. While the proportion of TFH cells was similar between asymptomatic ANA+ patients and HC, there was a significant expansion of TFH 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 TFH as compared to UCTD. Patients (n=50) with higher ANA titers (≥1:640) had a significant increase (p=001) in TFH when compared to individuals (n=13) with lower ANA levels (1:160). A positive correlation (p<0.0001) between the proportion of TFH and the number of auto-Abs within the patient population was found. No significant differences were noted in the iNKT or TFH cell proportions between SARD patients stratified by specific disease diagnosis.ConclusionsA 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 TFH 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 InterestNone declared

BackgroundNephritis 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.ObjectivesIn 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.MethodsAffymetrix 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.ResultsComparison of gene expression between healthy controls and all SLE patients revealed 27 genes that were increased >1 log2 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 log2 fold-change with a false discovery rate of q <0.25 (chosen due to the small number of samples). Twenty-two of the probes were overexpressed in patients with nephritis, representing 19 independent genes, the majority of which (15) are preferentially expressed in neutrophils, particularly low density granulocytes (LDGs). There was no difference in the levels of IFN-induced gene expression between active SLE patients with and without nephritis. Examination of gene expression in the validation cohort confirmed these findings and showed that the levels of LDG gene expression in renal remissions were similar to active patients without nephritis. In secondary analyses, the levels of LDG genes correlated positively with the total SLEDAI (p<0.001) and the hematuria (p<0.01) and proteinuria (p<0.001) components of the SLEDAI. There was no association between any of the IFN or LDG genes and vasculitis, treatment, and for those patients with paired renal biopsies, biopsy class, activity or chronicity scores. There was also no correlation between the levels of LDG genes and the neutrophil count in any of the patient subsets. On examination of a subset of patients longitudinally, the LDG signature did not appear to respond rapidly to treatment, remaining relatively consistent in several patients despite marked changes in the SLEDAI, treatment, and neutrophil count.ConclusionsPatients 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 InterestJ. 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