Nitrated nucleosome levels and neuropsychiatric events in systemic lupus erythematosus; a multi-center retrospective case-control study.

BACKGROUND: In patients with systemic lupus erythematosus (SLE) there is no serological test that will reliably distinguish neuropsychiatric (NP) events due to active SLE from those due to other causes. Previously we showed that serum levels of nitrated nucleosomes (NN) were elevated in a small number of patients with NPSLE. Here we measured serum NN in samples from a larger population of patients with SLE and NP events to see whether elevated serum NN could be a marker for NPSLE. METHODS: We obtained serum samples from patients in the Systemic Lupus International Collaborative Clinics (SLICC) inception cohort. This included 216 patients with NP events and two matched controls with SLE but no NP events for each of these patients. For the NP patients we tested samples taken before, during and after the NP event. RESULTS: Twenty-six patients had events attributed to SLE according to the most stringent SLICC attribution rule. In these patients there was no association between onset of event and elevated serum NN. In 190 patients in whom events were not attributed to SLE by the SLICC rules, median serum NN was elevated at the onset of event (P = 0.006). The predominant clinical features in this group of 190 patients were headache, mood disorders and anxiety. CONCLUSIONS: Serum NN levels rise at the time of an NP event in a proportion of patients with SLE. Further studies are needed to determine the value of serum NN as a biomarker for NPSLE.

Metabolic Profiling of Systemic Lupus Erythematosus and Comparison with Primary Sjögren's Syndrome and Systemic Sclerosis.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease which can affect most organ systems including skin, joints and the kidney. Clinically, SLE is a heterogeneous disease and shares features of several other rheumatic diseases, in particular primary Sjögrens syndrome (pSS) and systemic sclerosis (SSc), why it is difficult to diagnose The pathogenesis of SLE is not completely understood, partly due to the heterogeneity of the disease. This study demonstrates that metabolomics can be used as a tool for improved diagnosis of SLE compared to other similar autoimmune diseases. We observed differences in metabolic profiles with a classification specificity above 67% in the comparison of SLE with pSS, SSc and a matched group of healthy individuals. Selected metabolites were also significantly different between studied diseases. Biochemical pathway analysis was conducted to gain understanding of underlying pathways involved in the SLE pathogenesis. We found an increased oxidative activity in SLE, supported by increased xanthine oxidase activity and an increased turnover in the urea cycle. The most discriminatory metabolite observed was tryptophan, with decreased levels in SLE patients compared to control groups. Changes of tryptophan levels were related to changes in the activity of the aromatic amino acid decarboxylase (AADC) and/or to activation of the kynurenine pathway.

A Longitudinal Analysis of Outcomes of Lupus Nephritis in an International Inception Cohort Using a Multistate Model Approach.

OBJECTIVE: To study bidirectional change and predictors of change in estimated glomerular filtration rate (GFR) and proteinuria in lupus nephritis (LN) using a multistate modeling approach. METHODS: Patients in the Systemic Lupus International Collaborating Clinics inception cohort were classified annually into estimated GFR state 1 (>60 ml/minute), state 2 (30-60 ml/minute), or state 3 (<30 ml/minute) and estimated proteinuria state 1 (<0.25 gm/day), state 2 (0.25-3.0 gm/day), or state 3 (>3.0 gm/day), or end-stage renal disease (ESRD) or death. Using multistate modeling, relative transition rates between states indicated improvement and deterioration. RESULTS: Of 1,826 lupus patients, 700 (38.3%) developed LN. During a mean ± SD follow-up of 5.2 ± 3.5 years, the likelihood of improvement in estimated GFR and estimated proteinuria was greater than the likelihood of deterioration. After 5 years, 62% of patients initially in estimated GFR state 3 and 11% of patients initially in estimated proteinuria state 3 transitioned to ESRD. The probability of remaining in the initial states 1, 2, and 3 was 85%, 11%, and 3%, respectively, for estimated GFR and 62%, 29%, and 4%, respectively, for estimated proteinuria. Male sex predicted improvement in estimated GFR states; older age, race/ethnicity, higher estimated proteinuria state, and higher renal biopsy chronicity scores predicted deterioration. For estimated proteinuria, race/ethnicity, earlier calendar years, damage scores without renal variables, and higher renal biopsy chronicity scores predicted deterioration; male sex, presence of lupus anticoagulant, class V nephritis, and mycophenolic acid use predicted less improvement. CONCLUSION: In LN, the expected improvement or deterioration in renal outcomes can be estimated by multistate modeling and is preceded by identifiable risk factors. New therapeutic interventions for LN should meet or exceed these expectations.

The assessment of serum-mediated phagocytosis of necrotic material by polymorphonuclear leukocytes to diagnose and predict the clinical features of systemic lupus erythematosus: an observational longitudinal study.

BACKGROUND: Serum-mediated phagocytosis of antibody- and complement-opsonized necrotic cell material (NCM) by polymorphonuclear leukocytes can be quantified by using a flow cytometry-based assay. The phagocytosis of necrotic cell material (PNC) assay parallels the well-known lupus erythematosus cell test. In this study, we aimed to investigate the diagnostic accuracy of the assay and the relationship with clinical manifestations and disease activity in systemic lupus erythematosus (SLE). METHODS: The diagnostic accuracy for SLE diagnosis of the PNC assay was studied by cross-sectional assessment of blood samples from 148 healthy control subjects and a multicenter rheumatic group (MRG) of 529 patients with different rheumatic symptoms. A cohort of 69 patients with an established SLE diagnosis (SLE cohort) underwent longitudinal clinical and laboratory follow-up for analysis of the temporal relationships between PNC positivity and specific clinical manifestations. RESULTS: In 35 of 529 MRG patients, 13 of whom had SLE, the PNC assay result was positive. Combined positivity of the PNC assay and anti-double-stranded DNA antibodies increased specificity and positive predictive value for SLE diagnosis to 0.99 and 0.67, respectively. In the longitudinal study, 42 of 69 SLE cohort patients had positive results in the PNC assay at least once. PNC assay positivity was associated with current hematological manifestations and could predict mucocutaneous manifestations. When combined with hypocomplementemia, PNC positivity preceded increased Systemic Lupus Erythematosus Disease Activity Index 2000 score, glomerulonephritis, and alopecia. CONCLUSIONS: Serum-mediated PNC by polymorphonuclear leukocytes is commonly but not exclusively seen in patients with SLE. The PNC assay may be used in follow-up of patients with SLE and, especially in combination with other routinely assessed laboratory tests, may help to predict flares and different clinical manifestations, including glomerulonephritis. Our results encourage further development of the PNC assay as a complementary laboratory tool in management of patients with SLE.

The frequency and outcome of lupus nephritis: results from an international inception cohort study.

OBJECTIVE: To determine nephritis outcomes in a prospective multi-ethnic/racial SLE inception cohort. METHODS: Patients in the Systemic Lupus International Collaborating Clinics inception cohort (≤15 months of SLE diagnosis) were assessed annually for estimated glomerular filtration rate (eGFR), proteinuria and end-stage renal disease (ESRD). Health-related quality of life was measured by the Short Form (36 questions) health survey questionnaire (SF-36) subscales, mental and physical component summary scores. RESULTS: There were 1827 patients, 89% females, mean (s.d.) age 35.1 (13.3) years. The mean (s.d.) SLE duration at enrolment was 0.5 (0.3) years and follow-up 4.6 (3.4) years. LN occurred in 700 (38.3%) patients: 566/700 (80.9%) at enrolment and 134/700 (19.1%) during follow-up. Patients with nephritis were younger, more frequently men and of African, Asian and Hispanic race/ethnicity. The estimated overall 10-year incidence of ESRD was 4.3% (95% CI: 2.8%, 5.8%), and with nephritis was 10.1% (95% CI: 6.6%, 13.6%). Patients with nephritis had a higher risk of death (HR = 2.98, 95% CI: 1.48, 5.99; P = 0.002) and those with eGFR <30 ml/min at diagnosis had lower SF-36 physical component summary scores (P < 0.01) and lower Physical function, Physical role and Bodily pain scores. Over time, patients with abnormal eGFR and proteinuria had lower SF-36 mental component summary (P ≤ 0.02) scores compared to patients with normal values. CONCLUSION: LN occurred in 38.3% of SLE patients, frequently as the initial presentation, in a large multi-ethnic inception cohort. Despite current standard of care, nephritis was associated with ESRD and death, and renal insufficiency was linked to lower health-related quality of life. Further advances are required for the optimal treatment of LN.

Mood Disorders in Systemic Lupus Erythematosus: Results From an International Inception Cohort Study.

OBJECTIVE: To examine the frequency, characteristics, and outcome of mood disorders, as well as clinical and autoantibody associations, in a multiethnic/racial, prospective inception cohort of patients with systemic lupus erythematosus (SLE). METHODS: Patients were assessed annually for mood disorders (4 types, according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) and 18 other neuropsychiatric events. Global disease activity scores (SLE Disease Activity Index 2000 [SLEDAI-2K]), damage scores (Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index [SDI]), and Short Form 36 subscales, mental and physical component summary scores were collected. Time to event, linear and ordinal regressions, and multi-state models were used as appropriate. RESULTS: Among the 1,827 patients with SLE, 88.9% were female, and 48.9% were Caucasian. The mean ± SD age of the patients was 35.1 ± 13.3 years, disease duration was 5.6 ± 4.8 months, and the length of followup was 4.7 ± 3.5 years. During the course of the study, 863 (47.2%) of the 1,827 patients had 1,627 neuropsychiatric events. Mood disorders occurred in 232 (12.7%) of 1,827 patients, and 98 (38.3%) of 256 mood disorder events were attributed to SLE. The estimated cumulative incidence of any mood disorder after 10 years was 17.7% (95% confidence interval 15.1, 20.2%). A greater risk of mood disorder was associated with concurrent neuropsychiatric events (P ≤ 0.01), and a lower risk was associated with Asian race/ethnicity (P = 0.01) and treatment with immunosuppressive drugs (P = 0.003). Mood disorders were associated with lower mental health and mental component summary scores but not with the SLEDAI-2K, SDI, or lupus autoantibodies. Among the 232 patients with depression, 168 (72.4%) were treated with antidepressants. One hundred twenty-six (49.2%) of 256 mood disorders resolved in 117 (50.4%) of 232 patients. CONCLUSION: Mood disorders, the second most frequent neuropsychiatric event in patients with SLE, have a negative impact on health-related quality of life and improve over time. The lack of association with global SLE disease activity, cumulative organ damage, and lupus autoantibodies emphasizes the multifactorial etiology of mood disorders and a role for non-lupus-specific therapies.

Incidence and disease severity of anti-neutrophil cytoplasmic antibody-associated nephritis are higher than in lupus nephritis in Sweden.

OBJECTIVES: The objectives of this study were to compare incidence rates, renal and patient survival between lupus nephritis (LN) and anti-neutrophil cytoplasmic antibody-associated nephritis (AAN) during a 12-year period in two geographically defined populations in Sweden. METHODS: In the health care districts surrounding the Skåne University Hospital in Lund [mean population ≥18 years (1997-2008), 188 400] and the University Hospital in Linköping [mean population ≥18 years (1997-2008), 328 900] all patients with biopsy-proven LN and AAN during the period 1997-2008 were included in the study if they (i) were residing within the study areas at the time of onset of nephritis, (ii) had a clinical diagnosis of either SLE or ANCA-associated vasculitis (AAV) and (iii) experienced a first flare of biopsy-proven nephritis during the study period. RESULTS: Eighty-two patients (Lund 44 + Linköping 38) with biopsy-proven AAN were identified and 27 patients with LN (Lund 13 + Linköping 14). The annual incidence rate per million inhabitants aged ≥18 years in both study areas was estimated to be 13.2 (95% CI 10.4-16.1) for AAN and 4.3 (95% CI 2.7-6.0) for LN, P < 0.001. The patients were followed until January 2013. During the follow-up time 38 patients died (AAN 36, LN 2; P = 0.001), and 20 patients went into end-stage renal disease (AAN 19 and LN 1), P = 0.020. CONCLUSIONS: In Sweden, AAN was three times more common than LN, and the outcome was considerably worse. SLE is often diagnosed before the onset of nephritis leading to earlier treatment, while AAN is still often diagnosed at a later stage.

Factors associated with damage accrual in patients with systemic lupus erythematosus: results from the Systemic Lupus International Collaborating Clinics (SLICC) Inception Cohort.

BACKGROUND AND AIMS: We studied damage accrual and factors determining development and progression of damage in an international cohort of systemic lupus erythematosus (SLE) patients. METHODS: The Systemic Lupus International Collaborating Clinics (SLICC) Inception Cohort recruited patients within 15 months of developing four or more 1997 American College of Rheumatology (ACR) criteria for SLE; the SLICC/ACR damage index (SDI) was measured annually. We assessed relative rates of transition using maximum likelihood estimation in a multistate model. The Kaplan-Meier method estimated the probabilities for time to first increase in SDI score and Cox regression analysis was used to assess mortality. RESULTS: We recruited 1722 patients; mean (SD) age 35.0 (13.4) years at cohort entry. Patients with damage at enrolment were more likely to have further worsening of SDI (SDI 0 vs ≥1; p<0.001). Age, USA African race/ethnicity, SLEDAI-2K score, steroid use and hypertension were associated with transition from no damage to damage, and increase(s) in pre-existing damage. Male gender (relative transition rates (95% CI) 1.48 (1.06 to 2.08)) and USA Caucasian race/ethnicity (1.63 (1.08 to 2.47)) were associated with SDI 0 to ≥1 transitions; Asian race/ethnicity patients had lower rates of new damage (0.60 (0.39 to 0.93)). Antimalarial use was associated with lower rates of increases in pre-existing damage (0.63 (0.44 to 0.89)). Damage was associated with future mortality (HR (95% CI) 1.46 (1.18 to 1.81) per SDI point). CONCLUSIONS: Damage in SLE predicts future damage accrual and mortality. We identified several potentially modifiable risk factors for damage accrual; an integrated strategy to address these may improve long-term outcomes.

Electrocardiographic findings in systemic lupus erythematosus: data from an international inception cohort.

OBJECTIVE: To estimate the early prevalence of various electrocardiographic (EKG) abnormalities in patients with systemic lupus erythematosus (SLE) and to evaluate possible associations between repolarization changes (increased corrected QT [QTc] and QT dispersion [QTd]) and clinical and laboratory variables, including the anti-Ro/SSA level and specificity (52 or 60 kd). METHODS: We studied adult SLE patients from 19 centers participating in the Systemic Lupus International Collaborating Clinics (SLICC) Inception Registry. Demographics, disease activity (Systemic Lupus Erythematosus Disease Activity Index 2000 [SLEDAI-2K]), disease damage (SLICC/American College of Rheumatology Damage Index [SDI]), and laboratory data from the baseline or first followup visit were assessed. Multivariate logistic and linear regression models were used to asses for any cross-sectional associations between anti-Ro/SSA and EKG repolarization abnormalities. RESULTS: For the 779 patients included, mean ± SD age was 35.2 ± 13.8 years, 88.4% were women, and mean ± SD disease duration was 10.5 ± 14.5 months. Mean ± SD SLEDAI-2K score was 5.4 ± 5.6 and mean ± SD SDI score was 0.5 ± 1.0. EKG abnormalities were frequent and included nonspecific ST-T changes (30.9%), possible left ventricular hypertrophy (5.4%), and supraventricular arrhythmias (1.3%). A QTc ≥440 msec was found in 15.3%, while a QTc ≥460 msec was found in 5.3%. Mean ± SD QTd was 34.2 ± 14.7 msec and QTd ≥40 msec was frequent (38.1%). Neither the specificity nor the level of anti-Ro/SSA was associated with QTc duration or QTd, although confidence intervals were wide. Total SDI was significantly associated with a QTc interval exceeding 440 msec (odds ratio 1.38 [95% confidence interval 1.06, 1.79]). CONCLUSION: A substantial proportion of patients with recent-onset SLE exhibited repolarization abnormalities, although severe abnormalities were rare.

Impact of early disease factors on metabolic syndrome in systemic lupus erythematosus: data from an international inception cohort.

BACKGROUND: The metabolic syndrome (MetS) may contribute to the increased cardiovascular risk in systemic lupus erythematosus (SLE). We examined the association between MetS and disease activity, disease phenotype and corticosteroid exposure over time in patients with SLE. METHODS: Recently diagnosed (<15 months) patients with SLE from 30 centres across 11 countries were enrolled into the Systemic Lupus International Collaborating Clinics (SLICC) Inception Cohort from 2000 onwards. Baseline and annual assessments recorded clinical, laboratory and therapeutic data. A longitudinal analysis of factors associated with MetS in the first 2 years of follow-up was performed using random effects logistic regression. RESULTS: We studied 1150 patients with a mean (SD) age of 34.9 (13.6) years and disease duration at enrolment of 24.2 (18.0) weeks. In those with complete data, MetS prevalence was 38.2% at enrolment, 34.8% at year 1 and 35.4% at year 2. In a multivariable random effects model that included data from all visits, prior MetS status, baseline renal disease, SLICC Damage Index >1, higher disease activity, increasing age and Hispanic or Black African race/ethnicity were independently associated with MetS over the first 2 years of follow-up in the cohort. CONCLUSIONS: MetS is a persistent phenotype in a significant proportion of patients with SLE. Renal lupus, active inflammatory disease and damage are SLE-related factors that drive MetS development while antimalarial agents appear to be protective from early in the disease course.

Clinical phenotype associations with various types of anti-dsDNA antibodies in patients with recent onset of rheumatic symptoms. Results from a multicentre observational study.

UNLABELLED: Despite anti-dsDNA antibodies constitute a wide range of specificities, they are considered as the hallmark for systemic lupus erythematosus (SLE). OBJECTIVE: To identify clinical phenotypes associated with anti-dsDNA antibodies, independently of any clinical diagnoses. METHODS: Patients with recent onset of any rheumatic symptoms were screened for antinuclear antibodies (ANA). All ANA-positive and matching ANA-negative patients were examined, and their clinical phenotypes were registered, using a systematic chart formulated after consensus between the participating centres. All patients were tested for different anti-dsDNA antibody specificities with assays habitually used in each participating laboratory. Crithidia Luciliae Immuno Fluorescence Test (CLIFT) was performed three times (with two different commercial kits); solid and solution phase ELISA were performed four times. Associations between clinical phenotypes and results of anti-dsDNA assays were evaluated by linear regression analysis (LRA) and principal component analysis (PCA). RESULTS: Totally, 292 ANA-positive and 292 matching ANA-negative patients were included in the study. A full dataset for statistical analysis was obtained in 547 patients. Anti-dsDNA antibodies were most frequently detected by ELISA. LRA showed that overall positivity of anti-dsDNA antibodies was associated with proteinuria and pleuritis. Alopecia was significantly associated only with CLIFT-positivity. Besides confirming the same findings, PCA showed that combined positivity of CLIFT and ELISA was also associated with lymphopenia. CONCLUSIONS: Our results show that different anti-dsDNA antibody specificities are associated with nephropathy, pleuritis, alopecia and lymphopenia, regardless of the diagnosis. It may challenge the importance of anti-dsDNA antibodies as a diagnostic hallmark for SLE.

Platelet activation and anti-phospholipid antibodies collaborate in the activation of the complement system on platelets in systemic lupus erythematosus.

Anti-phospholipid (aPL) antibodies are important contributors to development of thrombosis in patients with the autoimmune rheumatic disease systemic lupus erythematosus (SLE). The underlying mechanism of aPL antibody-mediated thrombosis is not fully understood but existing data suggest that platelets and the complement system are key components. Complement activation on platelets is seen in SLE patients, especially in patients with aPL antibodies, and has been related to venous thrombosis and stroke. The aim of this study was to investigate if aPL antibodies could support classical pathway activation on platelets in vitro as well as in SLE patients. Furthermore, we investigated if complement deposition on platelets was associated with vascular events, either arterial or venous, when the data had been adjusted for traditional cardiovascular risk factors. Finally, we analyzed if platelet complement deposition, both C1q and C4d, was specific for SLE. We found that aPL antibodies supported C4d deposition on platelets in vitro as well as in SLE patients (p = 0.001 and p<0.05, respectively). Complement deposition on platelets was increased in SLE patients when compared with healthy individuals (p<0.0001). However, high levels of C4d deposition and a pronounced C1q deposition were also seen in patients with rheumatoid arthritis and systemic sclerosis. In SLE, C4d deposition on platelets was associated with platelet activation, complement consumption, disease activity and venous (OR = 5.3, p = 0.02), but not arterial, thrombosis, observations which were independent of traditional cardiovascular risk factors. In conclusion, several mechanisms operate in SLE to amplify platelet complement deposition, of which aPL antibodies and platelet activation were identified as important contributors in this investigation. Complement deposition on platelets was identified as a marker of venous, but not arterial thrombosis, in SLE patients independently of traditional risk factors and aPL antibodies. Further studies are needed to elucidate the role of complement deposition on platelets in development of venous thrombosis.

Bacteria in the adventitia of cardiovascular disease patients with and without rheumatoid arthritis.

The incidence of atherosclerosis is significantly increased in rheumatoid arthritis (RA). Infection is one factor that may be involved in the pathogenesis of both diseases. The cause of RA and atherosclerosis is unknown, and infection is one of the factors that may be involved in the pathogenesis of both diseases. The aims of this study were to identify bacteria in the aortic adventitia of patients with cardiovascular disease (CVD) in the presence and absence of RA, and to determine the effect of identified candidate pathogens on Toll-like receptor (TLR)-dependent signalling and the proinflammatory response. The aortic adventitia of 11 CVD patients with RA (RA+CVD) and 11 CVD patients without RA (CVD) were collected during coronary artery bypass graft surgery. Bacteria were detected in four samples from CVD patients and three samples from RA+CVD patients and identified by 16S rRNA gene sequencing. Methylobacterium oryzae was identified in all three RA+CVD samples, representing 44.1% of the bacterial flora. The effect of M. oryzae on TLR-dependent signalling was determined by transfection of HEK-293 cells. Although mild TLR2 signalling was observed, TLR4 was insensitive to M. oryzae. Human primary macrophages were infected with M. oryzae, and a TLDA qPCR array targeting 90 genes involved in inflammation and immune regulation was used to profile the transcriptional response. A significant proinflammatory response was observed, with many of the up-regulated genes encoding proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α) and chemokines (CCR7, IL-8). The aortic adventitia of CVD patients contains a wide range of bacterial species, and the bacterial flora is significantly less diverse in RA+CVD than CVD patients. M. oryzae may stimulate an proinflammatory response that may aggravate and perpetuate the pathological processes underlying atherosclerosis in RA patients.

American College of Rheumatology criteria at inception, and accrual over 5 years in the SLICC inception cohort.

OBJECTIVE: To determine the frequency of each American College of Rheumatology (ACR) criterion met at time of enrollment, and the increase in each of the criteria over 5 years. METHODS: In 2000 the Systemic Lupus International Collaborating Clinics (SLICC) recruited an international inception cohort of patients with systemic lupus erythematosus (SLE; ≥ 4 ACR criteria) who were followed at yearly intervals according to a standard protocol. Descriptive statistics were used to assess the total and cumulative number of ACR criteria met at each visit. Regression models were done to compare the increase of individual and cumulative criteria as a function of race/ethnicity group, and sex. RESULTS: In all, 768 patients have been followed for a minimum of 5 years. Overall, 59.1% of the patients had an increase in the number of ACR criteria they met over the 5-year period. The mean number of ACR criteria met at enrollment was 5.04 ± 1.13 and at year 5 was 6.03 ± 1.42. At enrollment, nonwhite patients had a higher number of ACR criteria (5.19 ± 1.23) than white patients. The total number of criteria increased in both white and nonwhite ethnicities, but increased more among whites. Males had a slightly lower number of criteria at enrollment compared to females and males accrued fewer criteria at 5 years. CONCLUSION: In this international inception cohort of SLE patients with at least 4 ACR criteria at entry, there was an accumulation of ACR criteria over the following 5 years. The distribution of criteria both at inception and over 5 years is affected by sex and ethnicity.

Multiplexing of miniaturized planar antibody arrays for serum protein profiling--a biomarker discovery in SLE nephritis.

In the quest to decipher disease-associated biomarkers, miniaturized and multiplexed antibody arrays may play a central role in generating protein expression profiles, or protein maps, of crude serum samples. In this conceptual study, we explored a novel, 4-times larger pen design, enabling us to, in a unique manner, simultaneously print 48 different reagents (antibodies) as individual 78.5 µm(2) (10 µm in diameter) sized spots at a density of 38,000 spots cm(-2) using dip-pen nanolithography technology. The antibody array set-up was interfaced with a high-resolution fluorescent-based scanner for sensitive sensing. The performance and applicability of this novel 48-plex recombinant antibody array platform design was demonstrated in a first clinical application targeting SLE nephritis, a severe chronic autoimmune connective tissue disorder, as the model disease. To this end, crude, directly biotinylated serum samples were targeted. The results showed that the miniaturized and multiplexed array platform displayed adequate performance, and that SLE-associated serum biomarker panels reflecting the disease process could be deciphered, outlining the use of miniaturized antibody arrays for disease proteomics and biomarker discovery.

Recombinant antibody microarray for profiling the serum proteome of SLE.

Systemic lupus erythematosus (SLE) is a severe autoimmune connective tissue disease. Our current knowledge about the serum proteome, or serum biomarker panels, reflecting disease and disease status is still very limited. Affinity proteomics, represented by recombinant antibody arrays, is a novel, multiplex technology for high-throughput protein expression profiling of crude serum proteomes in a highly specific, sensitive, and miniaturized manner. The antibodies are deposited one by one in an ordered pattern, an array, onto a solid support. Next, the sample is added, and any specifically bound proteins are detected and quantified. The binding pattern is then converted into a relative protein expression map, or protein map, deciphering the composition of the sample at the molecular level. The methodology provides unique opportunities for delineating serum biomarkers reflecting SLE, thus paving the way for improved diagnosis, classification, and prognosis.

Pathogenic mechanisms in lupus nephritis: Nucleosomes bind aberrant laminin ß1 with high affinity and colocalize in the electron-dense deposits.

OBJECTIVE: Apoptotic nucleosomes are structurally and immunologically involved in lupus nephritis. The purpose of this study was to examine the expression and function of laminins and their interactions with nucleosomes in the kidneys of patients with lupus nephritis, using surface plasmon resonance (SPR) analysis. METHODS: SPR interaction analysis was used to quantify the strength of laminin-nucleosome interactions. Electron microscopy techniques were used to determine in vivo colocalization of IgG, chromatin, and laminin ß1, as well as to characterize nucleosome-laminin interactions in vitro. RESULTS: Nucleosomes were found to possess high affinity for laminin ß1-containing laminins and to have the potential to form stable molecular complexes with these structures. In vivo, laminin ß1 was aberrantly expressed in the glomerular basement membrane (GMB) of lupus nephritis patients, and in situ, it acted as a nucleosome ligand, selectively colocalizing with nucleosomes within electron-dense deposits (EDDs). Normal adult laminin 11, which contains laminin ß2, did not bind nucleosomes, and it did not colocalize in vivo with the nucleosomes in the nephritic GBM. In addition, TGFß1 was expressed by the glomerular mesangium, glomerular endothelial cells, and by podocytes in patients with lupus nephritis. It was trapped in situ within EDDs by an as-yet-unknown ligand. As was recently described in a transgenic mouse model, paracrine kidney glomerular TGFß1 may thereby contribute to the development of glomerulopathy via the induction of laminin ß1 incorporation in the GBM, whereas systemic blood vessel-derived TGFß1 could be trapped during filtration. CONCLUSION: Our findings of the specific high-affinity binding of nucleosomes to aberrantly expressed laminin ß1 in the GBM and their colocalization in the GBM may explain important features of the initial steps in the pathogenesis of lupus nephritis, the planted antigen hypothesis.

25-hydroxyvitamin D and cardiovascular disease in patients with systemic lupus erythematosus: data from a large international inception cohort.

OBJECTIVE: An association between 25-hydroxyvitamin D (25[OH]D; vitamin D) deficiency and increased cardiovascular (CV) risk factors and CV disease (CVD) has been shown in general population studies. Vitamin D deficiency has been noted in systemic lupus erythematosus (SLE), and CVD is a major cause of morbidity and mortality in SLE. The objectives of this study were to estimate the associations of 25(OH)D levels with CV risk factors and to determine whether low baseline 25(OH)D levels predict future CV events in patients participating in an international inception cohort. METHODS: Data were collected on 890 participants, including demographics, SLE activity and damage assessments, CV risk factors and events, medications, laboratory assessments of 25(OH)D levels, and inflammatory markers. Multiple logistic and Cox regressions were used to estimate the associations of baseline 25(OH)D levels with baseline CV risk factors and CVD events. The models were adjusted for age, sex, race, season, and country, with and without body mass index. RESULTS: Patients in the higher quartiles of 25(OH)D were less likely to have hypertension and hyperlipidemia and were more likely to have lower C-reactive protein levels and lower Systemic Lupus Erythematosus Disease Activity Index 2000 scores at baseline when compared with the first quartile. Vitamin D levels were not independently associated with CVD event incidence; however, hazard ratios for CVD event incidence decreased with successively higher quartiles. CONCLUSION: Lower baseline 25(OH)D levels are associated with higher risk for CV risk factors and more active SLE at baseline. There may be a trend toward a lower likelihood of CVD events in those with higher baseline 25(OH)D levels.

Lymphoma risk in systemic lupus: effects of disease activity versus treatment.

OBJECTIVE: To examine disease activity versus treatment as lymphoma risk factors in systemic lupus erythematosus (SLE). METHODS: We performed case-cohort analyses within a multisite SLE cohort. Cancers were ascertained by regional registry linkages. Adjusted HRs for lymphoma were generated in regression models, for time-dependent exposures to immunomodulators (cyclophosphamide, azathioprine, methotrexate, mycophenolate, antimalarial drugs, glucocorticoids) demographics, calendar year, Sjogren's syndrome, SLE duration and disease activity. We used adjusted mean SLE Disease Activity Index scores (SLEDAI-2K) over time, and drugs were treated both categorically (ever/never) and as estimated cumulative doses. RESULTS: We studied 75 patients with lymphoma (72 non-Hodgkin, three Hodgkin) and 4961 cancer-free controls. Most lymphomas were of B-cell origin. As is seen in the general population, lymphoma risk in SLE was higher in male than female patients and increased with age. Lymphomas occurred a mean of 12.4 years (median 10.9) after SLE diagnosis. Unadjusted and adjusted analyses failed to show a clear association of disease activity with lymphoma risk. There was a suggestion of greater exposure to cyclophosphamide and to higher cumulative steroids in lymphoma cases than the cancer-free controls. CONCLUSIONS: In this large SLE sample, there was a suggestion of higher lymphoma risk with exposure to cyclophosphamide and high cumulative steroids. Disease activity itself was not clearly associated with lymphoma risk. Further work will focus on genetic profiles that might interact with medication exposure to influence lymphoma risk in SLE.