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Introduction: Antibodies against double-stranded DNA (anti-dsDNA) are a specific biomarker for systemic lupus erythematosus (SLE). The first WHO International Standard (IS) for anti-dsDNA (established in 1985), which was used to assign units to diagnostic tests, was exhausted over a decade ago.Methods: Plasma from a patient with SLE was first evaluated in 42 European laboratories. The plasma was thereafter used by the National Institute for Biological Standards and Control to prepare a candidate WHO reference preparation for lupus (anti-dsDNA) antibodies. That preparation, coded 15/174, was subjected to an international collaborative study, including 36 laboratories from 17 countries.Results: The plasma mainly contained anti-dsDNA, other anti-chromatin antibodies and anti-Ku. The international collaborative study showed that the field would benefit from 15/174 as a common reference reagent improving differences in performance between different assays. However, no statistically meaningful overall potency or assay parallelism and commutability could be shown.Conclusion: 15/174 cannot be considered equivalent to the first IS for anti-dsDNA (Wo/80) and was established as a WHO Reference Reagent for lupus (oligo-specific) anti-dsDNA antibodies with a nominal value of 100 units/ampoule. This preparation is intended to be used to align test methods quantifying levels of anti-dsDNA antibodies. [ABSTRACT FROM AUTHOR]

Introduction: Individual patients with rheumatoid arthritis (RA) show divergent specific anti-citrullinated protein/peptide antibodies (ACPA) patterns, but hitherto no individual ACPA specificity has consistently been linked to RA pathogenesis. ACPA are also implicated in immune complexes (IC)-associated joint pathology, but until now, there has been no method to investigate the role of individual ACPA in RA IC formation and IC-associated pathogenesis.Methods: We have developed a new technique based on IC binding to C1q-coated magnetic beads to purify and solubilise circulating IC in sera and synovial fluids (SF) from 77 patients with RA. This was combined with measurement of 19 individual ACPA in serum, SF and in the IC fractions from serum and SF. We investigated whether occurrence of individual ACPA as well as number of ACPA in these compartments was related to clinical and laboratory measures of disease activity and inflammation.Results: The majority of individual ACPA reactivities were enriched in SF as compared with in serum, and levels of ACPA in IC were regulated independently of levels in serum and SF. No individual ACPA reactivity in any compartment showed a dominating association to clinical and laboratory measures of disease activity and severity. Instead, the number of individual ACPA reactivities in the IC fraction from SF associated with a number of markers of joint destruction and inflammation.Conclusions: Our data highlight the polyclonality of ACPA in joint IC and the possibility that a broad ACPA repertoire in synovial fluid IC might drive the local inflammatory and matrix-degrading processes in joints, in analogy with antibody-induced rodent arthritis models. [ABSTRACT FROM AUTHOR]

Introduction: The second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients.Methods: We investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array.Results: The prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset.Conclusions: Multiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement. [ABSTRACT FROM AUTHOR]

Objectives: To study the effects of abatacept on disease activity and on muscle biopsy features of adult patients with dermatomyositis (DM) or polymyositis (PM).Methods: Twenty patients with DM (n=9) or PM (n=11) with refractory disease were enrolled in a randomised treatment delayed-start trial to receive either immediate active treatment with intravenous abatacept or a 3 month delayed-start. The primary endpoint was number of responders, defined by the International Myositis Assessment and Clinical Studies Group definition of improvement (DOI), after 6 months of treatment. Secondary endpoints included number of responders in the early treatment arm compared with the delayed treatment arm at 3 months. Repeated muscle biopsies were investigated for cellular markers and cytokines.Results: 8/19 patients included in the analyses achieved the DOI at 6 months. At 3 months of study, five (50%) patients were responders after active treatment but only one (11%) patient in the delayed treatment arm. Eight adverse events (AEs) were regarded as related to the drug, four mild and four moderate, and three serious AEs, none related to the drug. There was a significant increase in regulatory T cells (Tregs), whereas other markers were unchanged in repeated muscle biopsies.Conclusions: In this pilot study, treatment of patients with DM and PM with abatacept resulted in lower disease activity in nearly half of the patients. In patients with repeat muscle biopsies, an increased frequency of Foxp3+ Tregs suggests a positive effect of treatment in muscle tissue. [ABSTRACT FROM AUTHOR]

Objective: Antifibrillar collagen type II (anti-CII) antibody-positive patients with rheumatoid arthritis (RA) have early but not late signs of increased inflammation and joint erosions. We wanted to replicate this in a large RA cohort, and to relate to human leukocyte antigen (HLA)-DRB1* alleles.Methods: Anti-CII and anti-cyclic citrullinated peptide (CCP)2 were measured at baseline in 773 patients with RA from the Swedish Epidemiological Investigation in Rheumatoid Arthritis (EIRA) study with clinical follow-up data from the Swedish Rheumatology Quality Register (SRQ) registry, and 1476 with HLA-DRB1* information. Comparisons were done concerning C reactive protein (CRP), erythrocyte sedimentation rate (ESR), tender joint count (TJC), swollen joint count (SJC), Disease Activity Score encompassing 28 joints based on ESR (DAS28), DAS28CRP, pain-Visual Analogue Scale (VAS), global-VAS and Health Assessment Questionnaire Score (HAQ) at eight occasions during 5 years, and association with HLA-DRB1* alleles.Results: Anti-CII associated with elevated CRP, ESR, SJC, DAS28 and DAS28CRP at diagnosis and up to 6 months, whereas anti-CCP2 associated with SJC and DAS28 from 6 months to 5 years, but not earlier. The anti-CII-associated phenotype was strong, and predominated in anti-CII/anti-CCP2 double-positive patients. Anti-CII was associated with improvements in CRP, ESR, SJC, TJC and DAS28, whereas anti-CCP2 was associated with deteriorations in SJC and DAS28 over time. Anti-CII-positive patients achieved European League Against Rheumatism good or moderate response more often than negative patients. Anti-CII was positively associated with HLA-DRB1*01 and HLA-DRB1*03, with significant interaction, and double-positive individuals had >14 times higher mean anti-CII levels than HLA double negatives. Whereas smoking was associated with elevated anti-CCP2 levels, smokers had lower anti-CII levels.Conclusions: Anti-CII seropositive RA represents a distinct phenotype, in many respects representing the converse to the clinical, genetic and smoking associations described for anticitrullinated protein peptide autoantibodies. Although not diagnostically useful, early anti-CII determinations predict favourable inflammatory outcome in RA. [ABSTRACT FROM AUTHOR]

Objective Smoking can induce autoantibodies in persons who are genetically predisposed to rheumatoid arthritis. We investigated the association between smoking and antiphospholipid antibodies (aPL) in systemic lupus erythematosus (SLE), a question not previously addressed. Further, we explored the relationship between smoking, aPL and vascular events (arterial and venous, VE). Methods In this cross-sectional study, clinical evaluation and questionnaire data were collected from 367 prevalent SLE patients. At the same time, we measured aPL (anticardiolipin (aCL), anti-β2 glycoprotein- 1(aβ2GP1) antibodies IgG/IgM/IgA, and lupus anticoagulant (LA)), and a large set of other SLE- associated autoantibodies for comparison. Association analyses using logistic regression models with smoking, (ever, former and current with never as reference) and antibody status as outcome variable were performed. As a secondary outcome, we investigated the associations between aPL, smoking and VE. Results In multivariable-adjusted models ever, and in particular former, cigarette smoking was associated with the most pathogenic aPL; LA, aCL IgG and aβ2GP1 IgG. Other SLE-associated autoantibodies were not associated with smoking. The combination of smoking and aPL was strongly associated with VE. We noted a positive interaction between smoking-LA and smoking-'triple aPL' positivity for previous VE. Conclusions We investigated a large set of commonly occurring autoantibodies in SLE, but only aPL were positively associated with a history of smoking. This association was especially apparent in former smokers. Among ever regular smokers who were aPL positive, we observed a strikingly high frequency of former VE. The underlying mechanisms and temporality between smoking, aPL and VE need further investigations. [ABSTRACT FROM AUTHOR]

Objective: To analyse if predictors of radiographic progression differ between patients treated with or without prednisolone in early rheumatoid arthritis (RA). Radiographs of hands and feet were assessed using the modified Sharp/van der Heijde score and radiographic progression was defined as an increase in the total Sharp score above 5.8 (the smallest detectable change). Design: Prospective, randomised study of patients with early RA. Setting: Secondary level of care; six participating centres from southern Sweden; both urban and rural populations. Participants: In all, 225 patients, 64% women, with a diagnosis of RA according to the American College of Rheumatology criteria, were included if they were between 18 and 80 years of age and had a disease duration of less than 1 year. Intervention: The patients were randomised to 7.5 mg prednisolone daily for 2 years (P-group; n=108) or no prednisolone (NoP-group; n=117) when they started with their first disease-modifying anti-rheumatic drug and were prospectively followed for 2 years. Results: The frequency of patients with radiographic progression after 2 years was 26% in the P-group and 39% in the NoP-group ( p=0.033). Relevant interactions between treatment and rheumatoid factor (RF) (p=0.061) and between treatment and anti-cyclic citrullinated peptide 2 (anti-CCP) (p=0.096) were found. RF and anti-CCP independently predicted radiographic progression only in the NoP-group, OR (95% CI) 9.4 (2.5 to 35.2), p=0.001 and OR (95% CI) 8.7 (2.5 to 31.3), p=0.001, respectively. Conclusions: The presence of RF and anti-CCP predicted radiographic progression in patients not treated with prednisolone but failed to predict progression in patients treated with this drug. The data suggest that early treatment with prednisolone may modulate not only inflammation but also autoimmunity-associated pathogenetic mechanisms. [ABSTRACT FROM AUTHOR]

Background Rheumatoid factors (RFs) and antibodies against cyclic citrullinated peptides (CCPs) of IgG, IgA and IgM isotype have been shown to precede disease onset by years. Objective To evaluate serological risk markers in first- degree relatives from multicase families in relation to genetic and environmental risk factors. Methods 51 multicase families consisting of 163 individuals with rheumatoid arthritis (RA) (mean±SD age, 60±14 years; disease duration 21 years; 71.8% female) and with 157 first-degree relatives unaffected by RA (54±17 years; 59.9% female) were recruited. Isotypes of antibodies against CCPs (IgG, IgA and IgM) and RFs (IgM and IgA) were determined using automated enzyme immunoassays. Cut-off levels were established using receiver operating characteristic curves based on values for 100 unrelated healthy controls. Results The concentrations and frequencies of all anti-CCP and RF isotypes were significantly increased in first-degree relatives and patients with RA compared with unrelated healthy controls. The relative distribution of IgA and IgM isotypes was higher than IgG in the relatives, whereas the IgG isotype dominated in patients with RA. The patients carried human leucocyte antigen- shared epitope (HLA-SE) significantly more often than the relatives (71.4% vs 53.9%, p=0.01), while the frequency of the PTPN22 T variant was similar. HLA-SE, combined with smoking, was significantly related to all combinations of anti-CCP and RF isotypes in patients with RA. No such relationships were found for the first-degree relatives. Conclusions All anti-CCP and RF isotypes analysed occurred more commonly in unaffected first-degree relatives from multicase families than in controls, but with different isotype distribution from patients with RA. [ABSTRACT FROM AUTHOR]

Introduction The anticyclic citrullinated peptide 2 (anti-CCP2) assay is a generic test for antibodies to citrullinated proteins, among which there is a subset of about 50% with antibodies to citrullinated enolase peptide 1 (CEP-1). The anti-CEP-1 positive subset is strongly associated with the HLA-DRB1 shared epitope and its interaction with smoking. Objective To investigate whether anti-CEP-1 antibodies may be helpful in predicting outcome. Methods Anti-CEP-1 and anti-CCP2 antibodies were measured in two prospective cohorts of patients (Karolinska n=272, Norfolk Arthritis Register (NOAR) n=408) with early rheumatoid arthritis (RA). Outcomes measured were C-reactive protein, erythrocyte sedimentation rate, visual analogue scales for pain and global assessment of disease activity, Health Assessment Questionnaire, physician's assessment, swollen and tender joint counts and radiological progression. Results Anti-CCP2 antibodies were present in 57% and 50%, and anti-CEP-1 in 27% and 24% of the Karolinska and NOAR cohorts, respectively. Importantly, no statistically significant differences in clinical outcomes were demonstrated between the anti-CEP-1−/CCP2+ and the anti-CEP-1+/CCP2+ subsets in either cohort, or in radiological outcomes in the Karolinska cohort. Conclusion Although antibodies to specific citrullinated proteins may have distinct genetic and environmental risk factors, the similarity in clinical phenotype suggests that they share common pathways in the pathogenesis of joint disease in RA. [ABSTRACT FROM PUBLISHER]

Background Earlier studies have demonstrated that smoking and genetic risk factors interact in providing an increased risk of rheumatoid arthritis (RA). Less is known on how smoking contributes to RA in the context of genetic variability, and what proportion of RA may be caused by smoking. Objectives To determine the association between the amount of smoking and risk of RA in the context of different HLA-DRB1 shared epitope (SE) alleles, and to estimate proportions of RA cases attributed to smoking. Design, Setting and Participants Data from the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA) case–control study encompassing 1204 cases and 871 controls were analysed. Main Outcome Measure Estimated OR to develop RA and excess fraction of cases attributable to smoking according to the amount of smoking and genotype. Results Smoking was estimated to be responsible for 35% of anticitrullinated protein/peptide antibody (ACPA)-positive cases. For each HLA-DRB1 SE genotype, smoking was dose-dependently associated with an increased risk of ACPA-positive RA (p trend <0.001). In individuals carrying two copies of the HLA-DRB1 SE, 55% of ACPA-positive RA was attributable to smoking. Conclusions Smoking is a preventable risk factor for RA. The increased risk due to smoking is dependent on the amount of smoking and genotype. [ABSTRACT FROM PUBLISHER]

Background Rheumatoid arthritis (RA) can be divided into two major subsets based on the presence or absence of antibodies to citrullinated peptide antigens (ACPA). Until now, data from genome-wide association studies (GWAS) have only been published from ACPA-positive subsets of RA or from studies that have not separated the two subsets. The aim of the current study is to provide and compare GWAS data for both subsets. Methods and results GWAS using the Illumina 300K chip was performed for 774 ACPA-negative patients with RA, 1147 ACPA-positive patients with RA and 1079 controls from the Swedish population-based case–control study EIRA. Imputation was performed which allowed comparisons using 1 723 056 single nucleotide polymorphisms (SNPs). No SNP achieved genome-wide significance (2.9 × 10) in the comparison between ACPA-negative RA and controls. A case–case association study was then performed between ACPA-negative and ACPA-positive RA groups. The major difference in this analysis was in the HLA region where 768 HLA SNPs passed the threshold for genome-wide significance whereas additional contrasting SNPs did not reach genome-wide significance. However, one SNP close to the locus in chromosome 2 reached a p value of 2 × 10 and this locus can thus be considered as a tentative candidate locus for ACPA-negative RA. Conclusions ACPA-positive and ACPA-negative RA display significant risk allele frequency differences which are mainly confined to the HLA region. The data provide further support for distinct genetic aetiologies of RA subsets and emphasise the need to consider them separately in genetic as well as functional studies of this disease. [ABSTRACT FROM PUBLISHER]

Objective To study the association between silica exposure, separately as well as combined with smoking, and the risk of developing rheumatoid arthritis (RA) with or without the presence of antibodies against citrullinated peptide antigens (ACPA). Methods This Swedish population based case–control study analysed 577 incident RA cases and 659 randomly selected controls, all men aged 18–70 years, included during May 1996 to May 2006. Self-reported silica exposure, defined as exposure to stone dust, rock drilling or stone crushing and cigarette smoking was registered. ACPA status among cases was analysed. Results Silica-exposed subjects were found to have a moderately increased risk of ACPA-positive RA (odds ratio (OR) adjusted for age and residency=1.67 (95% CI 1.13 to 2.48), but not of ACPA-negative RA (OR=0.98 (95% CI 0.57 to 1.66)), compared with subjects unexposed to silica. Subjects exposed to rock drilling were found to have a somewhat more markedly increased risk of ACPA-positive RA (OR=2.34 (95% CI 1.17 to 4.68)). A high risk of developing ACPA-positive RA was observed among silica-exposed current smokers (OR=7.36 (95% CI 3.31 to 16.38)), exceeding the risk expected from the separate effects of silica exposure and current smoking, indicating an interaction between these exposures (attributable proportion due to interaction=0.60 (95% CI 0.26 to 0.95)). Conclusion Silica exposure combined with smoking among men is associated with an increased risk of developing ACPA-positive RA. These results suggest that different inhalation exposures may interact in the aetiology of ACPA-positive RA. [ABSTRACT FROM PUBLISHER]

Background The anticitrullinated protein/peptide antibody (ACPA) response in rheumatoid arthritis (RA) might be driven by key autoantigens citrullinated in the target organ. We have recently by using high definition mass spectrometry defined fibrinogen sequences which are citrullinated in RA synovial tissue. Objective To use a newly developed multiplexing assay to investigate the humoral immune response against such in vivo citrullinated fibrinogen epitopes. Methods The Phadia ImmunoCAP ISAC chip was used to bind four synthetic fibrinogen peptides containing citrulline as shown in vivo corresponding to the α chain positions 573 and 591, the β chain positions 72 and 74, as well as their arginine-containing control peptides. 848 RA patients and 150 controls from the Epidemiological Investigations in Rheumatoid Arthritis cohort were investigated. Peptide-specific ELISA tests were developed for validation of the microarray in a smaller group of RA sera (n=123) and controls (n=20). Results Using the 98th percentile among the controls, 13.3% (113/848) of the RA patients had antibodies against the citrullinated 573 peptide. There was a substantial reactivity also to the arginine control peptide both in RA patients and controls, not generally observed for other arginine-containing control peptides. After subtraction of the arginine response, the net immune response discriminated better between patients and controls, and using the 98th percentile for the net citrulline response among HC, 35.5% (301/848) of the RA patients were autoantibody positive. The area under the receiver operator characteristics (ROC) curve was 0.610 for uncorrected values, but after subtraction of the readout for arginine controls the AUC increased to 0.800. The microarray showed good correlation to autoantibody levels measured with ELISA (r=0.83). The four peptides showed considerably divergent responses in ROC curve analyses both before and after correction for arginine control peptides, and the remaining three citrullinated peptides are currently validated. Conclusion This study proves a substantial autoantibody reactivity against fibrinogen epitopes citrullinated in vivo in RA synovial tissue. Autoantibody multiplexing can be used for the screening humoral reactivities against large numbers of potential autoantigenic epitopes, for example, those found citrullinated in vivo. This technique also allows easy correction for autoantibody reactivity against the arginine-containing peptide backbone normally not performed with ACPA ELISA tests. Concerning the fibrinogen 573 peptide, such correction increased the diagnostic performance of autoantibody measurement considerably. [ABSTRACT FROM PUBLISHER]

Rheumatoid arthritis (RA) is characterised by chronic, erosive polyarthritis and by the presence of various autoantibodies. According to our knowledge there are no data published concerning serologic RA phenotypes in Sudan. Our aim in this study is to evaluate the presence of rheumatoid factor (RF) and anticyclic citrullinated peptide antibody (anti-CCP) isotypes in well-characterised cross-sectional hospital-based consecutive cohort of Sudanese RA patients. Serum samples were collected from 78 RA Sudanese patients (66 female, 12 males) from two rheumatology centers in Khartoum, Sudan between December 2008 and May 2010. Female and male mean age was 48.3 and 55.4, respectively. Median disease duration was 3 years (range 1 month to 58 years). Control groups included 101 Sudanese and 100 Swedish healthy individuals. Diagnosis of this RA cohort done by senior rheumatologists following the 1987 American College of Rheumatology criteria. All patients were on regular follow-up. 69/78 (88.5%) were treated with disease-modifying antirheumatic drugs (DMARDs): 21/78 (27%) with methotrexate, 1/78 (1.3%) with hydroxychlorokine, 5/78 (6.4%) with sulfasalazine and 1/78 (1.3%) with azathioprine; and 41/78 (53%) were treated with different DMARD combinations. 9/78 (11.5%) were treated with non-steroidal anti-inflammatory drugs only. All autoantibody analyses were performed by ImmunoCap EIA (Phadia AB, Uppsala, Sweden). Results No differences were found between the Swedish and Sudanese healthy controls for IgG anti-CCP or for for IgM and IgA RF. Among the Sudanese RA patients and their controls, 60.3%(47/78) of Sudanese RA patients and none of the controls were IgG anti-CCP positive (p <0.0001 compared to Sudanese controls). For RF, 31.2% (24/77) of Sudanese RA patients were IgA RF positive (p <0.0001) and 52.6% (41/78) of them were IgM RF positive (p <0.0001). When we looked for autoantibody combinations, 64.1% (50/78) has either IgG anti-CCP and/or IgM RF, 62.3% (48/77) has any of IgG anti-CCP or IgA RF, 57.1% (44/77) has any of IgM RF or IgA RF and 65.4% (51/78) had any of the three investigated autoantibodies. There were no statistical difference in erythrocyte sedimentation rate or number of swollen or tender joints between patients with or without IgG anti-CCP, IgM RF or IgA RF, respectively. Conclusions Our data represent an interim analysis of an ongoing investigation of a larger Sudanese hospital based RA cohort and show IgG anti-CCP positivity in a portion of Sudanese RA patients comparable to results obtained from Swedish RA cohorts. Somewhat fewer RA patients had IgM RF and substantially fewer IgA RF. There were no differences between Swedish and Sudanese control cohorts for any of the investigated autoantibodies. [ABSTRACT FROM PUBLISHER]

Background and objective We have earlier shown that polyethylene glycol (PEG)-precipitated circulating immune complexes (CIC) from systemic lupus erythematosus (SLE) patients induce cytokines in vitro, and that levels of both CIC and CIC-induced cytokines are dependent on (1) SLE disease activity and (2) the occurrence of anti-SSA but not anti-dsDNA autoantibodies. These findings implicated anti-SSA in immune complex formation, but the formal proof that anti-SSA participates in the formation of SLE CIC to a greater degree than did other antinuclear antibody-associated autoantibodies was lacking. The aim of this study was to investigate to what extent different autoantibodies accumulate in SLE immune complexes, and whether such accumulation is dependent on SLE disease activity. Methods Circulating immune complexes were isolated from SLE sera by both PEG precipitation (n=19) and C1q binding (n=8). Autoantibody specificities were determined with a semiquantitative lineblot assay quantified by densitometry. To compare the relative levels of autoantibodies they were normalised against total IgG measured by ELISA in sera and CIC in parallel. Samples were investigated both in a cross-sectional design (n=19), as well as in a paired design with samples obtained during active and inactive SLE (n=10). Results All autoantibodies were enriched in CIC compared to in serum. The group of antibodies against RNA-associated antigens showed higher median enrichment than anti-dsDNA (anti-RNP/Sm p=0.0228, anti-Sm p=0.0392, anti-SSA/Ro60 p=0.0539, anti-SSA/Ro52 p=0.0122, anti-SSB/La p=0.0342). Similarly, the other DNA-associated antigens (antihistones and antinucleosomes) were also less enriched as compared with the RNA-associated specificities. These findings were corroborated by analysis of autoantibody content in C1q-bound CIC. There was no difference in degree of accumulation of the investigated autoantibodies in CIC during active and inactive SLE. Conclusion Our findings demonstrate a difference in enrichment between autoantibodies against RNA- and DNA-associated autoantigens in isolated SLE CIC, suggesting that the RNA-associated autoantibodies are more prone to form complexes in the circulation in SLE, in contrast to antibodies against DNA-associated autoantigens including dsDNA. These finding have implications on by what mechanisms different autoantibodies accumulate in target organs in SLE. [ABSTRACT FROM PUBLISHER]

Background and objectives Autoantibodies, for example, directed against citrullinated proteins/peptides (ACPA) are highly specific and predictive of development of rheumatoid arthritis (RA). Different subgroups of RA patients have different autoantibody specificities relating to, for example, genetic subsets and disease prognosis. Further autoantibody subgrouping of RA patients can give further information about disease subsets, which may have different prognosis and require different treatments. We have developed a microarray for the simultaneous detection of multiple RA-associated autoantibodies. Materials and methods The microarray is based on Phadia's ImmunoCAP ISAC system, where reactivity to more than 100 antigens can be analysed simultaneously using small volumes (3 μl) of sera. Antigens are immobilised in an arrayed fashion onto a chemically modified glass slide allowing a 3-dimensional layer with high binding capacity. Thirty μl of diluted serum from RA patients and healthy controls are applied onto reactions sites on the glass slides, followed by fluorescent-labelled antihuman IgG antibody, and analysed by capturing fluorescence intensity images in a laser scanner. Results are analysed with a customised image analysis software. The assay has been optimised concerning serum dilution and choice of anti-IgG conjugate. Each individual antigen is optimised concerning antigen concentration, binding (biotinylation or not) and choice of spotting buffer A large number of antigens, citrullinated and non-citrullinated peptides as well as full proteins, are being investigated and sequentially added to the chip after individual validation against antigen-specific ELISAs. Results Until now 16 antigens representing citrullinated and non-citrullinated vimentin, fibrinogen, α enolase, collagen type II and filaggrin have been validated. Strong correlations between ISAC and ELISA results were found for the individual validated antigens (R between 0.73 and 0.93). Results from a pilot study using ISAC imply that subtraction of fluorescence for the corresponding arginine-containing peptide from ACPA results will both increase diagnostic sensitivity as well as strengthen the genetic association to the HLA-DRB1* shared epitope. Such correction for non-citrullinated antigen background is not done with conventional ACPA ELISAs. Conclusions We have developed a multiplex array for detection of RA-associated antibodies, and validated against ELISA results for the individual antibody specificities. Currently eight citrullinated antigens and their non-citrullinated counterparts have been individually validated, and new antigens are continuously added to the array. This platform might be of great benefit in studies of RA pathogenesis, diagnosis and potentially as a guide to individualised treatment. The first clinical study using the presently validated antigens is planned for December 2010. [ABSTRACT FROM PUBLISHER]