Anti-carbamylated Protein Research Articles

Auto-antibodies to post-translationally modified proteins in osteoarthritis

Autoantibodies (AutoAbs) have been observed in osteoarthritis (OA) with broad antigenicity, although their prevalence and role remain unclear. Post-translational modification (PTMs) of proteins (oxidation, carbamylation, citrullination) is associated with synovitis and can lead to AutoAb development. Given the prevalence of synovitis, we explored whether AutoAbs to PTM-antigens are common in OA compared with rheumatoid arthritis (RA). Serum (n=895) was obtained from healthy controls, OA and RA patients; and arthritic synovial fluid (SF, n=290). ELISAs were used to quantify anti-citrullinated peptide (ACPA), anti-carbamylated protein (anti-CarP), anti-oxidized collagen (anti-ROS-CI/CII) antibodies. In sera, positivity for PTM-antigens AutoAbs was observed at a lower frequency in OA with 64.1% (95%CI: 57.2-70.1%) more ACPA+ and 29.8% (21.0-37.3%) more anti-CarP+patients in RA (both P<0.0001). Levels of ACPA, anti-CarP were also lower in OA (P<0.0001). Anti-ROS-CII positivity was lower in OA compared to RA (16.6%, 4.8-28.6%) less frequent, P=0.033) but not anti-native-CII. There was no impact of age/gender on AutoAbs associations with diseases either looking at positivity or levels. In SF, OA patients were often ACPA+ (45.9%) although less frequently than in RA (P=0.004). Anti-CarP were rarely observed (<5% all samples). All collagen AutoAbs were more frequent in RA compared to OA (all P<0.010) but only levels of anti-CII and anti-ROS-CII were significantly higher in they RA (P<0.050). Although the frequency of AutoAbs for PTM proteins were lower in OA sera compared to RA, a higher proportion of OA SF were positive. The relative retention of AutoAbs in the OA joint requires further investigation.

Predictive value of anti-CarP and anti-PAD3 antibodies alone or in combination with RF and ACPA for the severity of rheumatoid arthritis.

The objective of this study was to analyse the predictive value of anti-carbamylated protein (anti-CarP) and anti-peptidyl-arginine deiminase type-3 (anti-PAD3) antibodies, alone or in combination with RF and ACPA, to identify patients at high risk of developing severe RA outcomes. Patients within the Swiss Clinical Quality Management registry with a biobank sample were tested for RF, ACPA, anti-CarP, and anti-PAD3 antibodies. We examined the association of each autoantibody with DAS28, HAQ and radiographic damage (Ratingen) at baseline and longitudinally. Analyses included 851 established RA patients and 516 disease controls [axial spondyloarthritis (axSpA = 320) and PsA (196)]. Anti-CarP and anti-PAD3 antibodies were, respectively, present in 22.4% and 10.7% of the whole RA population, and in 13.2% and 3.8% of the RF and ACPA double seronegative patients. At baseline, RA patients with anti-PAD3 had higher DAS28 (4.2 vs 3.7; P= 0.005) and significantly more radiographic damage (14.9 vs 8.8; P= 0.02) than anti-PAD3-negative patients. In the ACPA-negative subgroup, baseline Ratingen scores were significantly higher in anti-PAD3-positive patients (P= 0.01). The combination of anti-PAD3, RF IgM, and ACPA was associated with significantly higher baseline radiographic scores than the double seropositive group (P= 0.04). The presence of any two of the previous autoantibodies was associated with significantly greater radiographic progression over 10 years than if all were absent (P= 0.02). There were no differences in RA outcome measures with regards to anti-CarP. Anti-PAD3 antibodies are associated with higher disease activity and joint damage scores in RA patients.

From Rheumatoid Factor to Anti-Citrullinated Protein Antibodies and Anti-Carbamylated Protein Antibodies for Diagnosis and Prognosis Prediction in Patients with Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease mainly involving synovial inflammation and articular bone destruction. RA is a heterogeneous disease with diverse clinical presentations, prognoses and therapeutic responses. Following the first discovery of rheumatoid factors (RFs) 80 years ago, the identification of both anti-citrullinated protein antibodies (ACPAs) and anti-carbamylated protein antibodies (anti-CarP Abs) has greatly facilitated approaches toward RA, especially in the fields of early diagnosis and prognosis prediction of the disease. Although these antibodies share many common features and can function synergistically to promote disease progression, they differ mechanistically and have unique clinical relevance. Specifically, these three RA associating auto-antibodies (autoAbs) all precede the development of RA by years. However, while the current evidence suggests a synergic effect of RF and ACPA in predicting the development of RA and an erosive phenotype, controversies exist regarding the additive value of anti-CarP Abs. In the present review, we critically summarize the characteristics of these autoantibodies and focus on their distinct clinical applications in the early identification, clinical manifestations and prognosis prediction of RA. With the advancement of treatment options in the era of biologics, we also discuss the relevance of these autoantibodies in association with RA patient response to therapy.

Autoantibodies are major predictors of arthritis development in patients with anti-citrullinated protein antibodies and musculoskeletal pain

Objectives: Predictors of arthritis development are highly warranted among patients with anti-citrullinated protein antibodies (ACPAs) and musculoskeletal symptoms to optimize clinical management. We aimed to identify clinical and laboratory predictors of arthritis development, including biochemically assessed alcohol consumption, among ACPA-positive patients with musculoskeletal pain. Method: 82 ACPA-positive individuals with musculoskeletal pain but no clinical arthritis were followed for a median of 72 months (interquartile range 57–81 months). We evaluated the prognostic value of baseline clinical and laboratory factors including smoking, symptom duration, age, gender, shared epitope, rheumatoid factor (RF), anti-carbamylated protein antibodies, ACPA levels, erythrocyte sedimentation rate, C-reactive protein levels, tender joint count, patient-reported general well-being, 28-joint Disease Activity Score, and alcohol consumption as measured by phosphatidyl ethanol (PEth) levels in whole blood. Results: During follow-up, 48% developed at least one arthritis. Multivariable analysis revealed an increased risk of arthritis development with RF positivity [hazard ratio (HR) = 2.3, 95% confidence interval (CI) 1.1–4.8, p = 0.028] and higher ACPA levels (HR = 1.0, 95% CI 1.000–1.001, p = 0.002). High levels of RF (HR = 4.4, 95% CI 1.7–11) entailed the highest HR in this ACPA-positive population. Neither clinical characteristics nor alcohol consumption measured by PEth conferred significant prognostic value. Conclusions: ACPA levels and concurrent presence of RF are independent predictors of arthritis development among ACPA-positive patients with musculoskeletal pain. The results are compatible with a dose–response relationship between RA-related autoantibodies and risk of arthritis development.

Improved classification of rheumatoid arthritis with a score including anti-acetylated ornithine antibodies

The presence of rheumatoid factor (RF) or anti-cyclic citrullinated peptide (anti-CCP) autoantibodies contributes to the current rheumatoid arthritis (RA) classification criteria. These criteria involve stratification on antibody levels, which limits reproducibility, and underperform in the RA patients without RF and anti-CCP. Here, we have explored if two anti-acetylated peptide antibodies (AAPA), anti-acetylated lysine (AcLys) and anti-acetylated ornithine (AcOrn), could improve the performance of the current criteria. The analysis was done in 1062 prospectively-followed early arthritis (EA) patients. The anti-AcOrn were more informative than the anti-AcLys, the conventional RA antibodies and the anti-carbamylated protein antibodies. The anti-AcOrn produced a classification that did not require antibody levels and showed improved specificity (77.6% vs. 72.6%, p = 0.003) and accuracy (79.0% vs. 75.8%, p = 0.002) over the current criteria. These improvements were obtained with a scoring system that values concordance between anti-AcOrn, RF and anti-CCP. No significant gain was obtained in sensitivity (80.2% vs. 78.8%, p = 0.25) or in improving the classification of the RA patients lacking RF and anti-CCP, although the anti-AcOrn ranked first among the analysed new antibodies. Therefore, the anti-AcOrn antibodies could contribute to the improvement of RA classification criteria by exploiting antibody concordance.

Anti-carbamylated protein antibodies in premenopausal rheumatoid arthritis women: relation to disease activity and bone loss.

Anti-carbamylated protein antibodies (anti-CarP Abs) are present in patients with RA, however, their association with bone loss is not confirmed. The purpose of this study was to determine the relation between the serum level of anti-CarP Abs in premenopausal RA women and disease activity and bone loss. This case-control study was conducted on 48 premenopausal women with RA and 48 matched healthy premenopausal women. All RA women were subjected to clinical examination, disease activity assessment using the 28-joint DAS (DAS28) and Clinical Disease Activity Index (CDAI), functional assessment using the HAQ, physical activity assessment using the International Physical Activity Questionnaire (IPAQ), fatigue assessment using the Modified Fatigue Impact Scale (MFIS), serological tests as well as anti-CarP Abs using ELISA. Moreover, the BMD was measured by DXA and plain X-ray of both hands was done to assess juxta-articular osteopenia and erosions. The anti-CarP Abs level was significantly higher in RA patients than in healthy controls. The serum level of anti-CarP Abs had a significant positive correlation with the RA DAS28, CDAI, HAQ, MFIS and original Sharp score, while a significant negative correlation was present with the IPAQ. Anti-CarP Abs were negatively correlated with either spine BMD or Z-score and positively correlated with the original Sharp score. Anti-CarP Abs were higher in premenopausal RA women compared with older and BMI matched healthy women. Anti-CarP Abs are associated with higher RA disease activity, increased disability and fatigability and decreased physical activity. Moreover, anti-CarP Abs are associated with systemic trabecular bone loss as well as local bone loss.

The Evaluation of Some Biomarkers According to Rheumatoid Factor in Early Diagnosis of Rheumatoid Arthritis from Iraqi Patients

Rheumatoid arthritis (RA) was a chronic inflammatory autoimmune disease for long-term that primarily affects small joints and leads to chronic inflammation in synovial. The aimed of the study to identify the relationships among some serological markers (antibodies to citrullinated protein/peptide antigens (ACPAs), anti-mutated citrullinated vimentin (anti-MCV), anti-carbamylated protein (Anti-Carp), anti- heterogeneous nuclear ribonucleoproteins (anti-hnRNP) and Glucose-6-phosphate isomerase (GPI)) and early diagnosis of RA. The study involved (60) Patients of newly diagnosis with RA that divided in to two subgroups (30 RF positive and 30 RF negative) groups and 30 subjects as healthy control group. The serological data from serum concentration of (ACPAs, Anti-MCV, Anti-Carp, Anti-hnRNP, G6PI) estimated by ELISA methods, RF estimated by latex agglutination kits. The results revealed that ACPAs, Anti-MCV, Anti-Carp, Anti-hnRNP, G6PI having a statistical significants at the mean±SD titer different between the two RF groups, a higher value among the refractory RA patients in comparison with the control group. Also effectively distinguishing RA patients groups for RF+ve and RF-ve with showed the sensitivity and specificity of, ACPAs (90.8% , 94.1%, and 88.2%, 86.6%); anti-MCV (66.7% , 33.0% and 70.0%, 70.2%); anti-CarP (76.7% , 90.0% and 93.3%, 78.5%); anti-hnRNP (74.9% , 61.9% and 71.4%, 70.9%) and GPI (77.3% , 76.7% and 84.4%, 80.1%) respectively. This study confirm the importance of measuring multiple serum biomarkers and their com­binations with high diagnostic value for RA and provide sup­port for the early diagnosis of RA.

Mass-spectrometric identification of carbamylated proteins present in the joints of rheumatoid arthritis patients and controls

Antibodies targeting post-translationally modified proteins, such as anti-carbamylated protein antibodies (anti-CarP antibodies) are present in the sera of rheumatoid arthritis (RA) patients. These autoantibodies associate with increased risk of RA development and with severity of joint destruction. It is not known which proteins in the RA joint are recognised by anti-CarP antibodies. Therefore, we investigated the presence and identity of carbamylated proteins in the human (inflamed) joint. We obtained synovium, cartilage and synovial fluid from RA joints. Cartilage and synovium were obtained from controls. Samples were processed and used for immunohistochemistry or mass-spectrometric analysis to investigate the presence of carbamylated proteins. Anti-CarP antibody reactivity towards identified carbamylated proteins was tested by ELISA. Immunohistochemistry showed extensive staining of RA and control synovial tissue. Whole proteome analyses of the joint tissues revealed a large number of carbamylated peptidyllysine residues. We identified many carbamylated proteins in cartilage and were also able to detect carbamylation in synovial tissue and synovial fluid. Carbamylation was not exclusive to the RA joint and was also present in the joints of controls. Anti-CarP antibodies in the sera of RA patients were able to recognise the identified carbamylated proteins. We conclude that numerous carbamylated proteins are present in the RA joint. These carbamylated proteins can be recognised by anti-CarP antibodies, substantiating the notion that anti-CarP antibodies may play a role in the pathogenesis of RA.

Elevated levels of anti-carbamylated protein antibody in patients with rheumatoid arthritis: association with disease activity and bone destruction

To measure the serum levels of anticarbamylated protein (CarP) antibodies in patients with rheumatoid arthritis (RA) in China and to evaluate the association of anti-CarP antibodies with clinical parameters and...

Anti-carbamylated Protein Antibodies and Serum Level of 14-3-3 Protein for Early Detection of Rheumatoid Arthritis Patient in Correlation with Rheumatoid Factor, Anti-CCP Antibodies, Disease Activity and Joint Damage using High Frequency Musculoskeletal Ultrasound

Background: Rheumatoid arthritis (RA) is a common progressive chronic inflammatory autoimmune disease which affects mostly small joints, causing pain, swelling, deformity, and disability. Although progress has been made in exploring RA nature, still there is a lot to know about the disease pathogenesis, diagnosis, and treatment. Aim of the Work: To investigate the role of serum anti-carbamylated protein antibodies and 14-3-3η in the diagnosis of RA compared to rheumatoid factor (RF), anti-CCP antibodies, and highfrequency musculoskeletal ultrasound used to assess the disease activity and joint damage. Methods: Serum anti-carbamylated protein antibodies and 14-3-3η were measured using ELISA in 61 RA patients and 26 normal controls. RA Disease Activity Score (DAS 28), X-ray and musculoskeletal ultrasound (hands and feet), carotid ultrasound (Intima-Media Thickness IMT) were used in assessing the RA disease. Results: Anti-carbamylated protein antibodies were significantly elevated in RA patients 4.5 (4.1- 8.9 U⁄ml) compared to the control 3.2(1.9- 4.3 U⁄ml) (p&lt; 0.001) but 14-3-3η showed no significant difference. There was a significant positive correlation between anti-carbamylated protein antibodies, 14-3-3η levels and disease activity score assessed by DAS 28, increased IMT measured by carotid duplex, total synovitis and total erosion score were assessed by musculoskeletal ultrasound. There was no correlation between RF and anti-CCP antibodies. Anti-carbamylated protein antibodies were found to have 66.7% sensitivity and 85.2% specificity in RA diagnosis, while 14- 3-3η had 51.9% sensitivity and 72.1% specificity. Conclusion: Anti-carbamylated protein antibodies and 14-3-3η have a high sensitivity and specificity in RA diagnosis and had a correlation with the disease activity and joint damage.

Anti-carbamylated protein antibodies: are they useful for the diagnosis of rheumatoid arthritis?

ACR/EULAR-2010 classification criteria for rheumatoid arthritis (RA) rely heavily on the presence of anti-citrullinated peptide antibody (ACPA). The role of anti-carbamylated protein antibodies (anti-CarP) in this context is uncertain. We aimed to investigate the value of anti-CarP for RA classification in patients with early inflammatory arthritis. Patients (n=402) were recruited from an early arthritis clinic and followed for 24 months. Healthy controls (n=95) were included. An anti-CarP ELISA was performed (aU/mL). Statistical analysis used regression and AUC analysis. The criteria for RA were met by 195/402 patients at inclusion; 28 developed RA during follow-up and 179 had other diagnosis (non-RA). 97/195 (49%) RA patients were anti-CarP+ (median 250 uA/mL [IQR 25-762]). In the group that progressed to RA, 7/28 (25%) were positive (82 uA/mL [13-235]) compared to non-RA (p=0.001) with 13/179 (7%) positive (26 uA/mL [5-80]). Being anti-CarP+ alone was observed in 17 patients of whom 7 (41%) were RA. Levels/positivity were not associated with other parameters. Anti-CarP+ had an odds ratio (OR) 6.5 for predicting RA (OR=17.1 for ACPA+ and OR=2.5 for RF+). In ACPA- patients, anti-CarP+ was also predictive of RA (OR=2.39). Being ACPA+/anti-CarP+/RF+ had a high predictive value for RA (OR=29.9 sensitivity/specificity (sen/spe) 33%/99%, positive/negative predictive values (ppv/npv) 97%/54%), however, being ACPA+/anti-CarP+ was superior (OR=36.1 sen/spe=41%/99%, ppv/npv=98%/57%) while being ACPA+/RF+ was inferior (OR=11.9, sen/spe=54%/95%, ppv/npv=94%/62%). For RA classification, anti-CarP+ was less sensitive than ACPA, but more specific than RF. Anti-CarP+ may prove useful, classifying early arthritis patients, notably ACPA- patients.

SAT0030 A NOVEL ASSOCIATION BETWEEN ANTI-CARBAMYLATED ANTIBODIES AND INTERSTITIAL LUNG DISEASE IN PATIENTS WITH RHEUMATOID ARTHRITIS

Background:Interstitial lung disease (ILD) is associated with a significant increase in morbidity and mortality in patients with rheumatoid arthritis (RA). Therefore, an early diagnosis is fundamental. Anti-carbamylated proteins (Anti-CarP) have been described in different chronic respiratory diseases without a previous history of RA.Objectives:The aim of this study was to analyse the association between Anti-CarP and ILD in RA patients.Methods:We performed a cross-sectional study, including RA patients fulfilling the 2010 ACR/EULAR criteria. The main population comprised 2 groups: 1) RA patients diagnosed with ILD (RA-ILD group) and 2) RA patients without ILD (non-ILD RA group). ILD was diagnosed by high-resolution tomography and confirmed by a multidisciplinary committee. Three IgG Anti-CarP autoantibodies against fetal calf serum (Anti-FCS), fibrinogen (Anti-Fib), and fibrine/filagrine homocitrullinated peptide (Anti-CFFHP) and one IgA against FCS (Anti-FCS-IgA) were determined by home-made ELISA. Associations between Anti-CarPs and ILD were explored using multivariable logistic regression adjusted by a set of variables known to be related to the development of ILD: smoking, sex, age, RA disease duration, RF and ACPA. An independent replication sample was obtained to validate our findings from another hospital.Results:The main population included 179 patients: 37 were included in the RA-ILD group, and 142 in the non-ILD RA group. Most patients were female (79%), with a mean age of 59.7±13 years with a mean disease duration of 6.6±5 years. Baseline features are shown in table 1. The replication sample was composed of 25 patients in the RA-ILD group and 50 patients in the non-ILD RA group. We found that Anti-CarPs specificities were more frequent in RA-ILD patients (Anti-FCS 70% vs. 43%; Anti-Fib 73% vs. 51%; Anti-CFFHP 38% vs. 19%; Anti-CarP-IgA 51% vs. 20%, p&lt;0.05 for all comparisons). Serum mean titers of Anti-CarPs were higher in RA-ILD patients with significant statistical differences for all of them, except Anti-Fib. The multivariate analysis showed that Anti-CarPs specificities were independently associated with ILD (Anti-FCS (OR: 3.42; CI95%: 1.13-10.40), Anti-Fib (OR: 2.85; CI95%: 0.83-9.70), Anti-CFFHP (OR: 3.11; CI95%: 1.06-9.14) and Anti-FCS-IgA (OR: 4.30; CI95%: 1.41-13.04). In the replication sample our findings were validated only for Anti-FCS (OR: 10.42; CI95%: 1.68-64.46).TABLE 1.Main population demographic, clinical, therapeutic, and autoantibody status features.RA-ILDn:37Non-ILD RAn:142p valueFemale (%)25 (68)116 (82)NSAge mean (±SD)67.3 (10.1)57.7 (12.9)&lt;0.005Mean disease duration (±SD)11.6 (7.1)5.3 (13.3)&lt;0.005Ever smokers (%)21 (57)62 (44)NSSmoking cumulative dose (±SD)30.7 (11.1)21.8 (12)&lt;0.005Caucasian (%)31 (84)120 (85)NSTreatmentGlucocorticoids (%)25 (68)81 (57)NScsDMARDs (%)33 (89)132 (86)NSMTX (%)20 (54)95 (67)NSbDMARDs (%)11 (30)36 (25)NSMean DAS28 (±SD)3.71 (1.35)2.74 (1.05)&lt;0.005Erosive disease (%)26 (70)63 (44)&lt;0.005Mean HAQ-DI (CI-95%)0.69 (0.53-0.85)0.31 (0.24-0.38)&lt;0.005ACPA positive (%)29 (78)99 (70)NSMedian titer ACPA (IQR) CU674 (2,215)143 (1,132)NSRF positive (%)28 (76)83 (59)NSMedian titer RF (IQR) IU105 (298)34 (110)NSConclusion:A strong association between RA-ILD and Anti-CarP was found independently of cofounders, including RF and ACPA. Our findings suggest a possible link between Anti-CarP and the development of ILD.Disclosure of Interests:Raul Castellanos-Moreira Speakers bureau: Lilly, MSD, Sanofi, UCB, Sebastian Rodriguez-Garcia: None declared, Katherine Cajiao: None declared, Gabriela Jimenez: None declared, Maria Jose Gomara: None declared, Virginia Ruiz Speakers bureau: Lilly, Pfizer, Ivette Casafont-Solé: None declared, Julio Ramirez: None declared, José Gomez Puerta Speakers bureau: Abbvie, Eli Lilly, BMS, Roche and Pfizer, Susana Holgado Pérez: None declared, Juan de Dios Cañete: None declared, Isabel Haro: None declared, Raimón Sanmartí Speakers bureau: Abbvie, Eli Lilly, BMS, Roche and Pfizer

SAT0585 GEO-EPIDEMIOLOGY OF AUTOANTIBODIES IN RA: DIFFERENT PREVALENCES IN FOUR ETHNICALLY DIVERSE RA POPULATIONS

Background:Rheumatoid arthritis (RA) has been described in virtually every ethnic population. Most RA patients harbor anti-modified protein antibodies (AMPAs), including anti-citrullinated protein (ACPA), anti-carbamylated protein (anti-CarP), anti-malondialdehyde acetaldehyde (anti-MAA), and anti-acetylated protein antibodies (AAPA). However, it is unclear whether differences exist in the AMPA response between different ethnic groups. Such differences could provide new clues to genetic and environmental factors contributing to autoantibody development.Objectives:To investigate the prevalence of different AMPA in four ethnically diverse RA populations, and their association with smoking.Methods:Enzyme-linked immunosorbent assays were used to measure anti-CarP IgG, anti-MAA IgG (both in-house), and anti-acetylated vimentin IgG (Orgentec) in ACPA-positive sera of Dutch (NL, n=103), Japanese (JP, n=174), Canadian First Nations People (FN, n=100), and black South Africans (SA, n=67) fulfilling the 1987 ACR classification criteria for RA. Ethnicity-matched local healthy controls were used to calculate cohort-specific cut-offs. Logistic regression was used to identify whether ever-smoking was associated with AMPA seropositivity in each cohort, corrected for age, gender, and disease duration. Random-effects meta-analysis was used to pool the resulting odds ratios (OR).Results:For all three AMPAs, median levels were higher in FN and especially SA than NL and JP patients (Figure 1). The median autoantibody levels in arbitrary units (in % of patients positive) for NL, JP, FN and SA RA patients were: anti-CarP IgG: 1157 (47%), 994 (43%), 1642 (58%) and 2336 (76%) (p&lt;0.001); anti-MAA IgG: 131 (29%), 179 (22%), 251 (29%) and 257 (53%) (p&lt;0.001); AAPA: 133 (20%), 136 (17%), 153 (38%) and 316 (28%) (p&lt;0.001). Prevalence, meaning positivity, also differed significantly between cohorts for all AMPAs (p&lt;0.001).There were also marked differences in total IgG levels in mean (SD) g/L: 13 (4) for NL, 17 (6) for JP, 18 (6) for FN, and 25 (8) for SA (p&lt;0.001). When the autoantibody levels were normalized to total IgG, the differences in became less pronounced between cohorts (Figure 2). The median arbitrary units per g/L Total IgG for NL, JP, FN and SA RA patients were: anti-CarP IgG: 54, 25, 53, and 79; anti-MAA IgG: 6, 5, 8, and 9; and AAPA: 2, 2, 2, and 3, suggesting that autoantibody level differences may partly correspond to cohort-specific differences in total IgG, although the overall trend of higher levels in SA persisted. There was no association between smoking and anti-CarP or anti-MAA positivity, with pooled OR (95% CI) of 1.31 (0.79-2.18) and 0.85 (0.46-1.56), respectively. However, smoking was positively and consistently associated with AAPA positivity in each cohort: pooled OR (95% CI) of 2.01 (1.06-3.81).Conclusion:In these ACPA-positive ethnically diverse RA populations, levels and prevalence of various AMPAs differ, suggesting that ethnic background and environment may influence the development of the autoantibody response in RA. Despite these differences, our results imply smoking as a consistent risk factor for AAPA across different ethnic backgrounds.Disclosure of Interests:Emma C. de Moel: None declared, Veerle Derksen: None declared, Leendert A Trouw: None declared, Chikashi Terao: None declared, Mohammed Tikly: None declared, Hani El-Gabalawy: None declared, Holger Bang Grant/research support from: Employee of Orgentec Diagnostika, Thomas Huizinga Grant/research support from: Ablynx, Bristol-Myers Squibb, Roche, Sanofi, Consultant of: Ablynx, Bristol-Myers Squibb, Roche, Sanofi, Rene Toes: None declared, Diane van der Woude: None declared

THU0113 ANTI CARBAMYLATED PROTEIN ANTIBODY IN INDIAN POPULATION WITH RHEUMATOID ARTHRITIS – A BETTER PREDICTOR OF DISEASE OUTCOME

Background:While diagnosis of RA relies mainly on detection of RF and ACCP antibodies at present, anti carbamylated protein antibody which has strong association with severe joint damage and poorer disease outcome1can be found in even healthy individuals years before onset of symptoms of RA2,3thus enabling early initiation of DMARDs and reducing complications of RA.4Objectives:Assess prevalence of anti-CarP Ab in RA patients in India and assess correlation between joint involvement, X-ray erosions and extra articular features between anti-CarP Ab positive and negative RA patients.Methods:Cross-sectional study (2017-2019) conducted among 150 RA patients who met 2010 ACR/EULAR criteria in tertiary referral hospital. Tests for CRP, RF and ACCP were done, DAS28-CRP scores were calculated at presentation &amp; X-ray images of hands and feet obtained.Anti-CarP Ab was tested using ELISA. Cut-off level for positivity was defined using ROC curve as 13.48 U/mL, with specificity 92.73%, sensitivity 85.26%, PPV 95.29% and NPV 78.46%.Results:Venn diagram representing overlap of sero-markersStudy population had 18% males, 82% females, 72% RF+ cases (108), 78% ACCP+ cases (117) and 47.3% anti-CarP Ab+ cases (71).48% of the 25 seronegative (RF-ACCP-) cases tested positive for anti-CarP Ab, indicating its ability in diagnosing more RA cases in conjunction with present markers.Testing association between RF and anti-CarP Ab and between ACCP and anti-CarP Ab proved to be statistically insignificant showing that anti-CarP Ab is independent of the incumbent markers in diagnosing RA.69.4% of RA patients with high disease activity (DAS &gt;3.2) were positive for anti-CarP Ab and showed statistically significant association.Association between DAS28 CRP and anti-CarP AbAlso while 39 of 71 (54.9%) anti-CarP Ab positive RA cases had joint deformities and 23 of 71 (32.4%) of anti-CarP Ab positive cases had extra articular involvement and were statistically significant, 27 of 71 anti-CarP Ab positive cases showed joint erosions in X-ray but was not statistically significant.In comparison, only extra articular involvement showed a statistical significance in ACCP positive patients whereas joint deformities and joint erosions on X-rays showed no correlation to ACCP.Joint DeformityTotalΧ2p valueAbsentPresentanti-CarP antibodyNegative5029795.0090.02563.3%36.7%100.0%Positive32397145.1%54.9%100.0%Total8268150Extra articular featuresTotalΧ2p valueAbsentPresentanti-CarP antibodyNegative6811797.2780.00786.1%13.9%100.0%Positive48237167.6%32.4%100.0%Total11634150Conclusion:Anti-CarP antibody positivity is indicative of increased disease activity (DAS28-CRP score), joint deformities and extra articular involvement in RA patients. Aside from its presence demonstrated early in the course of RA through other studies, its ability in diagnosing RA cases in otherwise seronegative (RF and ACCP negative) population and the high sensitivity, specificity and diagnostic accuracy exhibited paves the way for its routine use as a diagnostic tool in conjunction with RF and ACCP for diagnosing RA and predicting disease outcome.

FRI0376 EFFECT OF CARBAMYLATED LOW-DENSITY LIPOPROTEINS ON BONE CELLS HOMEOSTASIS

Background:Carbamylation is a post-translational modification occurring under several conditions such as uremia, smoking and chronic inflammation as in rheumatoid arthritis (RA). Low-density lipoproteins (LDL) represent a target of carbamylation. Carbamylated-LDL (cLDL) have an increased inflammatory and atherogenic potential. Growing evidence supports an influence of modified lipids on bone cells homeostasis. However, the role of cLDL on bone cells physiology is still unknown.Objectives:Considering the rate of carbamylation and the role of anti-carbamylated proteins antibodies as markers of erosive disease in RA, the purpose of this study is to investigate the effect of cLDL on bone homeostasis.Methods:In-vitrocarbamylation of LDL was performed as previously described by Ok et al. (Kidney Int. 2005). Briefly, native LDL (nLDL) were treated with potassium cyanate (KOCN) for 4 hours, followed by excessive dialysis for 36 hours to remove KOCN. Both osteoclasts (OCs) and osteoblasts (OBLs) were treated at baseline with 20 μg/ml, 100 μg/ml and 200 μg/ml of cLDL or nLDL. To induce osteoclast differentiation, CD14+ monocytes were isolated from peripheral blood of healthy donors by magnetic microbeads separation and then cultured on a 96-wells plate in DMEM media supplemented with RANKL and M-CSF. After 10 days cells were fixed, stained for tartrate-resistant acid phosphatase (TRAP), a marker of OC differentiation, and counted. OBLs were isolated from bone specimens of 3 patients who had undergone to knee or hip arthroplasty for osteoarthritis and treated for 5 days with different concentrations of cLDL and nLDL. OBLs were fixed and stained for alkaline phosphatase positive activity (ALP), a marker of osteogenic differentiation. Total RNA was extracted from cell lysates. Copies of single-stranded complementary DNA (cDNA) were synthesized and analyzed by real-time PCR to evaluate RANKL and Osteoprotegerin (OPG) mRNA expression levels.Results:In OCLs culture, cLDL significantly decreased the number of OC compared to untreated cells (200 μg/ml p=0,0015) and nLDL treated cells (200 μg/ml p= 0,011; 20 μg/ml p= 0,0014) (Fig 1). Moreover, treatment with cLDL induced an increase of not terminally differentiated OCs, reduced dimensions of OCs, less intense TRAP staining and vacuolization (Fig 2). In OBLs culture, cLDL (20, 100 μg/ml) significantly reduced the ALP activity of OBLs compared with untreated cells (p&lt;0.05) (Fig 3). nLDL did not affect the ALP expression. Treatment with cLDL stimulated RANKL mRNA expression in osteoblasts increasing the RANKL/OPG ratio (Fig 4).Fig 1.Fig 2.Fig 3.Fig 4.Conclusion:cLDL induce a significant depression of OC and OBL differentiation. Moreover, cLDL increase RANKL expression in OBL, unbalancing bone tissue turnover towards bone resorption. Accordingly, cLDL could be implicated in the bone loss characterizing several conditions associated to an increased carbamylation, such as RADisclosure of Interests:Bruno Lucchino: None declared, Martina Leopizzi: None declared, Tania Colasanti: None declared, Valeria Di Maio: None declared, cristiano alessandri Grant/research support from: Pfizer, Guido Valesini: None declared, fabrizio conti Speakers bureau: BMS, Lilly, Abbvie, Pfizer, Sanofi, Manuela Di Franco: None declared, Francesca Romana Spinelli Grant/research support from: Pfizer, Consultant of: Novartis, Gilead, Lilly, Sanofi, Celgene, Speakers bureau: Lilly

Anti-carbamylated protein antibodies as a clinical response predictor in rheumatoid arthritis patients treated with abatacept.

Carbamylation is an irreversible post-translational modification of proteins. The presence of anti-carbamylated protein antibodies (anti-CarP) has been observed in rheumatoid arthritis (RA). This study was focused to verify whether anti-CarP antibodies can be used as a predictive factor of clinical response to abatacept (CTLA4-Ig) in RA patients. Sixty RA patients treated with abatacept were enrolled. A home-made ELISA for anti-CarP and a commercial anti-CCP3.1 kit for anti-citrullinated proteins antibodies (anti-CCP) were applied to determine serum levels every six months of therapy. Rheumatoid factor (RF) was also tested. Anti-CarP positive patients (n=18) were younger (p=0.01) and with a longer disease duration (p=0.05) when compared to anti-CarP negative patients (n=42) at baseline. Considering the entire cohort, a significant reduction of anti-CarP titre after twelve-months of treatment was shown (p<0.01). A significant reduction of Disease Activity Score (DAS) 28-C-reactive protein (CRP) in the first six months of therapy was found in the subgroup of anti-CarP positive patients in comparison with the negative ones (p=0.003). No significant results were found by dividing the cohort using the positivity to anti-CCP and/or RF. Earlier onset and a longer disease duration in anti-CarP positive patients might suggest they are specific risk factors for RA in this subgroup of patients. The correlation between the anti-CarP positivity at baseline and the reduction of disease activity during the first six months of treatment with abatacept allowed us to hypothesise that anti-CarP antibodies, but not anti-CCP and/or RF, could be used as a good clinical response predictor.

Anti-carbamylated proteins antibody repertoire in rheumatoid arthritis: evidence of a new autoantibody linked to interstitial lung disease

ObjectiveTo analyse the association between anti-carbamylated protein antibodies (Anti-CarP) and interstitial lung disease (ILD) in rheumatoid arthritis (RA) patients.MethodsCross-sectional study including RA patients fulfilling the 2010 ACR/EULAR criteria. The main...

A predominant involvement of the triple seropositive patients and others with rheumatoid factor in the association of smoking with rheumatoid arthritis

The major environmental risk factor for rheumatoid arthritis (RA) is smoking, which according to a widely accepted model induces protein citrullination in the lungs, triggering the production of anti-citrullinated protein antibodies (ACPA) and RA development. Nevertheless, some research findings do not fit this model. Therefore, we obtained six independent cohorts with 2253 RA patients for a detailed analysis of the association between smoking and RA autoantibodies. Our results showed a predominant association of smoking with the concurrent presence of the three antibodies: rheumatoid factor (RF), ACPA and anti-carbamylated protein antibodies (ACarPA) (3 Ab vs. 0 Ab: OR = 1.99, p = 2.5 × 10–8). Meta-analysis with previous data (4491 patients) confirmed the predominant association with the concurrent presence of the three antibodies (3 Ab vs. 0 Ab: OR = 2.00, p = 4.4 ×10–16) and revealed that smoking was exclusively associated with the presence of RF in patients with one or two antibodies (RF+1+2vs. RF−0+1+2: OR = 1.32, p = 0.0002). In contrast, no specific association with ACPA or ACarPA was found. Therefore, these results showed the need to understand how smoking favors the concordance of RA specific antibodies and RF triggering, perhaps involving smoking-induced epitope spreading and other hypothesized mechanisms.

Anti-carbamylated protein antibody isotype pattern differs between palindromic rheumatism and rheumatoid arthritis.

Background:A restricted response against citrullinated peptides/proteins, with less isotype usage, has been found in palindromic rheumatism (PR) in comparison with rheumatoid arthritis (RA). We hypothesized that this different antibody response may be observed for other post-translational modified proteins. We compared the prevalence and isotype usage of two specificities of anti-carbamylated peptide/protein antibodies (Anti-CarP) in patients with PR and RA.Methods:Cross-sectional study including 54 patients with pure PR and 53 patients with RA, matched by sex, age, disease duration and ACPA. Anti-CarP specificities were determined by home-made enzyme-linked immunosorbent assay tests using a synthetic chimeric fibrin/filaggrin homocitrullinated peptide (CFFHP) and fetal calf serum (FCS) homocitrullinated protein as antigens. IgG, IgA and IgM isotypes were measured.Results:Anti-CarP were positive (CFFHP or FCS) in 24% and 64% of patients with PR and RA, respectively (p < 0.005). All Anti-CarP isotype proportions were significantly lower in PR than in RA: Anti-CarP-IgG (24% versus 51%), Anti-CarP-IgA (7% versus 34%) and Anti-CarP-IgM (7% versus 36%). Mean titers of Anti-CarP isotypes were also lower in PR. In Anti-CarP positive patients, the isotype distribution differed between PR and RA: IgG Anti-CarP was used in all PR patients and in 79% of RA patients. By contrast, a significantly lower isotype usage of both IgA (31% versus 53%) and IgM (31% versus 56%) was observed in PR patients. No significant differences in clinical or demographic characteristics were observed according to Anti-CarP status in PR patients, except for a higher prevalence of ACPA and higher mean titers of ACPA and rheumatoid factor in Anti-CarP positive patients.Conclusion:Anti-CarP are found in patients with PR but in a lower proportion and with a different isotype usage from in RA, suggesting a distinct B cell response to homocitrullinated antigens in PR.

Significance of anti-carbamylated protein antibodies in patients with rheumatoid arthritis-associated intersitial lung disease

Objective To evaluate the value of anti-carbamylated protein (CarP) antibody in the diagnosis of rheumatoid arthritis-associated intersitial lung diseas (RA-ILD). Methods Clinical and laboratory data and serum samples of patients with RA between December 2017 and June 2019 in Department of Rheumatology, General Hospital of Ningxia Medical University were collected. The patients were subclassified as RA-ILD and RA-without ILD based on computed tomography scans of the chest, Enzyme 1inked immunosorbent assay (ELISA) was used to assess anti-CarP antibody in the serum of each group. The occurrence of ILD and other laboratory indexes were analyzed. Comparison of measurement data between the 2 groups was performed by two independent sample t-test or Mann-Whitney U nonparametric test, while the count data were compared by Chi square test; Receiver operating characteristic curve (ROC) was drawn to determine the cut-off value of anti-CarP antibody to RA-ILD diagnosis and to analyze its diagnostic efficacy. Results The anti-CarP antibody level in the RA-ILD group was 21.14 (12.29, 29.75), which was significantly higher than that in the RA-without ILD group 11.6 (6.66, 19.05) (P=0.000). The difference was statistically significant (P 0.05). The age and disease activity score (DAS28) in the RA-ILD group were significantly higher than those in the RA-withhout ILD group (P 0.05). Conclusion The anti-CarP antibody level in RA-ILD patients is higher than that in RA-without ILD, suggesting that anti-CarP antibody may have a role in the development of RA-ILD.