Mikael Brink

Multiplex analyses of antibodies against citrullinated peptides in individuals prior to development of rheumatoid arthritis.

OBJECTIVE The presence of antibodies against cyclic citrullinated peptides has been demonstrated to precede the onset of symptoms of rheumatoid arthritis (RA) by several years. The aim of this study was to analyze antibodies against 10 citrullinated autoantigen-derived peptides for reactivity before the onset of RA symptoms. METHODS A case-control study was conducted within the Medical Biobank of Northern Sweden. The study was performed in 409 individuals, 386 of whom donated 717 blood samples before the onset of symptoms of RA (pre-patients). The median period of time predating the onset of RA was 7.4 years. A total of 1,305 population-based control subjects were also studied. Antibodies to 10 citrullinated peptides, fibrinogen α573 (Fibα573), Fibα591, Fibβ36-52, Fibβ72, Fibβ74, α-enolase (citrullinated α-enolase peptide 1 [CEP-1]), triple-helical type II collagen peptide C1 (citC1III), filaggrin, vimentin 2-17 (Vim2-17), and Vim60-75, were analyzed using a microarray system. RESULTS The fluorescence intensity of antibodies against Fibβ36-52, Fibβ74, CEP-1, citC1III, and filaggrin was significantly increased in pre-patients compared with controls (P<0.001). The levels of the earliest-detectable antibodies (Fibα591 and Vim60-75) fluctuated over time, with only a slight increase after the onset of disease. The frequency of antibodies against Fibβ36-52, CEP-1, and filaggrin increased gradually, reaching the highest levels before symptom onset. The frequency of a cluster of antibodies, citC1III, Fibα573, and Fibβ74, increased only slightly before the onset of symptoms but increased prominently after disease onset. The odds ratio for the development of RA in individuals expressing both CEP-1 and Fibβ36-52 antibodies (using data from samples obtained <3.35 years predating symptom onset) was 40.4 (95% confidence interval 19.8-82.3) compared with having either antibody alone. CONCLUSION Development of an immune response toward citrullinated peptides is initially restricted but expands with time to induce a more specific response, with levels, particularly those of antibodies against CEP-1, Fibβ36-52, and filaggrin, increasing during the predating time period closer to the onset of symptoms.

The type I IFN signature as a biomarker of preclinical rheumatoid arthritis

Objectives To validate the presence and demonstrate the clinical value of the type I interferon (IFN)-signature during arthritis development. Method In 115 seropositive arthralgia patients who were followed for the development of arthritis (Amsterdam Reade cohort), and 25 presymptomatic individuals who developed rheumatoid arthritis (RA) later, and 45 population-based controls (Northern Sweden cohort), the expression levels of 7 type I IFN response genes were determined with multiplex qPCR and an IFN-score was calculated. The diagnostic performance of the IFN-score was evaluated using Cox regression and Receiver Operating Characteristics (ROC)-curve analysis. Results In 44 of the 115 at-risk individuals (38%) from the Amsterdam Reade cohort, arthritis developed after a median period of 8 months (IQR 5–13). Stratification of these individuals based on the IFN-score revealed that 15 out of 25 IFNhigh individuals converted to arthritis, compared with 29 out of 90 IFNlow individuals (p=0.011). In the Northern Sweden cohort, the level of the IFN-score was also significantly increased in presymptomatic individuals who developed RA compared with population-based controls (p=0.002). Cox regression analysis of the Amsterdam Reade cohort showed that the hazard ratio (HR) for development of arthritis was 2.38 (p=0.008) for IFNhigh at-risk individuals after correction for anticitrullinated protein antibodies (ACPA) and rheumatoid factor (RF). The ROC-curve area under the curve (AUC) for the IFN-score combined with ACPA and RF in the prediction of arthritis was 78.5% (p=0.0001, 95% CI 0.70 to 0.87). Conclusions The results demonstrated clinical utility for the IFN-signature as a biomarker in the prediction of arthritis development.

Associations of antibodies against citrullinated peptides with human leukocyte antigen-shared epitope and smoking prior to the development of rheumatoid arthritis

IntroductionIt has previously been shown that an increased number of antibodies against citrullinated peptides/proteins (ACPA) predate the onset of rheumatoid arthritis (RA). Over time antibody positivity expands, involving more specific responses when approaching the onset of symptoms. We investigated the impact of human leukocyte antigen-shared epitope (HLA-SE) alleles and smoking on the development of ACPA, as well as in combination with ACPA during the state of quiescent autoimmunity (before the onset of symptoms), on the development of RA.MethodsBlood samples donated to the Medical Biobank of Northern Sweden from individuals prior to the onset of symptoms of RA (n = 370) and after onset (n = 203) and from population-based controls (n = 585) were used. Antibodies against 10 citrullinated peptides, fibrinogen (Fibα561-583, α580-600, ß62-81a, ß62-81b, ß36-52), vimentin (Vim2-17, 60-75), filaggrin (CCP-1/Fil307-324), α-enolase (CEP-1/Eno5-21), collagen type II (citC1359-369), and anti-cyclic citrullinated peptide (CCP)2 antibodies were analysed.ResultsHLA-SE-positive individuals were more frequently positive for ACPA compared with HLA-SE-negative individuals prior to the onset of symptoms of RA, particularly for antibodies against CEP-1 and Fibß62-81a (72). Smoking was associated with antibodies against Vim2-17 and citC1359-369. HLA-SE and smoking showed increasing association to the presence of the antibodies closer to disease onset. The highest odds ratio (OR) for development of RA was for the combination of HLA-SE alleles and ACPA positivity, especially for antibodies against Fibß62-81b, CCP-1/Fil307-324, and Fibβ36-52. A gene-environment additive interaction between smoking and HLA-SE alleles for the risk of disease development was found, with the highest OR for individuals positive for antibodies against Fibβ36-52, CEP-1, and Fibα580-600.ConclusionsThe relationships between antibodies against the different ACPA specificities, HLA-SE, and smoking showed a variable pattern in individuals prior to the onset of RA. The combination of smoking and HLA-SE alleles was significantly associated with the development of some of the antibody specificities closer to onset of symptoms, and these associations remained significant at diagnosis. An additive gene-environment interaction was found for several of the antibodies for the development of RA.

The autoantibody repertoire in periodontitis: a role in the induction of autoimmunity to citrullinated proteins in rheumatoid arthritis? Antibodies against uncitrullinated peptides seem to occur prior to the antibodies to the corresponding citrullinated peptides

The autoantibody repertoire in periodontitis : a role in the induction of autoimmunity to citrullinated proteins in rheumatoid arthritis? Antibodies against uncitrullinated peptides seem to occur prior to the antibodies to the corresponding citrullinated peptides

Enriched environment increases spinophilin mRNA expression and spinophilin immunoreactive dendritic spines in hippocampus and cortex

Housing rodents in an enriched environment (EE) induces structural and functional plasticity in the adult brain, including increased dendritic sprouting and number of dendritic spines. However, the molecular mechanisms behind EE-induced brain plasticity remain largely unknown. Circadian rhythm plays an important role in memory processing but the neurobiological mechanisms of how circadian rhythm affects memory and brain plasticity remain controversial. In the current study, we studied the expression of spinophilin, a protein highly enriched in dendritic spines and involved in spine morphology and synaptic plasticity, to examine the effects of EE and circadian rhythm in rats housed in EE for different periods of time. Spinophilin mRNA expression was studied by in situ hybridization and the density of spinophilin immunoreactive puncta was quantified after immunohistochemical staining. Compared to rats living in a deprived environment (DE), we found a transient increase in the density of spinophilin immunoreactive puncta in hippocampus and cortex after 1 week of EE housing and persistent elevations of spinophilin mRNA expression during 1-4 weeks of environmental enrichment. Increased spinophilin expression was found during the light phase of the diurnal cycle, but not the dark phase. Thus, enriched housing altered the diurnal variation in spinophilin mRNA expression, suggesting that circadian modulation is likely to be important for experience dependent plasticity. The current results suggest a possible role for spinophilin in neuronal plasticity induced by environmental enrichment, but further studies are needed to establish a cause-effect relation.

Anti-carbamylated protein antibodies precede the onset of symptoms of rheumatoid arthritis in a Swedish biobank cohort

Background Antibodies against citrullinated peptides of several specificities (ACPAs) have been found to predate the onset of symptoms of rheumatoid arthritis (RA) by years. The presence of a new auto-antibody system, anti-carbamylated protein (anti-CarP) antibodies has been identified in rheumatoid arthritis (RA) even predating the disease onset. Anti-CarP antibodies were associated with RA irrespectively of ACPA status. Objectives The presence of anti-CarP antibodies was evaluated in samples from individuals who subsequently developed RA before symptoms of joint disease and also after disease onset. Methods 223 individuals, with 423 samples, who were identified before onset of symptoms of RA (median 5.2years IQR 2.6 – 8.9 years), as donors to the Medical Biobank of Northern Sweden. 192 of them were also sampled at the time of diagnose, and 197 population controls were identified from the Medical Biobank for analysis of anti-CarP IgG antibodies with ELISA as previously described (1). All samples were also analysed for anti-CCP2 antibodies with ELISA (Euro-Diagnostica). Cut-off levels were identified by receiver operating characteristic (ROC) curves with 97% specificity for anti-CarP. Results The concentration of anti-CarP was significantly increased in the pre-symptomatic individuals compared with controls (median (IQR) 44.0 (142.6) AU/mL vs. 7.9 (43) AU/mL, p<0.001) and increased significantly after disease onset to 198.9 (379.1) AU/mL. The sensitivity for anti-CarP antibodies in the pre-symptomatic individuals including all samples was 13.9% (95%CI 11-17.6%,) as compared to 42.2% (95%CI 35.4-49.3,) after RA was diagnosed. The concentration of antibodies increased significantly the closer to disease onset the sample was collected. Sensitivity, less than 3 years before symptom onset, was 18.6% (95%CI 12.8-26.3). 5.1% of the anti-CCP negative pre-symptomatic individuals were anti-CarP ab positive and 10.4% of the anti-CCP negative RA-patients were anti-CarP positive. The frequencies of anti-CCP antibodies was in the pre-sympomatic individuals 35.7% (95%CI 31.3-40.4) and 74.6% (95%CI 67.9-80.3) in the RA patients Conclusions These results indicate that the anti-CarP antibodies could be involved in the pathogenesis of RA independent of anti-CCP antibodies References Shi J, Knevel R, Suwannalai P, van der Linden MP, Janssen GMC, van Veelen PA, et al. Autoantibodies recognizing carbamylated proteins are present in sera of patients with rheumatoid arthritis and predict joint damage. Proc. Natl. Acad. Sci. U. S. A. 2011;108(42):17372-7. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4530

Antibodies against native collagen and citrullinated proteins precede the development of rheumatoid arthritis with a consecutive pattern

Background and objective Presence of antibodies against cyclic citrullinated peptides (anti-CCP2) has been demonstrated to precede the development of rheumatoid arthritis (RA) by several years. The RR for developing subsequent RA was increased by the combination of HLA- shared epitopes (SE) and also presence of PTPN22 T variant. The underlying process of why RA-patients develop antibodies against certain citrullinated peptides is largely unknown and here the authors have investigated antibody concentration against thirteen citrullinated proteins, besides anti-CCP2, to elucidate their predictive value in the prepatient phase of RA together with information about HLA-SE, and cigarette smoking. Material and methods This study comprised 406 individuals, with 717 samples, who were identified before onset of symptoms of RA (median 7.4 years IQR 3.3–12.6 years), as donors to the Medical Biobank of Northern Sweden. 204 of them were also sampled at the time of diagnose, and have been analysed together with 1305 population controls from the Medical Biobank for concentrations of antibodies towards thirteen different citrullinated peptides in plasma; fibrinogen 36–50, fibrinogen 72, fibrinogen 74, fibrinogen α 36–50, fibrinogen α 621–635, fibrinogen β 60–74, fibrinogen 573, fibrinogen 591, α-enolase 1, collagen Type II C1 arginine and citrulline and U1, CCP-1, vimentin 2–17, vimentin 60–75 using the microarray based ImmunoCAP ISAC system (Phadia Diagnostics, Uppsala, Sweden). All samples were also analysed for anti-CCP2 antibodies with ELISA (Euro-Diagnostics). Cutoff levels were set with 98% specificity. Results The three antibodies with highest sensitivity for predicting RA was anti-CCP2 (33.8%), fibrinogen 36–52 (24.3%) and α enolase 1 (24.1%) counting ever being positive in all prepatient samples. On individual and group level, the concentrations of the citrullinated antibodies increased significantly the closer to onset of symptoms the samples were collected. The number of positive samples also increased the closer to disease onset. The predating time for the antibodies to appear (with concentrations above cut-off) varied significantly between them. Conclusion Citrullinated antibodies and antibodies against native collagen appeared many years before onset of symptoms and at different time points. The concentrations increased gradually with few exceptions until onset of symptoms and there was an epitope spreading. Citrullination was an important preceding process for disease development maybe initiated by an earlier non-citrullinated antibody stimulation.

The combination of three autoantibodies, ACPA, RF and anti-CarP antibodies is highly specific for rheumatoid arthritis: implications for very early identification of individuals at risk to develop rheumatoid arthritis

In rheumatoid arthritis (RA), anti–citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are commonly used to aid in the diagnosis. Although these autoantibodies are mainly found in RA, their specificity is not optimal. It is therefore difficult to identify RA patients, especially in very early disease, based on the presence of ACPAs and RF alone. In addition, anti–carbamylated protein (anti‐CarP) antibodies have diagnostic and prognostic value, since their presence is associated with joint damage in RA patients and also associated with the future development of RA in patients with arthralgia. Therefore, the aim of the present study was to investigate the value of combined antibody testing in relation to prediction and diagnosis of (early) RA.

An increased concentration of receptor activator of nuclear factor kappa-B ligand pre-dates the onset of rheumatoid arthritis

Objectives RANK ligand (RANKL) is involved in destruction and osteoporosis in RA. In this study, the relationships between RANKL and ACPA, anti-carbamylated protein antibodies (anti-CarP), cytokines and chemokines were analysed in individuals before the onset of RA symptoms, and their associations with radiological findings at disease onset were assessed. Methods This was a case-control study performed within the Medical Biobank of Northern Sweden that included 470 pre-symptomatic individuals [334 women and 136 men; mean (s.d.) age 52.3 (9.4) years] using blood samples donated before symptom onset (pre-dating time; 5.0 years) and 96 controls (60 women and 36 men). Plasma was analysed for RANKL (BioVendor, Karasek, Brno, Czech Republic), anti-CCP2 antibodies (Eurodiagnostics, Malmö, Sweden), anti-CarP antibodies (in-house ELISA), ACPA specificities (ISAC-platform, Phadia AB, Uppsala, Sweden) and cytokines/chemokines (Meso Scale Discovery methods, Rockville, MD, USA). Radiographs of hands and feet were graded using the Larsen score. Results The concentration of RANKL was higher in the pre-symptomatic individuals compared with controls; mean (s.e.m.): 0.50 (0.03) vs 0.22 (0.02) nmol/l (P < 0.001). The concentration increased gradually over time until symptom onset but appeared later than ACPA/RF/anti-CarP antibodies. Positivity for these antibodies yielded higher levels of RANKL compared with seronegativity (P < 0.001). RANKL concentrations were significantly associated with IL-6 and IL-10 concentrations. The combination of positivity for RANKL and anti-CarP antibodies resulted in a higher Larsen score at diagnosis β = 6.18 (95% CI: 0.93, 11.43; P = 0.022). Conclusion RANKL concentrations were increased several years before symptom onset for RA, particularly in ACPA/RF/anti-CarP-positive individuals, all detectable earlier than RANKL. Positivity for RANKL and anti-CarP antibodies yielded the highest Larsen score at disease onset.

SAT0065 Acpa against different citrullinated peptides identify specific phenotypes of rheumatoid arthritis

Background Anti-citrullinated protein/peptide antibodies (ACPA) have been suggested to identify a more severe phenotype of rheumatoid arthritis (RA). Objectives In this study in an inception cohort of early RA we have analysed a number of antibodies against different citrullinated and/or mutated peptides using a multiplex platform in relation to the patients disease inflammation and radiological destruction Methods Patients with early RA (≤12 m of symptoms) fulfilling the 1987 ARA criteria (n=1022, 692f/330m, mean age56.7±14.0 years) were sampled at the time of diagnosis and assessed using disease activity score (DAS28) at baseline, 6, 12, 18 and 24 months. Radiographs were graded using Larsen score (baseline and at 24 m). Plasma sampled at baseline was analysed for presence of antibody reactivities against 21 different citrullinated peptides/proteins; Fibrinogen (Fib) α36–50, Fibα573, Fibα591, Fibα621–635, Fibβ36–52, Fibβ60–74, Fibβ62–78 (72), Fibβ62–78 (74), Filaggrin (Fil307–324), α-Enolase peptide 5–21 (CEP-1), Vimentin (Vim) 2–17, Vim60–75, F4-R-Cit, F4-Cit-Cit, F4- Cit-R), or mutated proteins (Bla26, Pept1, Pept5, PeptZ1, PeptZ2) and type II Collagen citrullinated or not using a custom-made microarray assay based on the ImmunoCAP ISAC system (Phadia AB, Sweden). Cut-off levels were at the 98th percentile of controls (n=477). Anti-CCP2 was analysed using ELISA (Euro Diagnostica, Sweden). Results The most frequent appearing ACPA were; Fibβ60–74 (63%), Vim60–75 (56.6%), Fibβ36–52 (55.1%), Fil307–324 (54.9%), CEP-1 (53.7%) and Pept5 (52.0%) besides CCP2 (67.5%). Adding all ACPAs gave additional 13.1% of positivity in the anti-CCP2 negative group, yielding a positivity of 77.5%. The median (IQR) number of positive ACPA-peptide was 8 (11). There was a high degree of correlation between the antibodies, e.g., anti-Fibβ60–74 vs. -Vim60–75, -Fibβ36–52 or anti-CCP2 antibodies and also anti-Fil307–324 vs. -Fibβ36–52, -F4 R-Cit or anti-CCP2 antibodies (rs 0.692–0.79). Positivity for all antibodies was associated with higher ESR (baseline and AUC24). A number of antibodies were associated with both high DAS28 (baseline and AUC24) and radiological findings/progression (anti-CCP2, - Fil307–324, -Vim60–75 and Vim 2–17, and -CEP1 antibodies), whilst some others were more associated with inflammation (DAS28, baseline and AUC24) (anti-Fibβ60–74, -Pept5 and -F4R-cit antibodies) and others more with radiological destruction/progression (anti-Fibβ36–52, -Fibβ74, -PeptZ1, -F4 Cit-R antibodies). Partial least squares regression analyses confirmed the results with significant correlation between radiological progression and antibodies against Vim2–17, Fibβ36–52, CEP1, Fibα621–635, and CCP2 and between DAS28AUC24 and Vim60–75, Vim2–17, Fibα621–635 and F4-R-Cit. Patients treated with biologics during the first 24 months (11.2%) were significantly more frequent positive for anti-CCP2, -Vim60-75, -Fibα36–50, -PeptZ1 and -PeptZ2 antibodies vs. being negative. Conclusions Analyses at baseline, of the ACPA specificity profiles allowed different patterns of disease activity and radiological progression during the first 24 months of the disease to be identified. Disclosure of Interest None declared