Anti-citrullinated Protein/peptide Antibodies Research Articles

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

ObjectivesRheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and...

Rheumatoid Arthritis Serology in Macedonian Patients with Rheumatoid Arthritis: Rheumatoid Factor, Anti-Cyclic Citrulinated Peptide Antibodies or both?

Introduction : In the last 70 years, Rheumatoid Factor (RF) was considered the most useful laboratory marker in patients with rheumatoid arthritis (RA). The next very important milestone for the RA diagnosis was the discovery of anti-citrullinated peptide/protein antibodies (ACPA). The detection of ACPA is usually done with the CCP test of the second generation, CCP2. Objective : To evaluate the performances of RF and CCP2 tests and to see whether or not the performance of both tests together is better then the performances of either of the tests alone. Materials and Methods : We performed a cross-sectional study with 380 participants of which 155 RA patients who fulfilled the American College of Rheumatology (ACR) 1987 classification criteria for RA, 120 patients with inflammatory and other connective tissue diseases (Non-RA) and 105 healthy controls (HC), at the Rheumatology Clinic in Skopje, Macedonia. The patients and controls were tested for RF and ACPA. Results : The mean age of the RA patients was 50 years, and 87 % of the patients were female. The average duration of the disease has been 6 years. In this group of patients, CCP2 test showed identical sensitivity of 0,69 and better specificity than RF (0,95 vs. 0,87 respectively). For the patients who tested positive for both tests, the sensitivity was lower 0,60 and for the patients who were either ACPA or RF positive, sensitivity was slightly higher 0,73. The specificity of 0,88 for both or either of the tests was identical with that of RF. The positive likelihood ratio was 13,8 for the CCP2 test, 5,3 for RF and 5,1 for both tests done together. Ninety four out of 155 RA patients were positive for both ACPA and RF, 20 patients were positive for either one of the antibodies of which 7 (4,5%) RA patients were only anti-CCP positive and 13 (8,3%) patients were only RF positive. Conclusion : The results from our study showed that the sensitivity and specificity of both tests, done in parallel, does not differ much from the sensitivity of either of the tests alone and from the specificity of RF, respectfully. Still, CCP2 test showed the highest specificity and positive likelihood ratio as was expected. The results from our study support the idea that in countries like Macedonia, which can not afford enough CCP2 antibody kits, we may use RF first, especially in patients who are not likely to meet any clinical criteria for RA. In patients with early undifferentiated arthritis or early RA we may use both antibodies in order to select the patients who will need more aggressive treatment.

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens.

IntroductionWe have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).MethodsThe autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit.ResultsTwo peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency.ConclusionsHere we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.

In antisynthetase syndrome, ACPA are associated with severe and erosive arthritis: an overlapping rheumatoid arthritis and antisynthetase syndrome.

Anticitrullinated peptide/protein antibodies (ACPA), which are highly specific for rheumatoid arthritis (RA), may be found in some patients with other systemic autoimmune diseases. The clinical significance of ACPA in patients with antisynthetase syndrome (ASS), a systemic disease characterized by the association of myositis, interstitial lung disease, polyarthralgia, and/or polyarthritis, has not yet been evaluated with regard to phenotype, prognosis, and response to treatment. ACPA-positive ASS patients were first identified among a French multicenter registry of patients with ASS. Additionally, all French rheumatology and internal medicine practitioners registered on the Club Rhumatismes et Inflammation web site were asked to report their observations of ASS patients with ACPA. The 17 collected patients were retrospectively studied using a standardized questionnaire and compared with 34 unselected ACPA-negative ASS patients in a case–control study. All ACPA-positive ASS patients suffered from arthritis versus 41% in the control group (P < 0.0001). The number of swollen joints was significantly higher (7.0 ± 5.0 vs 2.9 ± 3.9, P < 0.005), with a distribution resembling that of RA. Radiographic damages were also more frequent in ACPA-positive ASS patients (87% vs 11%, P < 0.0001). Aside from a significantly higher transfer factor for carbon monoxide in ACPA–ASS patients, lung, muscle, and skin involvements had similar incidences, patterns, and severity in both groups. Although Nonbiologic treatments were similarly used in both groups, ACPA-positive patients received biologics more frequently (59% vs 12%, P < 0.0008), mostly due to refractory arthritis (n = 9). Eight patients received anti-Cluster of differentiation 20 (CD20) monoclonal antibodies (mAbs) with good efficacy and tolerance, whereas 2 of the 5 patients treated with antitumor necrosis factor drugs had worsened myositis and/or interstitial lung disease. After a >7-year mean follow-up, extra-articular outcomes and survival were not different. ACPA-positive ASS patients showed an overlapping RA–ASS syndrome, were at high risk of refractory erosive arthritis, and might experience ASS flare when treated with antitumor necrosis factor drugs. In contrast, other biologics such as anti-CD20 mAb were effective in this context, without worsening systemic involvements.

Anti Citrullinated Protein / Peptide Antibody Assay, Rheumathoid Factor or Both as Shifted Test in Diagnostic and Prognostic Evaluation in Patients with Rheumathoid Arthritis

Aim: The aim of this study was to compare the diagnostic values of laboratory variables, to present quantitative evaluations of the anti citrullinated protein / peptide antibody (ACPA), or anti CCP ( anti-cyclic citrullinated peptide, anti-CCP 2) antibodies in second generation antibody assay diagnostic test with reference to sensitivity and specificity, the predictive value of the positive and negative test and precision of the test for ACPA antibodies, rheumatoid factor, C-reactive protein and DAS 28 index, in the early diagnosis of untreated rheumatoid arthritis. Materials and methods: 70 participants (35 patients with rheumatoid arthritis not treated, 35 individuals as healthy controls) took part in the study. Their serum was examined using ELISA technology of DIA-STAT TM Anti-CCP (Axis–Shield Diagnostics). Rheumatoid factor was examined with the test for agglutination (Latex RF test). Results: We found the presence of ACPA antibodies (sensitivity of the test 65.71%) in 23 of the 35 examined patients with rheumatoid arthritis while rheumatoid factor appeared in 17 patients (sensitivity of the test 48.57%). Twelve patients were ACPA and rheumatoid factor positive, 11 were ACPA positive, but rheumatoid factor negative. Five patients were ACPA negative and rheumatoid factor positive. In 17 rheumatoid factor positive patients, ACPA antibodies were positive in 12 patients. Of 18 rheumatoid factor negative patients, 11 were ACPA positive. In the healthy control group, 1 patient was anti-CCP 2 positive, while 2 patients were rheumatoid factor positive. Conclusion: ACPA antibodies have higher sensitivity and specificity than rheumatoid factor in rheumatoid arthritis.

Smokeless Tobacco (Moist Snuff) Use and the Risk of Developing Rheumatoid Arthritis: Results From a Case–Control Study

To investigate the association between snuff use (smokeless tobacco containing nicotine) and the risk of anti–citrullinated protein/peptide antibody (ACPA)–positive and ACPA-negative rheumatoid arthritis (RA). Data from the Swedish Epidemiological Investigation of Rheumatoid Arthritis, a population-based case–control study including 1,998 incident cases and 2,252 randomly selected controls (matched on age, sex, and residential area) ages 18–70 years, were analyzed. Ever, current, and past moist snuff users were compared with never users. We calculated odds ratios (ORs) with 95% confidence intervals (95% CIs) by means of unconditional logistic regression models. All analyses were adjusted for cigarette smoking, alcohol consumption, and the matching variables. In total, 254 (13%) cases were ever moist snuff users compared with 290 (13%) controls, resulting in an OR of 1.0 (95% CI 0.8–1.2) of RA overall. When exposure to moist snuff was analyzed in relation to ACPA-positive and ACPA-negative disease, no associations were observed. Neither current nor past moist snuff use was related to the risk of any of the 2 RA subgroups. Analyses restricted to never smokers provided similar results. The use of moist snuff was not associated with the risk of either ACPA-positive or ACPA-negative RA. The increased risk of RA associated with smoking is most probably not due to nicotine.

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides—viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein–Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4—were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10−6M>KD>10−7M) and interacted with HCP1 with higher apparent affinity (KD=10−8M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.

Diagnostic value of anti-MCV antibodies for ankylosing spondylitis

Objective To investigate the diagnostic value of anti-mutated citrullinated vimentin（anti-MCV） and anti-cyclic citrullinated peptide（anti-CCP） antibodies for ankylosing spondylitis (AS), and to establish a new diagnostic method of AS based on anti-citrullinated protein/peptide antibodies（ACPAs） detection. Methods Totally 121 AS patients(AS group), 97 rheumatoid arthritis（RA） patients(RA group)and 103 healthy people(control group) were enrolled for the detection of serum levels of anti-MCV and anti-CCP antibodies using ELISA method as a diagnostic test. HLA-B27 in AS patients was tested by flow cytometry, and RF-IgM in RA and AS patients was detected by immune rate nephelometry. Receiver operating characteristics (ROC) curve was used to analyze the diagnostic value and to determine the cut-off value. Anti-MCV and anti-CCP antibodies among each group were compared by Mann-Whitney U test, Chi-square test or Fisher′s exact test was used to compare positive rate. Results The anti-MCV levels in AS group ［11.60 (12.36-25.83) U/ml］were significantly higher than control group ［11.60(8.41-13.54）U/ml ］,U=2 413,P<0.001, while the levels of anti-CCP had no difference between the two groups ［AS group 6.22 （4.30-8.07）U/ml］, and control group ［6.01(3.77-7.58)U/ml］, respectively; U=5 421,P=0.094. The calculated area under the ROC curve of anti-MCV was 0.806,and 14.67 U/ml was the optimal cut-off value with sensitivity of 0.645 and specificity of 0.942. In both HLA-B27 positive and negative AS patients, anti-MCV antibodies levels and positive rate were significantly higher than control group using new cut-off value above （U=133.5, P=0.001; U=2 279.5,P<0.001). Sensitivity of the combination detection of anti-MCV and anti-CCP (MCV+CCP－) or RF-IgM (MCV+RF－) were 60.3% (73/121), 62.8% (76/121) and specificity were 89.7% (87/97), 90.7% (88/97) respectively in differential diagnosis of AS and RA, which were significantly higher than anti-MCV detection alone in specificity (16.5%,16/97). Conclusions Anti-MCV could be a new biomarker for the diagnosis of AS. With high sensitivity and specificity, anti-MCV has an equal diagnostic efficiency in HLA-B27 positive and negative AS patients using our new cut-off value. Additionally， the combination detection of anti-MCV and anti-CCP or RF-IgM could be an effective method for differential diagnosis of AS and RA.（Chin J Lab Med,2014,37: 455-458） Key words: Spondylitis,ankylosing; Peptides,cyclic; Vimentin; Mutation; Rheumatoid factor

SAT0239 T Cell CD80/Cd86 Co-Stimulatory Blockade Effectively Suppresses CD25 (+) in CD4 (+) T Cell Subpopulation but not the ACPA Titers in the Course of 48-Week Treatment of Patients with Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is a common immflamatory autoimmune disease characterized by persistent synovitis and progressive destruction of cartilage and bone in multiple joints. Among the autoantibodies detected in RA patients,...

THU0462 Anti-Citrullinated Peptide/Protein Antibody (ACPA)-Negative RA Shares Large Proportion of Susceptibility Genes with Acpa-Positive RA

BackgroundSusceptibility genes for anti-citrullinated peptide/protein antibodies (ACPA)-positive rheumatoid arthritis (RA) have been successfully discovered by the development of genome-wide association studies (GWAS). However, little is known about the genetic background...

Laboratory biomarkers or imaging in the diagnostics of rheumatoid arthritis?

Rheumatoid arthritis (RA) is a common autoimmune disease in which a heterogeneous course and different pathogenic mechanisms are implicated in chronic inflammation and joint destruction. Despite the diagnostic contribution of anti-citrullinated protein/peptide antibodies (ACPAs) and rheumatoid factors, about one-third of RA patients remain seronegative. ACPAs belong to a heterogeneous family of autoantibodies targeting citrullinated proteins, including myelin-basic protein, several histone proteins, filaggrin and fibrin, fibrinogen or vimentin. In addition to ACPAs, antibodies directed against other post-translationally modified-carbamylated proteins (anti-CarP) were detected in up to 30% of ACPA-negative patients. Using phage display technology, further autoantibodies were recently discovered as candidate biomarkers for seronegative RA patients. Furthermore, in clinical practice, ultrasound may reveal subclinical synovitis and radiographically undetected bone erosions. To improve diagnostic certainty in undifferentiated arthritis and seronegative patients, ultrasound imaging and several new biomarkers may help to identify at risk patients and those with early disease. In this commentary we summarize recent advances in joint ultrasound and future potential of serological biomarkers to improve diagnosis of RA.

A6.8 Elevated antibody levels to porphyromonas gingivalis detected in rheumatoid arthritis patients with a specific anti-citrullinated protein/peptide antibody profile

Background and ObjectivesPorphyromonas gingivalis, a major cause of chronic periodontitis (PD), has been implicated also in the aetiology of rheumatoid arthritis (RA), specifically in anti-citrullinated protein/peptide antibody (ACPA) positive RA,...

A1.41 Effect of multiple cycles of B-cell depletion therapy in patients with rheumatoid arthritis on serological evidence of plasmablast activation, serum baff levels and autoantibody specificity

Background and ObjectivesRituximab induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA), however, all patients eventually relapse. Although B cell return precedes clinical relapse absolute B...

Serum Cytokine Profile of Unaffected First-degree Relatives of Patients with Rheumatoid Arthritis

Various cytokines have been implicated in the pathogenesis of rheumatoid arthritis (RA) and it is known that elevations in multiple cytokines occur prior to disease onset. The objective of our study is to determine whether the cytokine profile in unaffected first-degree relatives (FDR) of patients with RA is distinct from healthy controls. The sera of patients with RA, their unaffected FDR, and healthy controls were measured for 27 cytokines using a luminex-based multiplexed immunoassay. Subjects were also tested for rheumatoid factor and 6 different anticitrullinated protein/peptide antibodies (ACPA). Discriminant analysis was performed to define the cytokine profile of the 3 study groups. Fourteen patients with RA, 37 unaffected FDR, and 27 healthy controls were enrolled. All patients with RA and 49% of FDR were ACPA-positive. Patients with RA had elevated levels of inflammatory cytokines compared to FDR and controls. Lower interleukin 13 (IL-13) levels were independently associated with RA. IL-4 was significantly lower in FDR compared to controls. Using discriminant analysis based on the levels of all cytokines measured, RA, FDR, and healthy controls could be distinguished with 91% accuracy. ACPA-positive subjects had higher levels of IL-9, but lower levels of IL-12p70 and macrophage inflammatory protein-1β. No individual cytokine was associated with ACPA-positivity in FDR, but the entire cytokine profile accurately distinguished ACPA-positive from ACPA-negative FDR. The cytokine profiles of patients with RA, healthy controls, unaffected ACPA-positive FDR, and ACPA-negative FDR appear to be distinct.

Recognition of new citrulline‐containing peptide epitopes by autoantibodies produced in vivo and in vitro by B cells of rheumatoid arthritis patients

Anti-citrullinated peptide/protein antibodies (ACPAs) are highly sensitive and specific markers of rheumatoid arthritis (RA). Identification of peptide epitopes that may detect different subgroups of RA patients might have diagnostic and prognostic significance. We have investigated citrulline- and arginine-containing peptide pairs derived from filaggrin, collagen or vimentin, and compared this citrulline-peptide panel with the serological assays conventionally used to detect ACPAs. Furthermore, we studied if the same citrulline-peptides identify antibody-secreting cells in in vitro cultures of RA B cells. Recognition of citrulline- and arginine-containing filaggrin, vimentin and collagen peptide epitopes were tested by Multipin ELISA system, by indirect ELISA and by a peptide-specific microarray. B cells were purified from blood by negative selection; antibody-producing cells were enumerated by ELISPOT assay. The panel composed of citrulline-peptide epitopes of filaggrin, collagen and vimentin was recognized by RA sera with a sensitivity and specificity comparable with the currently used tests. Moreover, the combined citrulline-peptide panel including the new short epitope peptide of filaggrin, fil311-315, also identified nearly one-third of RA cases that were negative for antibodies against cyclic citrullinated peptides, mutated citrullinated vimentin or for rheumatoid factor. The results with the peptide-specific microarray have shown that although most ACPAs recognizing the four citrulline peptides are IgG, some of them specifically recognizing citrulline-containing filaggrin peptides (fil311-315 and fil306-326) are IgM, and so may be produced either by newly formed activated B cells or by unswitched B memory cells. Furthermore, the citrulline-peptides of filaggrin and vimentin detect ACPA-producing cells, and so could also be applied to study the B cells of RA patients.

Serum C-X-C motif chemokine 13 is elevated in early and established rheumatoid arthritis and correlates with rheumatoid factor levels.

IntroductionWe hypothesized that serum levels of C-X-C motif chemokine 13 (CXCL13), a B-cell chemokine, would delineate a subset of rheumatoid arthritis (RA) patients characterized by increased humoral immunity.MethodsSerum from patients with established RA (the Dartmouth RA Cohort) was analyzed for CXCL13, rheumatoid factor (RF) levels, anticitrullinated peptide/protein antibody (ACPA) and total immunoglobulin G (IgG); other parameters were obtained by chart review. A confirmatory analysis was performed using samples from the Sherbrooke Early Undifferentiated PolyArthritis (EUPA) Cohort. The Wilcoxon rank-sum test, a t-test and Spearman’s correlation analysis were utilized to determine relationships between variables.ResultsIn both the Dartmouth and Sherbrooke cohorts, CXCL13 levels were selectively increased in seropositive relative to seronegative RA patients (P = 0.0002 and P < 0.0001 for the respective cohorts), with a strong correlation to both immunoglobulin M (IgM) and IgA RF levels (P < 0.0001). There was a weaker relationship to ACPA titers (P = 0.03 and P = 0.006, respectively) and total IgG (P = 0.02 and P = 0.14, respectively). No relationship was seen with regard to age, sex, shared epitope status or inclusion high-sensitivity C-reactive protein (hsCRP) in either cohort or regarding the presence of baseline erosions in the Sherbrooke Cohort, whereas a modest relationship with Disease Activity Score in 28 joints CRP (DAS28-CRP) was seen in the Dartmouth cohort but not the Sherbrooke cohort.ConclusionUsing both established and early RA cohorts, marked elevations of serum CXCL13 levels resided nearly completely within the seropositive population. CXCL13 levels exhibited a strong relationship with RF, whereas the association with clinical parameters (age, sex, DAS28-CRP and erosions) or other serologic markers (ACPA and IgG) was either much weaker or absent. Elevated serum CXCL13 levels may identify a subset of seropositive RA patients whose disease is shaped by or responsive to RF production.

The effect of B-cell depletion therapy on serological evidence of B-cell and plasmablast activation in patients with rheumatoid arthritis over multiple cycles of rituximab treatment

B-cell depletion therapy (BCDT) based on rituximab (RTX) induces clinical remission in a majority of seropositive patients with Rheumatoid arthritis (RA). However, all patients eventually relapse. The aim of this study was to determine whether dynamic changes in combinations of serological measures of B-cell activation were associated over up to three cycles of BCDT. We included only RA patients who gave an adequate clinical response, as measured by DAS28. Twenty three patients were studied over 1 cycle, 21 over 2, and 15 over 3 cycles of BCDT. Serum analytes including isotypes of Rheumatoid factors (RhF) and anti-citrullinated protein/peptide antibodies (ACPA), B-cell activating factor (BAFF), serum free light chains (SFLC), soluble CD23 (sCD23), antibodies to tetanus toxoid (TT) and to pneumococcal capsular polysaccharide (PCP) were measured by ELISA at 4 key points in each cycle, namely: Baseline (pre-RTX in each cycle); when B-cell depleted (CD19+B-cells < 5/μl); at B-cell return (CD19+B-cells ≥ 5/μl); and at clinical relapse (ΔDAS28 > 1.2). SFLC were used as a measure of plasmablast activity. As sCD23 is cleaved from naïve B-cells coincident with attaining CD27 expression, levels were used as a novel measure of maturation of B-cells to CD27+. The most consistent changes between baseline and B-cell depletion within all 3 cycles were in SFLC, sCD23 and IgM-RhF which fell and in BAFF levels which rose. After 3 complete cycles of BCDT, both IgM autoantibodies and IgG-CCP had decreased, BAFF levels were higher (all p < 0.05); other analytes remained unchanged compared with baseline. Dynamic changes in λSFLC, sCD23, IgM-RhF and BAFF were also consistently associated with relapse in patients with longer clinical responses after B-cell return. Incremental rises in sCD23 levels in cycles 2 and 3 were correlated with time to relapse. Repopulation of the periphery after BCDT is initiated by naïve B-cells and precedes relapse. Our study showed that differentiation into plasmablasts, attended by sCD23 and SFLC production and IgM-RhF specificity may be required to precipitate relapse in patients experiencing longer responses after RTX. These studies also provide novel information related to the resumption of autoimmune responses and their association with B-cell kinetics following BCDT.

Negative regulation of the peptidylarginine deiminase type IV promoter by NF-κB in human myeloid cells

High titers of anti-citrullinated protein/peptide antibodies (ACPAs) have been detected in sera of rheumatoid arthritis (RA) patients, implicating citrullinating enzymes in the pathogenesis of RA. Peptidylarginine deiminase type IV (PAD4) is a member of the PAD family of citrullinating enzymes and has been linked to RA. Therefore, our aim was to determine how transcription of PAD4 is regulated in the human myeloid lineage. We located the PAD4 transcription start site and promoter and phylogenetic comparisons of the area identified a 200bp conserved region. Bioinformatics analysis predicted the presence of a NF-κB binding site and we tested this via luciferase assays. Intriguingly, mutation of the predicted NF-κB site significantly increased biological activity. We used RT-qPCR to quantify PAD4 expression in HL-60 cells treated with TNF-α to activate the canonical NF-κB pathway and found that PAD4 mRNA was reduced in response to TNF-α treatment. Finally, we used chromatin immunoprecipitation (ChIP) to determine NF-κB enrichment at the PAD4 promoter and the p50 subunit of NF-κB was more highly enriched than p65 at the PAD4 promoter. These results suggest that the p50 subunit of NF-κB may play a role in the repression of PAD4 transcription during inflammation.

Relatives Without Rheumatoid Arthritis Show Reactivity to Anti–Citrullinated Protein/Peptide Antibodies That Are Associated With Arthritis‐Related Traits: Studies of the Etiology of Rheumatoid Arthritis

To examine reactivity to anti-citrullinated protein/peptide antibodies (ACPAs) and determine associations between ACPAs and other rheumatoid arthritis (RA)-related autoantibodies and clinically assessed swollen or tender joints in unaffected first-degree relatives of RA patients. Serum samples were obtained from first-degree relatives without RA according to the 1987 American College of Rheumatology (ACR) and the 2010 ACR/European League Against Rheumatism classification criteria. A bead-based assay was used to measure 16 separate ACPAs in sera from 111 antibody-positive first-degree relatives who were positive on at least 1 visit for any of 5 RA-related autoantibodies (rheumatoid factor [RF], anti-cyclic citrullinated peptide 2 [anti-CCP-2], and RF isotypes), and sera from 99 antibody-negative first-degree relatives who were never autoantibody positive. Cutoffs for positivity for each ACPA were determined using receiver operating characteristic curves derived from data on 200 RA patients and 98 blood donor controls, in which positivity for ≥9 ACPAs had 92% specificity and 62% sensitivity for RA. In first-degree relatives, ACPA reactivity was assessed, and associations between ACPAs (number positive, and positivity for ≥9 ACPAs) and RA-related characteristics were examined. Fifty-seven percent of anti-CCP-2-positive first-degree relatives and 8% of anti-CCP-2- negative first-degree relatives were positive for ≥9 ACPAs. After adjusting for age, sex, ethnicity, and pack-years of smoking, an increasing number of ACPAs was directly associated with the presence of ≥1 tender joint on examination (odds ratio [OR] 1.18, 95% confidence interval [95% CI] 1.04-1.34), with the greatest risk of having ≥1 tender joint seen in first-degree relatives positive for ≥9 ACPAs (OR 5.00, 95% CI 1.37-18.18). RA-free first-degree relatives (even those negative for RF and anti-CCP-2) demonstrate reactivity to multiple ACPAs, and the presence of an increasing number of ACPAs may be associated with signs of joint inflammation. Prospective evaluation of the relationship between these findings and the progression of classifiable RA is warranted.

AB0155 Anti-VCA and EBNA1 antibodies are produced in the rheumatoid synovium in the presence of ectopic lymphoid structures and correlate with ACPA production

BackgroundEctopic lymphoid structures (ELS) are preferential sites for Epstein-Barr virus (EBV) latency and reactivation in the brain of multiple sclerosis (1) and are functional sites of autoreactive B cell activation...