[Application value of anti-carbamylated protein antibody in the diagnosis of rheumatoid arthritis].

OBJECTIVE: To investigate the expression level and application value of anti-carbamylated protein (CarP) antibody in rheumatoid arthritis (RA). METHODS: Demographic data and laboratory test results of RA patients, non-RA patients and healthy controls in the physical examination center were reviewed from December 2018 to June 2019 in the Rheumatology and Immunology Department of the People' s Hospital of Xinjiang Uygur Autonomous Region. The serum concentrations of anti-CarP antibodies in all the subjects were measured by ELISA and statistically analyzed. RESULTS: A total of 259 subjects were included in this study, including 158 in the RA group (45 serum-negative RA patients), 59 in the non-RA group and 42 in the healthy control group. The concentration of anti-CarP antibody in RA group [8.31 (5.22, 15.26) U/mL] was higher than that in non-RA group [4.50 (3.35, 5.89) U/mL] and healthy control group [3.46 (2.76, 4.92) U/mL]. The concentration of anti-CarP antibody in non-RA group was not significantly different from that in healthy control group (P=0.10). Receiver operating characteristic (ROC) curve analysis showed that the sensitivity of anti-CarP antibody in the diagnosis of RA was 58.2%, and the specificity was 93.1%. The sensitivity of the combined detection of anti-CarP antibody, anti-cyclic peptide containing citrulline (CCP) antibody and rheumatoid factor (RF) was 82.3%, and the specificity was 96.5%. The positive rate of anti-CarP antibody in serum-negative RA patients was 44.4% (20/45). Univariate Logisitic regression analysis showed that age, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), RF, glucose-6-phosphate isomerase (GPI), anti-CCP antibody and anti-CarP antibody were risk factors for RA. Multivariate Logisitic regression analysis showed that anti-CCP antibody and anti-CarP antibody were independent risk factors for RA. Spearman correlation analysis showed that there was no significant correlation between anti-CarP antibody and swollen joint count (SJC), tenderness joints count (TJC), ESR, disease activity score for 28 joints (DAS28), clinical disease activity index (CDAI), simplified disease activity index (SDAI). The concentration of anti-CarP antibody in RA with bone erosion (n=88) was higher than that in RA without bone erosion (n=70), and there was significant difference between the two groups (P < 0.05). CONCLUSIONS: Anti-CarP antibody is an effective serological marker for the diagnosis of RA. The combined detection of RF, anti-CCP antibody and anti-CarP antibody can improve its diagnostic value, and anti-CarP antibody may be an effective assistant diagnostic tool for serum negative RA. The high serum concentration of anti-CarP antibody in patients with RA may indicate an increased risk of bone erosion and should be treated early, but further cohort studies are needed for follow-up observation.

Impact of Posttranslational Modification in Pathogenesis of Rheumatoid Arthritis: Focusing on Citrullination, Carbamylation, and Acetylation.

Rheumatoid arthritis (RA) is caused by prolonged periodic interactions between genetic, environmental, and immunologic factors. Posttranslational modifications (PTMs) such as citrullination, carbamylation, and acetylation are correlated with the pathogenesis of RA. PTM and cell death mechanisms such as apoptosis, autophagy, NETosis, leukotoxic hypercitrullination (LTH), and necrosis are related to each other and induce autoantigenicity. Certain microbial infections, such as those caused by Porphyromonasgingivalis, Aggregatibacter actinomycetemcomitans, and Prevotella copri, can induce autoantigens in RA. Anti-modified protein antibodies (AMPA) containing anti-citrullinated protein/peptide antibodies (ACPAs), anti-carbamylated protein (anti-CarP) antibodies, and anti-acetylated protein antibodies (AAPAs) play a role in pathogenesis as well as in prediction, diagnosis, and prognosis. Interestingly, smoking is correlated with both PTMs and AMPAs in the development of RA. However, there is lack of evidence that smoking induces the generation of AMPAs.

Increased disease activity in early arthritis patients with anti-carbamylated protein antibodies.

The initial management of rheumatoid arthritis (RA) has a high impact on disease prognosis. Therefore, we need to select the most appropriate treatment as soon as possible. This goal requires biomarkers of disease severity and prognosis. One such biomarker may be the presence of anti-carbamylated protein antibodies (ACarPA) because it is associated with adverse long term outcomes as radiographic damage and mortality. Here, we have assessed the ACarPA as short-term prognostic biomarkers. The study was conducted in 978 prospective early arthritis (EA) patients that were followed for two years. Our results show the association of ACarPA with increased levels of all the disease activity measures in the first visit after arthritis onset. However, the associations were more significant with the high levels in local measures of inflammation and physician assessment than with the increases in systemic inflammation and patient-reported outcomes. More notably, disease activity was persistently increased in the ACarPA positive patients during the two years of follow-up. These differences were significant even after accounting for the presence of other RA autoantibodies. Therefore, the ACarPA could be considered short-term prognostic biomarkers of increased disease activity in the EA patients.

Complementary Effects of Carbamylated and Citrullinated LL37 in Autoimmunity and Inflammation in Systemic Lupus Erythematosus.

LL37 acts as T-cell/B-cell autoantigen in Systemic lupus erythematosus (SLE) and psoriatic disease. Moreover, when bound to "self" nucleic acids, LL37 acts as "danger signal," leading to type I interferon (IFN-I)/pro-inflammatory factors production. T-cell epitopes derived from citrullinated-LL37 act as better antigens than unmodified LL37 epitopes in SLE, at least in selected HLA-backgrounds, included the SLE-associated HLA-DRB1*1501/HLA-DRB5*0101 backgrounds. Remarkably, while "fully-citrullinated" LL37 acts as better T-cell-stimulator, it loses DNA-binding ability and the associated "adjuvant-like" properties. Since LL37 undergoes a further irreversible post-translational modification, carbamylation and antibodies to carbamylated self-proteins other than LL37 are present in SLE, here we addressed the involvement of carbamylated-LL37 in autoimmunity and inflammation in SLE. We detected carbamylated-LL37 in SLE-affected tissues. Most importantly, carbamylated-LL37-specific antibodies and CD4 T-cells circulate in SLE and both correlate with disease activity. In contrast to "fully citrullinated-LL37," "fully carbamylated-LL37" maintains both innate and adaptive immune-cells' stimulatory abilities: in complex with DNA, carbamylated-LL37 stimulates plasmacytoid dendritic cell IFN-α production and B-cell maturation into plasma cells. Thus, we report a further example of how different post-translational modifications of a self-antigen exert complementary effects that sustain autoimmunity and inflammation, respectively. These data also show that T/B-cell responses to carbamylated-LL37 represent novel SLE disease biomarkers.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: Anti-PAD3 antibodies are associated with higher disease activity and joint damage scores in RA patients.

Association Between Bone Mineral Density and Autoantibodies in Patients With Rheumatoid Arthritis.

OBJECTIVE: Autoantibodies, such as anti-citrullinated protein antibodies (ACPAs), have been described as inducing bone loss in rheumatoid arthritis (RA), which can also be reflected by bone mineral density (BMD). We therefore examined the association between osteoporosis and autoantibodies in two independent RA cohorts. METHODS: Dual x-ray absorptiometry (DXA) of the lumbar spine and left hip was performed in 408 Dutch patients with early RA during 5 years of follow-up and in 198 Swedish patients with early RA during 10 years of follow-up. The longitudinal effect of ACPAs and other autoantibodies on several BMD measures was assessed using generalized estimating equations. RESULTS: In the Dutch cohort, significantly lower BMD at baseline was observed in ACPA-positive patients compared to ACPA-negative patients, with an estimated marginal mean BMD in the left hip of 0.92 g/cm2 (95% confidence interval [95% CI] 0.91-0.93) versus 0.95 g/cm2 (95% CI 0.93-0.97) (P = 0.01). In line with this, significantly lower Z scores at baseline were noted in the ACPA-positive group compared to the ACPA-negative group (estimated marginal mean Z score in the left hip of 0.18 [95% CI 0.08-0.29] versus 0.48 [95% CI 0.33-0.63]) (P < 0.01). However, despite clear differences at baseline, ACPA positivity was not associated with greater decrease in absolute BMD or Z scores over time. Furthermore, there was no association between BMD and higher levels of ACPAs or other autoantibodies (rheumatoid factor and anti-carbamylated protein antibodies). In the Swedish cohort, ACPA-positive patients tended to have a higher prevalence of osteopenia at baseline (P = 0.04), but again, ACPA positivity was not associated with an increased prevalence of osteopenia or osteoporosis over time. CONCLUSION: The presence of ACPAs is associated with significantly lower BMD at baseline, but not with greater BMD loss over time in treated RA patients. These results suggest that ACPAs alone do not appear to contribute to bone loss after disease onset when disease activity is well-managed.

HLA-B*08 Identified as the Most Prominently Associated Major Histocompatibility Complex Locus for Anti-Carbamylated Protein Antibody-Positive/Anti-Cyclic Citrullinated Peptide-Negative Rheumatoid Arthritis.

OBJECTIVE: Previously, only the HLA-DRB1 alleles have been assessed in rheumatoid arthritis (RA). The aim of the present study was to identify the key major histocompatibility complex (MHC) susceptibility factors showing a significant association with anti-carbamylated protein antibody-positive (anti-CarP+) RA. METHODS: Analyses were restricted to RA patients who were anti-cyclic citrullinated peptide antibody negative (anti-CCP-), because the anti-CCP status dominated the results otherwise. Therefore, we studied samples from 1,821 anti-CCP- RA patients and 6,821 population controls from Spain, Sweden, and the Netherlands. The genotypes for ~8,000 MHC biallelic variants were assessed by dense genotyping and imputation. Their association with the anti-CarP status in RA patients was tested with logistic regression and combined with inverse-variance meta-analysis. Significance of the associations was assessed according to a study-specific threshold of P < 2.0 × 10-5 . RESULTS: The HLA-B*08 allele and its correlated amino acid variant Asp-9 showed a significant association with anti-CarP+/anti-CCP- RA (P < 3.78 × 10-7 ; I2 = 0). This association was specific when assessed relative to 3 comparator groups: population controls, anti-CarP-/anti-CCP- RA patients, and anti-CCP- RA patients who were positive for other anti-citrullinated protein antibodies. Based on these findings, anti-CarP+/anti-CCP- RA patients could be separated from other antibody-defined subsets of RA patients in whom an association with the HLA-B*08 allele has been previously demonstrated. No other MHC variant remained associated with anti-CarP+/anti-CCP- RA after accounting for the presence of the HLA-B*08 allele. Specifically, the reported association of HLA-DRB1*03 was observed at a level comparable to that reported previously, but it was attributable to linkage disequilibrium. CONCLUSION: These results identify HLA-B*08 carrying Asp-9 as the MHC locus showing the strongest association with anti-CarP+/anti-CCP- RA. This knowledge may help clarify the role of the HLA in susceptibility to specific subsets of RA, by shaping the spectrum of RA autoantibodies.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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 disease activity. 260 Chinese patients with RA, 40 patients with osteoarthritis (OA), 88 patients with spondyloarthritis (SpA) and 77 healthy controls were included. The serum levels of anti-CarP antibodies were detected by ELISA. Blood tests to detect the anticyclic citrullinated peptide (CCP) antibody level, rheumatoid factor (RF) level, erythrocyte sedimentation rate, C reactive protein level and Disease Activity Score in 28 joints using the erythrocyte sedimentation rate (DAS28-ESR) were performed by standard methods. Bone erosion was assessed by colour Doppler ultrasonography. A total of 18.8% of patients with RA and 9.4% of anti-CCP antibody and RF-double-negative patients were positive for anti-CarP antibody. The anti-CarP antibody level was significantly higher in patients with RA than in patients with OA or SpA and in healthy controls. Univariate and multivariate analyses showed that the level of anti-CarP antibody was positively correlated with DAS28-ESR; the higher a level of serum anti-CarP antibody, the higher the DAS28-ESR score. Anti-CarP-positive patients had higher disease activity scores than anti-CarP-negative patients. Moreover, anti-CarP-positive patients had a higher risk of developing bone erosion. The anti-CarP antibody was found to play an important role in the diagnosis of RA, especially in anti-CCP antibody and RF-double-negative patients. The anti-CarP antibody is a potential marker of disease activity and bone erosion in RA.

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+2 vs. 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.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

OBJECTIVE: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs. METHODS: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated. RESULTS: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens. CONCLUSIONS: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis.

Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

Diagnostic Accuracy of Anti-Carbamylated Protein Antibodies in Rheumatoid Arthritis: a Systematic Review and Meta-Analysis.

BACKGROUND: The purpose of this study was to estimate the diagnostic accuracy of anti-carbamylated protein (anti-CarP) antibodies in rheumatoid arthritis. METHODS: We searched the PubMed, EMBASE, Cochrane Library, Web of Science, and Scopus databases for studies published before January 1, 2019. Two investigators independently evaluated studies to determine their inclusion in the analysis, assess their quality, and extract the relevant data. The articles were assessed with the Quality Assessment of Diagnostic Accuracy Studies tool, and a bivariate mixed effects model was used to estimate the diagnostic indexes across studies. RESULTS: We included 16 published studies in this meta-analysis. The pooled sensitivity and specificity of anti-CarP were 43.1% and 94.4%, respectively. The area under the summary receiver operator characteristic curve was 0.55. The specificity estimates were highly heterogeneous, which could be partly explained by the higher specificity in the healthy control group (43.0%, 96.8%) than in the other disease group (43.4%, 89.8%). CONCLUSIONS: Anti-CarP antibodies have a relatively low sensitivity and high specificity for rheumatoid arthritis. However, the specificity was lower in the other disease subgroups than in the healthy controls.

Post-Translational Modifications of Proteins: Novel Insights in the Autoimmune Response in Rheumatoid Arthritis.

Post-translational modifications (PTM) are chemical changes mostly catalyzed by enzymes that recognize specific target sequences in specific proteins. These modifications play a key role in regulating the folding of proteins, their targeting to specific subcellular compartments, their interaction with ligands or other proteins, and eventually their immunogenic properties. Citrullination is the best characterized PTM in the field of rheumatology, with antibodies anticyclic citrullinated peptides being the gold standard for the diagnosis of rheumatoid arthritis (RA). In recent years, growing evidence supports not only that a wide range of proteins are subject to citrullination and can trigger an autoimmune response in RA, but also that several other PTMs such as carbamylation and acetylation occur in patients with this disease. This induces a wide spectrum of autoantibodies, as biomarkers, with different sensitivity and specificity for diagnosis, which may be linked to peculiar clinical manifestations and/or response to treatment. The purpose of this review article is to critically summarize the available literature on antibodies against post-translationally modified proteins, in particular antibodies against citrullinated proteins (ACPA) and antibodies against modified proteins (AMPA), and outline their diagnostic and prognostic role to be implemented in clinical practice for RA patients.

Clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune/inflammatory disease affecting 0.5 to 1% of adults worldwide and frequently leads to joint destruction and disability. Early diagnosis and early and effective therapy may prevent joint damage and lead to better long-term results. Therefore, reliable biomarkers and outcome measures are needed. Refinement of the understanding of molecular pathways involved in disease pathogenesis have been achieved by combining knowledge on RA-associated genes, environmental factors and the presence of serological elements. The presence of autoantibodies is a distinctive feature of RA. Rheumatoid Factor and Anti-Citrullinated Protein Antibodies are the two most remarkable autoantibodies in RA and provide different clinical and pathophysiological information. They precede the onset of disease symptoms and predict a more severe disease course, indicating a pathogenetic role in RA. Therefore, they promote a more accurate prognosis and contribute for a better disease management. Several RA-associated autoantibody systems have been identified: Anti-Carbamylated Antibodies, Anti-BRAF, Anti-Acetylated, Anti-PAD4 antibodies and others. Hopefully, the characterization of a comprehensive array of novel autoantibody systems in RA will provide unique pathogenic insights of relevance for the development of diagnostic and prognostic approaches compatible with an effective personalized medicine.

Specific Association of HLA-DRB1*03 With Anti-Carbamylated Protein Antibodies in Patients With Rheumatoid Arthritis.

OBJECTIVE: Recognition of a new type of rheumatoid arthritis (RA)-specific autoantibody, the anti-carbamylated protein antibodies (anti-CarP), has provided an opportunity to improve the management and understanding of RA. The current study was undertaken to assess the relationship between anti-CarP antibodies and HLA-DRB1 alleles in RA. METHODS: Serum samples were obtained from 3 different collections, comprising a total of 1,126 RA patients. Serum reactivity against in vitro carbamylated fetal calf serum proteins was determined by enzyme-linked immunosorbent assay. HLA-DRB1 alleles were determined using either hybridization techniques or imputation from HLA-dense genotypes. Results of these analyses were combined in a meta-analysis with data from 3 previously reported cohorts. The carrier frequencies of the common HLA-DRB1 alleles were compared between the antibody-positive RA subgroups and the double-negative subgroup of RA patients stratified by anti-citrullinated protein antibody (ACPA)/anti-CarP antibody status, and also between the 4 RA patient strata and healthy controls. RESULTS: Meta-analysis was conducted with 3,709 RA patients and 2,305 healthy control subjects. Results revealed a significant increase in frequency of HLA-DRB1*03 carriers in the ACPA-/anti-CarP+ subgroup as compared to ACPA-/anti-CarP- RA patients and healthy controls; this was consistently found across the 6 sample collections. This association of HLA-DRB1*03 with ACPA-/anti-CarP+ RA was independent of the presence of the shared allele (SE) and any other confounders analyzed. No other allele was specifically associated with the ACPA-/anti-CarP+ RA patient subgroup. In contrast, frequency of the SE was significantly increased in the ACPA+/anti-CarP- and ACPA+/anti-CarP+ RA patient subgroups, without a significant distinction between them. Furthermore, some alleles (including HLA-DRB1*03) were associated with protection from ACPA+ RA. CONCLUSION: These findings indicate a specific association of HLA-DRB1*03 with ACPA-/anti-CarP+ RA, suggesting that preferential presentation of carbamylated peptides could be a new mechanism underlying the contribution of HLA alleles to RA susceptibility.

Biomarkers of erosive arthritis in systemic lupus erythematosus: Application of machine learning models.

OBJECTIVE: Limited evidences are available on biomarkers to recognize Systemic Lupus erythematosus (SLE) patients at risk to develop erosive arthritis. Anti-citrullinated peptide antibodies (ACPA) have been widely investigated and identified in up to 50% of X-ray detected erosive arthritis; conversely, few studies evaluated anti-carbamylated proteins antibodies (anti-CarP). Here, we considered the application of machine learning models to identify relevant factors in the development of ultrasonography (US)-detected erosive damage in a large cohort of SLE patients with joint involvement. METHODS: We enrolled consecutive SLE patients with arthritis/arthralgia. All patients underwent joint (DAS28, STR) and laboratory assessment (detection of ACPA, anti-CarP, Rheumatoid Factor, SLE-related antibodies). The bone surfaces of metacarpophalangeal and proximal interphalangeal joints were assessed by US: the presence of erosions was registered with a dichotomous value (0/1), obtaining a total score (0-20). Concerning machine learning techniques, we applied and compared Logistic Regression and Decision Trees in conjunction with the feature selection Forward Wrapper method. RESULTS: We enrolled 120 SLE patients [M/F 8/112, median age 47.0 years (IQR 15.0); median disease duration 120.0 months (IQR 156.0)], 73.3% of them referring at least one episode of arthritis. Erosive damage was identified in 25.8% of patients (mean±SD 0.7±1.6), all of them with clinically evident arthritis. We applied Logistic Regression in conjunction with the Forward Wrapper method, obtaining an AUC value of 0.806±0.02. As a result of the learning procedure, we evaluated the relevance of the different factors: this value was higher than 35% for ACPA and anti-CarP. CONCLUSION: The application of Machine Learning Models allowed to identify factors associated with US-detected erosive bone damage in a large SLE cohort and their relevance in determining this phenotype. Although the scope of this study is limited by the small sample size and its cross-sectional nature, the results suggest the relevance of ACPA and anti-CarP antibodies in the development of erosive damage as also pointed out in other studies.

Anti-LL37 Antibodies Are Present in Psoriatic Arthritis (PsA) Patients: New Biomarkers in PsA.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis associated with psoriasis. A third of psoriatic patients develop PsA via unknown mechanisms. No reliable diagnostic markers are available for PsA, or prognostic biomarkers for PsA development in psoriasis. We previously uncovered a pro-inflammatory role for cathelicidin LL37 in lesional psoriasis skin. LL37 binds nucleic acids and stimulates plasmacytoid/myeloid dendritic cells (pDC, mDCs) to secrete type I interferon (IFN-I) and pro-inflammatory factors. LL37 becomes an autoantigen for psoriatic Th1-Th17/CD8 T cells. Anti-LL37 antibodies were detected in systemic lupus erythematosus, an autoimmune disease characterized by neutrophil-extracellular-traps release (NETosis) in target organs. LL37 can be substrate of irreversible post-translational modifications, citrullination or carbamylation, linked to neutrophil activity. Here we analyzed inflammatory factors, included LL37, in PsA and psoriasis plasma and PsA synovial fluids (SF)/biopsies. We show that LL37 (as a product of infiltrating neutrophils) and autoantibodies to LL37 are elevated in PsA, but not OA SF. Anti-LL37 antibodies correlate with clinical inflammatory markers. Anti-carbamylated/citrullinated-LL37 antibodies are present in PsA SF/plasma and, at lower extent, in psoriasis plasma, but not in controls. Plasma anti-carbamylated-LL37 antibodies correlate with PsA (DAS44) but not psoriasis (PASI) disease activity. Ectopic lymphoid structures, and deposition of immunoglobulin-(Ig)G-complexes (IC) co-localizing with infiltrating neutrophils, are observed in PsA and not OA synovial tissues (ST). Activated complement (C5a, C9), GM-CSF and IFN-I are up-regulated in PsA and not OA synovia and in PsA and psoriasis plasma but not in HD. C9 and GM-CSF levels in PsA SF correlate with clinical inflammatory markers and DAS44 (C9) and with anti-carbamylated/citrullinated-LL37 antibodies (GM-CSF and IFN-I). Thus, we uncover a role for LL37 as a novel PsA autoantibody target and correlation studies suggest participation of anti-LL37 antibodies to PsA pathogenesis. Notably, plasma antibodies to carbamylated-LL37, which correlate with DAS44, suggest their use as new disease activity markers. GM-CSF and complement C5a and C9 elevation may be responsible for autoantigens release by neutrophils and their modification, fueling inflammation and autoreactivity establishment. Finally, targeting GM-CSF, C5a, C9 can be beneficial in PsA.

Association of high titers of anti-carbamylated protein antibodies with decreased bone mineral density in early arthritis patients.

Rheumatoid arthritis (RA) has a negative impact on bone that is partly mediated by anti-citrullinated proteins antibodies (ACPA). These antibodies are associated with erosions, and with juxta-articular and systemic bone loss. Other RA autoantibodies, the anti-carbamylated protein antibodies (anti-CarPA), are independently associated with erosions. However, we do not know if they are also associated with juxta-articular and systemic bone loss. Here, we have addressed this question with data from 548 early arthritis (EA) patients. Bone mineral density (BMD) was assessed by dual-energy x-ray absorptiometry at the lumbar spine (LS), total hip (TH) and metacarpophalangeal joints (MCP). The 25.9% anti-CarPA positive patients did not show significant differences in BMD Z-scores with the negative patients. Nevertheless, this result was due to the similarity between negative and low-positive (below the median of the positive) patients, whereas the high-positive patients showed significant decrease of BMD at LS (ß = -0.39, p = 0.01) and TH (ß = -0.30, p = 0.02); but not at the juxta-articular bone of MCP. Given the overlap between anti-CarPA and ACPA, we included the two autoantibodies in an analysis that showed significantly lower BMD Z-scores at LS and TH (p< 0.01) only in the ACPA positive/anti-CarPA high-positive subgroup. However, the similar coefficients of regression between the ACPA positive/anti-CarPA high-positive and the ACPA negative/anti-CarPA high-positive subgroups (ß = -0.50 vs. -0.52 at LS, and ß = -0.37 vs. -0.30 at TH) suggested an independent association. Overall, these results support a contribution of anti-CarPA to systemic bone loss in EA patients.