Seronegative Rheumatoid Arthritis Patients Research Articles

Identification of autoantibodies against PsoP27 in synovial fluid derived from psoriatic arthritis and rheumatoid arthritis patients

PsoP27 is an antigen expressed in psoriatic lesions. It plays an inflammatory role in psoriasis. This study objective was to characterize antibodies (Abs) against PsoP27 in patients with psoriatic arthritis (PsA) and rheumatoid arthritis (RA). Levels of Abs against native and citrullinated PsoP27 in PsA and RA patients’ synovial fluid (SF) and sera were determined by ELISA. SF of osteoarthritis (OA) patients and sera of healthy donors were used as controls. Levels of Abs against PsoP27 were correlated with disease activity scores. Abs against native and citrullinated PsoP27 levels in SF of PsA (n = 48; 0.38 ± 0.03 and 0.44 ± 0.04, respectively) and RA (n = 22; 0.57 ± 0.1 and 0.62 ± 0.09, respectively) were significantly higher than in OA patients (n = 23; 0.14 ± 0.01 and 0.15 ± 0.01, respectively) (p < .0001). For both Abs, there were no significant differences between their level in PsA and RA patients. There was no difference in the level of Abs against citrullinated PsoP27 in SF of seronegative versus seropositive RA patients. Levels of Abs against both native and citrullinated PsoP27 in the SF and level of systemic C-reactive protein in PsA correlated positively, while in RA there were no significant correlations with disease activity scores. No differences in level of Abs against PsoP27 were found in the sera of all three study groups. Abs against native and citrullinated PsoP27 are present in PsA and RA SF but not in those of OA patients, suggesting a potential role of those Abs in inflammatory joint diseases.

Serum and urine lipidomic profiles identify biomarkers diagnostic for seropositive and seronegative rheumatoid arthritis.

Seronegative rheumatoid arthritis (RA) is defined as RA without circulating autoantibodies such as rheumatoid factor and anti-citrullinated protein antibodies; thus, early diagnosis of seronegative RA can be challenging. Here, we aimed to identify diagnostic biomarkers for seronegative RA by performing lipidomic analyses of sera and urine samples from patients with RA. We performed untargeted lipidomic analysis of sera and urine samples from 111 RA patients, 45 osteoarthritis (OA) patients, and 25 healthy controls (HC). These samples were divided into a discovery cohort (n = 97) and a validation cohort (n = 84). Serum samples from 20 patients with systemic lupus erythematosus (SLE) were also used for validation. The serum lipidome profile of RA was distinguishable from that of OA and HC. We identified a panel of ten serum lipids and three urine lipids in the discovery cohort that showed the most significant differences. These were deemed potential lipid biomarker candidates for RA. The serum lipid panel was tested using a validation cohort; the results revealed an accuracy of 79%, a sensitivity of 71%, and a specificity of 86%. Both seropositive and seronegative RA patients were differentiated from patients with OA, SLE, and HC. Three urinary lipids showing differential expression between RA from HC were identified with an accuracy of 84%, but they failed to differentiate RA from OA. There were five lipid pathways that differed between seronegative and seropositive RA. Here, we identified a panel of ten serum lipids as potential biomarkers that can differentiate RA from OA and SLE, regardless of seropositivity. In addition, three urinary lipids had diagnostic utility for differentiating RA from HC.

Ultrasonographic Evaluation of the Ankle Joint in Relation to Rheumatoid Factor Status and Disease Activity in Patients with Rheumatoid Arthritis

Abstract Background: Rheumatoid arthritis (RA) is a form of inflammatory disease whose clinical pattern is largely dependent on the presence of both anti-citrullinated protein antibodies and rheumatoid factor (RF). Although this is still debatable, seronegative RA seems to be a somewhat less serious condition. The present study aimed to evaluate ankle joint ultrasound in relation to RF status and disease activity in RA patients. Methods: A cross-sectional study involving RA patients from a single center was conducted. Laboratory test evaluations and clinical activity assessments were carried out. The ankle joint was examined using musculoskeletal ultrasound (US). Results: The study included 100 patients with established RA in total. Eighty-two patients tested positive for RF with a mean age of 42.3, whereas only 18 tested negative with a mean age of 39.6. Patients who tested positive for RF had a longer duration of illness (9.39 ± 5.39 vs. 4.56 ± 3.24). There were no differences in clinical activity scores between the seropositive and seronegative groups. The pathological US findings of any ankle joint showed no differences between the seropositive and seronegative groups. Patients with US findings of tibialis posterior tenosynovitis in the left ankle and synovitis and erosion in the right ankle, particularly in the tibiotalar and talonavicular joints, had significantly high Disease Activity Score 28-Erythrocyte sedimentation rate-scores. The increased disease activity was accompanied by significant erosions on both ankles. Conclusion: In terms of disease activity, there is no clinically significant difference between seropositive and seronegative RA patients. Sonographic ankle joint abnormalities do not appear to be associated with the patients’ RF status. High RA disease activity, on the other hand, is associated with synovitis and erosions, particularly in the talonavicular and tibiotalar joints, as well as tibialis posterior tenosynovitis.

Effectiveness of biological targeted therapies may discriminate seronegative from seropositive rheumatoid arthritis.

To assess the real-world effectiveness of targeting biologic drugs (bDMARD) in rheumatoid arthritis (RA) patients negative for rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). We retrospectively selected 81 seronegative and 404 seropositive RA patients receiving treatment with abatacept, anti-tumor necrosis factor (TNF) alpha, or tocilizumab. Effectiveness was evaluated by analyzing drug survival using Kaplan-Meyer analysis over 10-year follow-up. Survival rates were compared by log rank test, and hazard ratios (HRs) of therapy discontinuation were estimated through multivariate Cox-regression. Clinical characteristics were similar between the two groups, except for a significantly higher percentage of inadequate responders to prior bDMARDs in the seronegative RA patients (p= 0.02). Among seronegative RA, tocilizumab demonstrated a survival rate of 73.9% with a mean survival time (MST) of 76.8 months (95% CI 61-92), which was significantly higher than abatacept (37.5%, MST 37.1 months (95% CI 22-51; p= 0.01). Anti-TNF alpha therapy fell in the middle (50.0%, MST 63.5 months (95% CI 47-79) but the difference was not significant. Nevertheless, seropositive RA patients did not show significantly different drug survival rates. Negative predictors of drug discontinuation were RF/ACPA positivity (HR 0.56) and sex male (HR 0.58), but treatment with abatacept (HR 1.88) or anti-TNF alpha (HR 1.79), no co-therapy with cDMARD (HR 1.74), absence of bone erosions (HR 1.41), and higher HAQ (HR 1.58) were positive predictors. To confirm these preliminary findings and to explore the hypothesis of a distinctive therapeutic algorithm in seronegative RA, prospective studies on larger cohorts are needed.

A Comparative Analysis of Inflammatory Markers and Disease Activity Score between Seropositive and Seronegative Rheumatoid Arthritis

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disorder that is characterized by persistent inflammation in the joints and other tissues, leading to significant joint damage irrespective of the serogroups i.e. seropositive and seronegative RA. C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) are commonly used biomarkers to monitor inflammation in RA and to inform therapeutic decision-making. Methods: The present study aimed to investigate the relationship between CRP and ESR levels and the severity of disease among seronegative and seropositive patients. ACR/EULAR 2010 criteria were used to recruit RA patients in the study. Clinical assessments were performed to estimate the RF, ACCP antibodies, ESR and CRP. Swollen and tender joints were also taken into account for the calculation of disease activity score-28. Results: The results indicated that significantly elevated CRP levels were consistently observed in seropositive RA, in conjunction with seronegative RA patients (p=0.036). ESR levels were found elevated between the two groups but were not found to be statistically different (p=0.885). DAS28-CRP and DAS28-ESR results showed high severity of disease (&gt;5.1) in both seronegative and seronegative RA patients. Conclusion: The findings of this study highlight the diagnostic and prognostic significance of ESR and CRP levels in RA among seronegative and seropositive patients, particularly in differentiating seropositive and seronegative cases and in guiding therapeutic decisions.

Soluble CD24 is an inflammatory biomarker in early and seronegative rheumatoid arthritis

Introduction: Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by autoantibody production, joint inflammation and bone destruction. Nearly 1/3 of RA patients with the active disease also exhibit a normal range of ESR and CRP. Here we assessed the performance and clinical significance of soluble CD24 (sCD24) as a biomarker of disease activity in RA. Methods: A total of 269 RA patients, 59 primary Sjogren’s syndrome (SS) patients, 81 systematic lupus erythematosus (SLE) patients, 76 osteoarthritis (OA) patients and 97 healthy individuals (HC) were included in this study. Soluble CD24 in sera were detected by ELISA. Therefore, the concentration of sCD24 was analyzed in RA patients with different disease activity statuses. Results: The sCD24 was significantly increased in RA (2970 pg/mL), compared to other rheumatic diseases (380-520 pg/mL) and healthy individuals (320 pg/mL). Moreover, sCD24 was elevated in 66.67% of early RA and 61.11% of seronegative RA patients. In addition, sCD24 was significantly correlated with the disease duration and inflammatory indicators. Conclusion: The sCD24 could be an inflammatory biomarker in RA patients, especially in early and seronegative patients.

In silico analysis of serum miRNA profiles in seronegative and seropositive rheumatoid arthritis patients by small RNA sequencing.

Rheumatoid arthritis (RA) is a refractory autoimmune disease, affecting about 1% of the world's population. RA is divided into seronegative RA and seropositive RA. However, biomarkers for discriminating between seronegative and seropositive RA have not been reported. In this study, we profiled serum miRNAs in seronegative RA patients (N-RA), seropositive RA patients (P-RA) and healthy controls (HC) by small RNA sequencing. Results indicated that compared with HC group, there were one up-regulated and four downregulated miRNAs in N-RA group (fold change ≥ 2 and P value < 0.05); compared with P-RA group, there were two up-regulated and four downregulated miRNAs in N-RA group; compared with HC group, there were three up-regulated and four downregulated miRNAs in P-RA group. Among them, the level of hsa-miR-362-5p in N-RA group was up-regulated compared with that in HC group and P-RA group, and the level of hsa-miR-6855-5p and hsa-miR-187-3p in P-RA group was upregulated compared with that in N-RA group and HC group. Validation by qPCR confirmed that serum hsa-miR-362-5p level was elevated in N-RA group. Subsequently, by analyzing the target genes using RNAhybrid, PITA, Miranda and TargetScan and functions of differential miRNAs utilizing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), we found that the target genes and molecular pathways regulated by miRNAs in seronegative RA and seropositive RA were roughly the same, and miRNAs in these two diseases may participate in the occurrence and development of diseases by regulating the immune system. In conclusion, this study revealed the profiles of serum miRNAs in seronegative and seropositive RA patients for the first time, providing potential biomarkers and targets for the diagnosis and treatment of seronegative and seropositive RA.

Aberrant B-cell activation and B-cell subpopulations in rheumatoid arthritis: analysis by clinical activity, autoantibody seropositivity, and treatment.

Few studies analyze the role of B-cell subpopulations in rheumatoid arthritis (RA) pathophysiology. Therefore, this study aimed to analyze the differences in B-cell subpopulations and B-cell activation according to disease activity, RA subtype, and absence of disease-modifying antirheumatic drugs (DMARDs) therapy. These subgroups were compared with control subjects (CS). One hundred and thirty-nine subjects were included, of which 114 were RA patients, and 25 were controls. Patients were divided into 99 with seropositive RA, 6 with seronegative RA, and 9 without DMARDs. The patients with seropositive RA were subclassified based on the DAS28 index. A seven-color multicolor flow cytometry panel was used to identify B-cell immunophenotypes and cell activation markers. There were no changes in total B-cell frequencies between RA patients and controls. However, a lower frequency of memory B cells and pre-plasmablasts was observed in seropositive RA compared to controls (P < 0.0001; P = 0.0043, respectively). In contrast, a higher frequency of mature B cells was observed in RA than in controls (P = 0.0002). Among patients with RA, those with moderate activity had a higher percentage of B cells (P = 0.0021). The CD69+ marker was increased (P < 0.0001) in RA compared to controls, while the CD40+ frequency was decreased in patients (P < 0.0001). Transitional, naïve, and double-negative B-cell subpopulations were higher in seronegative RA than in seropositive (P < 0.01). In conclusion, in seropositive and seronegative RA patients, there are alterations in B-cell activation and B-cell subpopulations, independently of clinical activity and DMARDs therapy.

Distinct innate and adaptive immunity phenotypic profile at the circulating single-cell level in Psoriatic Arthritis

Mass cytometry was employed to investigate 47 circulating leukocyte subsets in patients with active psoriatic arthritis (PsA, n = 16) compared to healthy controls (n = 13), seropositive (RF and/or anti-CCP, n = 12) and seronegative (n = 9) RA patients. Comparing PsA to controls, different cell frequencies were found in both innate and adaptive immunity cell subsets, as well as in cells bridging innate and adaptive immunity. In some T cell subsets increased costimulatory molecules' expression in PsA, was also noted.No changes were observed in patients who remained disease-active after 3 months of treatment, in contrast to those who achieved remission/low-disease activity. Comparing PsA to seropositive RA, elevated frequencies of naïve and activated CD8+ T cells, B cells, MAIT/iNKT and ILCs were found, while the opposite was the case for terminal effector, senescent, and Th2-like cells. Strikingly, the composition of the leukocyte pool in PsA was comparable to seronegative RA, providing evidence for the pathogenetic similarities between these two entities.

Autoantibodies to Disease-Related Proteins in Joints as Novel Biomarkers for the Diagnosis of Rheumatoid Arthritis.

This study was undertaken to develop and characterize a multiplex immunoassay for detection of autoantibodies against peptides derived from proteins known to play a role in development of arthritis and that are also expressed in joints. We selected peptides from the human counterpart of proteins expressed in the joints, based on mouse models that showed these to be targeted by pathogenic or regulatory antibodies in vivo. Using bead-based flow immunoassays measuring IgG antibodies, we selected triple helical or cyclic peptides, containing the epitopes, to avoid collinear reactivity. We characterized the analytical performance of the immunoassay and then validated it in 3 independent rheumatoid arthritis (RA) cohorts (n=2,110), Swedish age- and sex-matched healthy controls, and patients with osteoarthritis (OA), patients with psoriatic arthritis (PsA), and patients with systemic lupus erythematosus (SLE). Screening assays showed 5 peptide antigens that discriminated RA patients from healthy controls with 99% specificity (95% confidence interval [CI] 98-100%). In our validation studies, we reproduced the discriminatory capacity of the autoantibodies in 2 other RA cohorts, showing that the autoantibodies had high discriminatory capacity for RA versus OA, PsA, and SLE. The novel biomarkers identified 22.5% (95% CI 19-26%) of early RA patients seronegative for anti-cyclic citrullinated peptide and rheumatoid factor. The usefulness of the biomarkers in identifying seronegative RA patients was confirmed in validation studies using 2 independent cohorts of RA patients and cohorts of patients with OA, PsA, and SLE. A multiplex immunoassay with peptides from disease-related proteins in joints was found to be useful for detection of specific autoantibodies in RA serum. Of note, this immunoassay had high discriminatory capacity for early seronegative RA.

Hand and Wrist Involvement in Seropositive Rheumatoid Arthritis, Seronegative Rheumatoid Arthritis, and Psoriatic Arthritis—The Value of Classic Radiography

The hand and wrist are among the most common anatomical areas involved in rheumatic diseases, especially seropositive and seronegative rheumatoid arthritis (RA) and psoriatic arthritis (PsA). The purpose of this study was to identify the most differentiating radiographic characteristics of PsA, seropositive RA, and seronegative RA, particularly in the early stages. A retrospective analysis of radiographic hand findings was performed on 180 seropositive RA patients (29 males, 151 females, mean age at the point of acquisition of the analyzed radiograph of 53.4 y/o, SD 12.6), 154 PsA patients (45 males, 109 females, age median of 48.1 y/o, SD 12.4), and 36 seronegative RA patients (4 males, 32 females, age median of 53.1 y/o, SD 17.1) acquired during the period 2005-2020. Posterior-anterior and Nørgaard views were analyzed in all patients. The radiographs were evaluated for three radiographic findings: type of symmetry (asymmetric/bilateral/changes in corresponding joint compartments/'mirror-image' symmetry), anatomic location (e.g., wrist, metacarpophalangeal (MCP), proximal interphalangeal (PIP), distal interphalangeal (DIP) joints), and type of lesions (e.g., juxta-articular osteoporosis, bone cysts, erosions, proliferative bone changes). The study showed that symmetric distribution of lesions defined as 'lesions present in corresponding compartments' was more suggestive of seropositive or seronegative RA than PsA. Lesions affecting the PIP joints, wrist, or styloid process of the radius; juxta-articular osteoporosis, joint space narrowing, joint subluxations, or dislocations were more common in patients with seropositive RA than in those with PsA, whereas DIP joints' involvement and proliferative bone changes were more likely to suggest PsA than seropositive RA. Lesions in PIP, MCP, and wrist joints, as well as erosions, advanced bone damage, joint subluxations, dislocations, and joint space narrowing, were more common in seropositive RA patients than in seronegative RA patients. The ulnar styloid was more commonly affected in seronegative RA patients than in PsA patients. The study confirmed that types of bone lesions and their distribution in the hands and wrists can be useful in differentiating seropositive RA from PsA and suggests that seronegative RA varies in radiological presentation from seropositive RA and PsA.

Clinical Utility of Pro-inflammatory Oligomeric Glycoprotein Tenascin-C in the Diagnosis of Seropositive and Seronegative Rheumatoid Arthritis.

Owing to limited usefulness of Rheumatoid Factor and anti-CCP in rheumatoid arthritis, there is a need to identify a more sensitive and specific biomarker to detect rheumatoid arthritis (RA), particularly seronegative RA cases. Tenascin-C is an extracellular matrix glycoprotein, which has been implicated in the pathophysiology of RA. The objective of our study was to evaluate the diagnostic utility of serum Tenascin-C in seropositive and seronegative rheumatoid arthritis patients. We conducted a cross-sectional case control study. Sixty patients who fulfilled the ACR 2010 criteria for rheumatoid arthritis were included in the study. Thirty patients were found to be positive for RF and/or anti-CCP and 30 were negative for both RF and anti-CCP. Thirty age and gender-matched healthy subjects were taken as controls. Serum Tenascin-C was measured by quantitative sandwich enzyme immunoassay technique. The mean serum concentration of Tenascin-C in controls, seronegative and seropositive cases was 0.66ng/ml, 20.54ng/ml and 23.42ng/ml, respectively. Tenascin-C levels were significantly higher in RA cases compared to controls (p < 0.0001). There was no significant difference in Tenascin-C between seropositive and seronegative cases (p = 0.603). ROC curve analysis showed a sensitivity of 96.6% and specificity of 100% with AUC of 0.98 at 2.21ng/ml as cut-off value for diagnosing RA. Tenascin-C is elevated in both seronegative and seropositive RA, which indicates that it can be used as a sensitive marker for RA. The addition of Tenascin-C to the existing RF and anti-CCP may help in identifying a large number of patients with RA, particularly seronegative rheumatoid arthritis cases.

Cerebrovascular reactivity depending on rheumatoid factor and anticitrullinated protein antibody positivity in hypertensive patients with rheumatoid arthritis

Objective – to investigate cerebrovascular reactivity (CVR) depending on rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) positivity in hypertensive patients with rheumatoid arthritis (RA).Subjects and methods. A single cross-sectional study included 61 patients (mean age 59.8±7.7 years; 6 men and 55 women) with combined RA and grade 1–2 hypertension (HTN). The duration of RA was 11.2±7.4 years. The duration of HTN was 12.1±8.6 years. All patients were treated with methotrexate. RA patients were categorized into RF/ACPA seronegative and RF/ACPA seropositive subgroups. CVR was evaluated by bilateral transcranial Doppler sonography of the middle cerebral arteries (MCA) in a hyperoxic test (O2 CVR) and in a hypercapnic test (CO2 CVR). We measured MCA mean blood flow velocity (Vmn), time average maximal blood flow velocity (TAMX), peak systolic velocity (Vps) at baseline, within 2 minutes of 100% oxygen inhalation and within 3 minutes of recovery phase (hyperoxic test). We calculated the following indicators for assessing CVR: index changes of flow velocity mean (IFVm), speed modification of velocity (SMFVm) and normalized answer of reserve (NAR). Then, according to the same scheme, we performed a hypercapnic test with the inhalation of a 4% mixture of carbon dioxide with air. Values are presented as Me [Q1 ; Q3 ].Results and discussion. Hypertensive patients with RA had a decrease in response power of MCA blood flow to hyperoxia. RF-seropositive RA patients had a more pronounced decrease in the power of the response to hyperoxia compared with RF-seronegative RA patients. The values of IFVm in the hyperoxic test were –13.4 [–19.9; –0.9] versus –16.2 [–22.7; –13.4]% (р=0.0453), respectively. ACPA-seropositive RA patients had not only a more pronounced decrease in the power of the response of MCA blood flow to hyperoxia, but also a more pronounced slowdown in the response velocity of MCA blood flow to hyperoxia compared with ACPA-seronegative RA patients. The values of IFVm in the hyperoxic test were –9.74 [–15.9; 2.84] versus –20.9 [–25.0; –14.7]% (р=0.0062), the values of SMFVm were –0.05 [–0.09; 0.02] versus –0.09 [–0.20; –0.05] sm/s2 (р=0.0488) respectively. Combined RA and HTN patients had a decrease in response power of MCA blood flow to hypercapnia. However, no statistical differences were found in the state of CO2 CVR between patients with seropositive RA and seronegative RA.Conclusion. Hypertensive patients with seropositive RA have a more pronounced O2 CVR disorder in compared to seronegative RA patients.

The immune dysregulations in inflammatory arthritis immune related adverse events

Abstract The immune checkpoint inhibition therapy induces various immune related adverse events (irAEs). The immunological basis of irAEs remains unclear. We recruited a cohort of patients with inflammatory arthritis irAE (IA-irAE), the most common form of rheumatic irAEs, together with matched patients with conventional rheumatoid arthritis (RA) and cancer patients without irAEs. Comprehensive clinical and immunological investigations reveal an imbalance of T cell exhaustion and senescence in IA-irAE patients, and immunological similarities between IA-irAE patients and seronegative RA patients. These data suggest that IA-irAE and seronegative RA may have shared immunological mechanisms. Thus, our study identifies potential immunological underpinnings of IA-irAE and the relationship between IA-irAE and conventional RA. The results may have implications for managing irAE and seronegative RA patients. Supported byR01AR0077518 Arthritis National Research Foundation

Anti-CCP Antibody and Rheumatoid Factor among Clinically Diagnosed Rheumatoid Arthritis Patients Attending a Tertiary Hospital at Chitwan, Nepal

INTRODUCTION: Rheumatoid Arthritis (RA) is a common, chronic inflammatory multisystem autoimmune disorder of undetermined aetiology involving primarily the synovial membranes and articular structures of many joints. Rheumatoid factor (RF) is most frequently used serum biomarkers for the diagnosis of RA; however, these antibodies are detectable in many other pathological conditions and even in 5% in healthy population. Anti-Cyclic Citrullinated Peptide (Anti-CCP) antibodies is another serological biomarker having high sensitivity and specificity than RF which present in pre-clinical stage and seronegative rheumatoid arthritis patients. MATERIALS AND METHODS: This is a cross-sectional study conducted among the confirmed RA patients visiting orthopaedic OPD. Anti-CCP antibody was determined quantitively by CLIA on Siemen ADVIA Centaur CP immunoassay system and RA-factor were determined quantitively by Nephelometry assay on MISPA I3 and comparison was done between two serological tests, i.e., Anti-CCP and RF. RESULTS: Out of 112 clinically diagnosed RA patients, 23 (20.5%) were males and 89(79.5%) were females with the ratio of 1:3.9 and the mean age ± SD of the patients was 50 ± 14.56. Out of 112 clinically diagnosed RA patients, 55 patients, i.e., 49.1% were RF antibodies positive and 57 patients, i.e., 50.9% were RF antibodies negative. Among RF positive, 32 (58.2%) patients were positive for anti-CCP antibody and among RF negative, 10 (17.5%) patients were positive for anti-CCP antibody and the differences between Anti-CCP and RF groups was statistically highly significant (p&lt;0.001). CONCLUSIONS: Females are at high risk of developing Rheumatoid arthritis than males because male to female ratio is found to be 1:3.9. This study also showed that Anti-CCP antibody is more beneficial and better marker than RF for the diagnosis of Rheumatoid arthritis.

Serum metabolomic and lipidomic profiling identifies diagnostic biomarkers for seropositive and seronegative rheumatoid arthritis patients

BackgroundDiagnosing seronegative rheumatoid arthritis (RA) can be challenging due to complex diagnostic criteria. We sought to discover diagnostic biomarkers for seronegative RA cases by studying metabolomic and lipidomic changes in RA patient serum.MethodsWe performed comprehensive metabolomic and lipidomic profiling in serum of 225 RA patients and 100 normal controls. These samples were divided into a discovery set (n = 243) and a validation set (n = 82). A machine-learning-based multivariate classification model was constructed using distinctive metabolites and lipids signals.ResultsTwenty-six metabolites and lipids were identified from the discovery cohort to construct a RA diagnosis model. The model was subsequently tested on a validation set and achieved accuracy of 90.2%, with sensitivity of 89.7% and specificity of 90.6%. Both seropositive and seronegative patients were identified using this model. A co-occurrence network using serum omics profiles was built and parsed into six modules, showing significant association between the inflammation and immune activity markers and aberrant metabolism of energy metabolism, lipids metabolism and amino acid metabolism. Acyl carnitines (20:3), aspartyl-phenylalanine, pipecolic acid, phosphatidylethanolamine PE (18:1) and lysophosphatidylethanolamine LPE (20:3) were positively correlated with the RA disease activity, while histidine and phosphatidic acid PA (28:0) were negatively correlated with the RA disease activity.ConclusionsA panel of 26 serum markers were selected from omics profiles to build a machine-learning-based prediction model that could aid in diagnosing seronegative RA patients. Potential markers were also identified in stratifying RA cases based on disease activity.

ITRAQ-based proteomic analysis of differentially expressed proteins in sera of seronegative and seropositive rheumatoid arthritis patients.

ObjectiveThe diagnosis of seronegative rheumatoid arthritis (SNRA) is often difficult due to the unavailability of reliable laboratory markers. The aim of this study was to identify differentially expressed proteins in sera of SNRA, seropositive RA (SPRA), and healthy donors (HD).MethodsA total of 32 seropositive RA patients, 32 SNRA patients, and 35 HD were enrolled in our study. Differentially expressed proteins between 3 groups were identified via isobaric tags for relative and absolute quantitation (iTRAQ)‐based proteomic analysis, and an ELISA test was used for the validation test. Correlation analysis was conducted by GraphPad Prism.ResultsUsing iTRAQ quantitative proteomics, we identified 14 proteins were significantly different between SPRA and SNRA, including 4 upregulated proteins and 10 downregulated proteins. Four differentially expressed proteins were validated by ELISA test, and the results showed that SAA1 protein was significantly higher in SPRA and SNRA patients compared with HD, and PSME1 was elevated in SPRA patients. What's more, SAA1 was increased in the anti‐CCP or RF high‐level group in RA patients, and PSME1 was increased in the RF high‐level group. Alternatively, SAA1 was positively correlated with inflammation indicators in RA patients, while PSME1 showed no correlation with inflammation indicators.ConclusionsiTRAQ proteomic approaches revealed variations in serum protein composition among SPRA patients, SNRA patients, and HD and provided new idea for advanced diagnostic methods and precision treatment of RA.

Human carbamylome description identifies carbamylated α2-macroglobulin and hemopexin as two novel autoantigens in early rheumatoid arthritis.

ObjectivesAnti-carbamylated protein antibodies (anti-CarPAs) are present in RA sera and have been associated with erosive disease. The exact targets of anti-CarPAs in vivo are currently not well known; we used a proteomic approach on serum and SF of RA patients to assess the human carbamylome and to identify carbamylated autoantigens as potential biomarkers in early RA.MethodsMass spectrometry was performed on SF and serum from RA patients. Carbamylated proteins present in both sample types were selected as candidate autoantigens for the establishment of ELISAs. A cohort of early RA patients was tested for positivity for specific anti-CarPAs.ResultsEleven novel carbamylated proteins were identified, and five were selected as potential autoantigens for detection of anti-CarPAs. Among them, antibodies against carbamylated hemopexin (anti-CaHPX) and alpha-2-macroglobulin (anti-CaA2M) showed comparable diagnostic value to the established carbamylated foetal calf serum–based ELISA. A cohort of 189 early RA patients was studied. The combination of these new biomarkers with anti-citrullinated protein antibodies and RF identified 89% of early RA patients in our cohort. There was little correlation between the tested biomarkers, and each one of the tested antigens could identify a different subset of seronegative RA patients. Anti-CaA2M positivity showed clinical potential, being associated with higher disease disability.ConclusionWe highlight the detection of novel carbamylated autoantigens in vivo using a combined proteomics approach in the SF and serum of RA patients. Anti-CaHPX and anti-CaA2M are promising clinical biomarkers, especially in seronegative RA.

Identification of a Novel Serological Marker in Seronegative Rheumatoid Arthritis Using the Peptide Library Approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation mainly affecting the joints leading to cartilage and bone destruction. The definition of seropositive or seronegative RA is based on the presence or absence of rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs). Other autoantibodies have been identified in the last decade such as antibodies directed against carbamylated antigens, peptidyl-arginine deiminase type 4 and v-Raf murine sarcoma viral oncogene homologue B. In order to identify relevant autoantigens, we screened a random peptide library (RPL) with pooled IgGs obtained from 50 patients with seronegative RA. Patients’ sera were then used in an ELISA test to identify the most frequently recognized peptide among those obtained by screening the RPL. Sera from age- and sex-matched healthy subjects were used as controls. We identified a specific peptide (RA-peptide) recognized by RA patients’ sera, but not by healthy subjects or by patients with other immune-mediated diseases. The majority of sera from seronegative and seropositive RA patients (73.8% and 63.6% respectively) contained IgG antibodies directed against the RA-peptide. Interestingly, this peptide shares homology with some self-antigens, such as Protein-tyrosine kinase 2 beta, B cell scaffold protein, Liprin-alfa1 and Cytotoxic T lymphocyte protein 4. Affinity purified anti-RA-peptide antibodies were able to cross react with these autoantigens. In conclusion, we identified a peptide that is recognized by seropositive and, most importantly, by seronegative RA patients’ sera, but not by healthy subjects, conferring to this epitope a high degree of specificity. This peptide shares also homology with other autoantigens which can be recognized by autoantibodies present in seronegative RA sera. These newly identified autoantibodies, although present also in a percentage of seropositive RA patients, may be considered as novel serum biomarkers for seronegative RA, which lacks the presence of RF and/or ACPAs.

The citrullinated/native index of autoantibodies against hnRNP-DL predicts an individual \u201cwindow of treatment success\u201d in RA patients

BackgroundThere is a need for biomarker to identify patients “at risk” for rheumatoid arthritis (risk-RA) and to better predict the therapeutic response and in this study we tested the hypothesis that novel native and citrullinated heterogeneous nuclear ribonucleoprotein (hnRNP)-DL autoantibodies could be possible biomarkers.MethodsUsing protein macroarray and ELISA, epitope recognition against hnRNP-DL was analysed in sera from different developed RA disease and diagnosed SLE patients. Toll-like receptor (TLR) 7/9 and myeloid differentiation primary response gene 88 (MyD88)-dependency were studied in sera from murine disease models. HnRNP-DL expression in cultivated cells and synovial tissue was analysed by indirect immunofluorescence, immunoblot and immunohistochemistry.ResultsHnRNP-DL was highly expressed in stress granules, citrullinated in the rheumatoid joint and targeted by autoantibodies either as native or citrullinated proteins in patient subsets with different developed RA disease. Structural citrullination dependent epitopes (SCEs) of hnRNP-DL were detected in 58% of the SLE patients although 98% of these sera were α-CCP-2-negative. To obtain a specific citrullinated signal value, we subtracted the native antibody value from the citrullinated signal. The citrullinated/native index of autoantibodies against hnRNP-DL (CNDL-Index) was identified as a new value for an “individual window of treatment success” in early RA and for the detection of RF IgM/α-CCP-2 seronegative RA patients (24–46%). Negative CNDL-index was found in SLE patients, risk-RA and early RA cohorts such as EIRA where the majority of these patients are DAS28-responders to methotrexate (MTX) treatment (87%). High positive CNDL-values were associated with more severe RA, shared epitope and parenchymal changes in the lung. Specifically, native α-hnRNP-DL is TLR7/9-dependent, associated with pain and ROC analysis revealed an association to initial MTX or etanercept treatment response, especially in seronegative RA patients.ConclusionCNDL-index defines people at risk to develop RA and the “window of treatment success” thereby closing the sensitivity gap in RA.