New-onset Rheumatoid Arthritis Patients Research Articles

Incident rheumatoid arthritis in patients living in Turkey and in Denmark: a comparative clinical, genetic, and serological study

Objective The north–south gradient hypothesis proposes that individuals with rheumatoid arthritis (RA) residing in southern regions manifest a younger age of onset and milder disease compared to their northern counterparts. This study aimed to compare treatment-naïve, new-onset RA patients in Denmark and Turkey, examining demographic, clinical, laboratory, and genetic parameters. Method Prospective data collection was conducted, with all patients meeting the 2010 American College of Rheumatology/European League Against Rheumatism criteria. Shared epitope (SE) allele carrier frequencies were examined for genetic comparisons between patients and normal controls. Results Out of 223 RA patients, 109 were Danish and 114 Turkish. Danish patients exhibited a median age at onset of 60 years, whereas Turkish patients were younger at 51 years (p = 0.0007). The Danish cohort displayed significantly more swollen and tender joints, resulting in higher Disease Activity Score based on 28-joint count–C-reactive protein (DAS28-CRP). Danish RA patients and controls possessed more RA risk-enhancing alleles (S2 + S3P) and fewer risk-protective (S1 + S3D) alleles than Turkish patients and controls. Conclusion This study substantiates the north–south gradient hypothesis, highlighting that new-onset RA patients in Denmark tend to experience an older age of onset and more severe disease activity than their Turkish counterparts. Variations in risk-enhancing alleles and fewer risk-protective alleles in Danish patients and controls are associated with these distinctions. Future research should investigate the genetic and environmental factors underlying these regional disparities, exploring their persistence in the long-term course of the disease through follow-up studies.

JAK inhibitors attenuate hyperactivation of nonswitched memory B cells in rheumatoid arthritis patients in remission

ObjectiveTo investigate the distribution and activation of B-cell subpopulations in rheumatoid arthritis (RA) patients treated with Janus kinase inhibitors (JAKis) and to analyze their correlation with disease remission.MethodsPeripheral blood samples were collected from 23 adult healthy controls and 58 RA patients, 31 of whom were treated with JAKis and assessed during a 24-month follow-up. The number of peripheral B-cell subpopulations (including naive B cells, nonswitched memory B (NSMB) cells, switched memory B cells, and double-negative B cells), their activation, and phosphorylation of SYK and AKT upon B-cell receptor (BCR) stimulation in each population were analyzed by flow cytometry.ResultsCompared with that in healthy controls, the frequency of NSMB cells was significantly lower in new-onset untreated RA patients. However, expression of CD40, CD80, CD95, CD21low and pAKT significantly increased in these NSMB cells. Additionally, the number of NSMB cells correlated negatively with DAS28-ESR and IgG and IgA levels in these patients; expression of CD80, CD95 and CD21low on NSMB cells correlated positively with DAS28-ESR and IgG and IgA levels. After treatment with JAKis, the serum IgG concentration significantly decreased in RA patients in remission, but CD40, CD95 and pAKT levels in NSMB cells significantly decreased.ConclusionRA patients present different B-cell subpopulations, in which the frequency of NSMB cells is negatively associated with disease activity. However, treatment with JAKis can inhibit activation of NSMB cells, restore the balance of kinase phosphorylation, and facilitate disease remission in RA patients.

Impaired immune tolerance mediated by reduced Tfr cells in rheumatoid arthritis linked to gut microbiota dysbiosis and altered metabolites

BackgroundPatients with rheumatoid arthritis (RA) showed impaired immune tolerance characterized by reduced follicular regulatory T (Tfr) cells, and they also exhibited altered gut microbiotas and their metabolites in RA. However, the association of gut microbiotas and their metabolites with the immune tolerance mediated by Tfr cells in RA remains unclear.MethodsPeripheral blood and stool samples were collected from 32 new-onset RA patients and 17 healthy controls (HCs) in the Second Hospital of Shanxi Medical University between January 2022 and June 2022. The peripheral blood was used to detect the circulating regulatory T (Treg), helper T(Th)17, Tfr, and follicular helper T (Tfh) cells by modified flow cytometry. The stool samples were used to analyze the gut microbiotas and their metabolites via 16S rDNA sequencing and metabolomic profiling. We aimed to characterize the gut microbiotas and their metabolites in RA and identified their association with Tfr cell-mediated immune tolerance.ResultsThe new-onset RA demonstrated reduced Treg and Tfr cells, associated with the disease activity and autoantibodies. There were significant differences in gut microbiotas between the two groups as the results of β diversity analysis (P = 0.039) including 21 differential gut microbiotas from the phylum to genus levels. In which, Ruminococcus 2 was associated with the disease activity and autoantibodies of RA, and it was identified as the potential biomarker of RA [area under curve (AUC) = 0.782, 95% confidence interval (CI) = 0.636–0.929, P = 0.001]. Eleven differential metabolites were identified and participated in four main pathways related to RA. Arachidonic acid might be the potential biomarker of RA (AUC = 0.724, 95% CI = 0.595–0.909, P = 0.038), and it was the core metabolite as the positive association with six gut microbiotas enriched in RA. The reduced Tfr cells were associated with the altered gut microbiotas and their metabolites including the Ruminococcus 2, the arachidonic acid involved in the biosynthesis of unsaturated fatty acid pathway and the 3-methyldioxyindole involved in the tryptophan metabolism pathway.ConclusionThe breakdown of immune tolerance mediated by reduced Tfr cells was associated with the altered gut microbiotas and their metabolites implying the possible mechanism of RA pathogenesis from the perspective of microecology-metabolism-immune.

Differential expression and correlation of immunoregulation related piRNA in rheumatoid arthritis.

Although PIWI-interacting RNAs (piRNAs) have recently been associated with germline development and many human diseases, their expression pattern and relationship in autoimmune diseases remain indistinct. This study aimed to investigate the presence and correlation of piRNAs in rheumatoid arthritis (RA). We first analyzed the expression profile of piRNAs using small RNA sequencing in peripheral leukocytes of three new-onset untreated RA patients and three healthy controls (HCs). We then selected piRNAs related to immunoregulation by bioinformatics analysis and verified them in 42 new-onset RA patients and 81 HCs by RT-qPCR. Furthermore, a receiver operating characteristic curve was generated to quantify the diagnostic performance of these piRNAs. A correlation analysis was conducted to observe the link between piRNA expression and RA clinical characteristics. A total of 15 upregulated and 9 downregulated piRNAs among 1,565 known piRNAs were identified in peripheral leukocytes of RA patients. Dysregulated piRNAs were enriched in numerous pathways related to immunity. After selection and validation, two immunoregulation piRNAs (piR-hsa-27620 and piR-hsa-27124) were significantly elevated in RA patients and have good abilities to distinguish patients from controls, which have the potential to serve as biomarkers. PIWI and other proteins implicated in the piRNA pathway were also associated with RA.

P137 First-line tumour necrosis factor inhibitor (TNFi) treatment is associated with high multi-criteria remission early on compared to treat-to-target in a treatment naïve RA cohort

Abstract Background/Aims In rheumatoid arthritis (RA) disease assessment, SDAI and Boolean remission criteria are known to be more stringent in comparison to DAS28-ESR. Comparing remission rates across multiple assessment indices provides a more comprehensive evaluation of disease control. In an early RA trial cohort, we aimed to investigate stringent and multi-criteria remission (MCR) rates and evaluate their proportional change in response to treatment. Methods The ‘VEDERA’ trial randomised 120 treatment-naïve, new-onset RA patients to first-line etanercept+methotrexate (ETN+MTX) or methotrexate treat-to-target (MTX-TT) with escalation to ETN+MTX if not in DAS28-ESR remission at week24. Clinical assessments were completed at baseline, weeks12/24/48 (protocolised phase) up to week96 (standard-of-care). Remission cut-points included: DAS28-ESR ≤2.6, SDAI ≤3.3 and Boolean score for individual components≤1. MCR was defined as patients who were in DAS28-ESR, SDAI and Boolean remission concurrently. Descriptive analyses were undertaken to address the above aims. Results Overall, at week12, 26%(31/120) were in SDAI and 13%(16/120) in Boolean remission. This increased to 41%(49/120) and 23%(28/120) respectively by week48. In the ETN+MTX group at week12, 30%(18/60) were in SDAI and 17%(10/60) in Boolean remission. This increased to 43% (26/60) and 32%(19/60) respectively by week48. Stringent remission rates were lower in MTX-TT compared to ETN+MTX and significantly different for Boolean at week48 (15% vs 32%, odds ratio 2.6, 95% confidence interval 1.1 to 6.5, nominal p < 0.05) (Table 1). Of those in remission by the three different criteria, 36%(14/39) were in MCR at week12, with a greater proportion in ETN+MTX group [42%(10/24) compared to 27%(4/15) in MTX-TT]. MCR remained unchanged up to week48 but improved with treatment by week 96 to 45%(27/60). MCR rates did not converge between the 2 treatment groups at week 96 (ETN+MTX 55%; MTX-TT 38%). Conclusion First-line ETN+MTX is associated with higher rates of stringent remission from week 12 compared to MTX-TT, with improvement gradually over time with both strategies. Significantly higher proportion of patients treated with first line ETN+MTX compared to MTX-TT achieved Boolean and MCR. Early TNFi treatment associates with more complete and better cumulative disease activity profile. Patients not meeting MCR early following treat-to-target approach should be considered for more aggressive treatment strategies. Disclosure R. Shukla: None. D. Plant: None. P. Emery: None. M.H. Buch: None.

P215 In a treatment-naïve, early rheumatoid arthritis cohort, baseline ultrasound power Doppler synovitis and tenosynovitis are highly associated with remission when treated with first line etanercept + methotrexate (MTX) but not MTX alone

Abstract Background/Aims Clinical remission is the treatment target in rheumatoid arthritis (RA). In a treatment-naïve, early RA trial (VEDERA: ‘Very early Etanercept and Methotrexate versus Methotrexate with/without Delayed Etanercept in RA’), we previously reported the presence of ultrasound power Doppler synovitis (PDUS) at diagnosis that improved in the majority with treatment. For this study, we aimed to investigate specifically for an association of baseline PDUS and PD tenosynovitis (PDTS) with subsequent clinical remission (DAS28ESR ≤ 2.6) in VEDERA, also comparing the two treatment groups. Methods The ‘VEDERA’ trial randomised 120 treatment-naïve, new-onset RA patients to either first-line etanercept+methotrexate (ETN+MTX) or methotrexate treat-to-target (MTX-TT) regime with escalation to ETN+MTX if not in DAS28ESR remission at week 24. Clinically symptomatic and dominant hand (MCP 1-5, wrist, flexor tendons 1-5 and extensor carpii ulnaris tendon) ultrasound assessments were completed at baseline, weeks 12, 24 and 48. PDUS and PDTS were assessed semi-quantitatively and converted to dichotomous (absent = total score 0, present = total score ≥ 1) for this logistic regression analysis. Results At baseline, 68% (81/120) demonstrated PDUS and 68% also demonstrated PDTS overall. In the MTX-TT group, 63% (38/60) had PDUS and 62% (37/60) had PDTS at baseline. In ETN+MTX group, 72% (43/60) had PDUS and 75% (45/60) had PDTS at baseline. As per randomised trial, there were no differences in baseline demographics, DAS28ESR and HAQ between the groups. Overall, baseline PDUS was associated with week 24 remission and baseline PDTS was associated with week 24 and 48 remission (table 1). Baseline PDUS was particularly associated with week 24 remission in the ETN+MTX group, with a high OR (95% CI) of 6.83(1.2 - 38.91) compared to the MTX-TT group [1.75(0.5 - 6.14)]. Baseline PDTS was also associated with a high OR for week 48 remission in the ETN+MTX group (OR [95% CI] 9.13[1.77 - 47.02]) in contrast to an absence of association in the MTX-TT group. Conclusion Baseline PDUS and PDTS are associated with remission in early RA patients treated with TNF-inhibitor treatment. These measures may be useful to stratify patients early for TNF bDMARD to attain the target of clinical remission in RA. Disclosure R. Shukla: None. R. Hum: Other; R.H holds an Academic Clinical Fellowship funded by the National Institute for Health Research (NIHR) through the Integrated Academic Training (IAT) Programme. P. Ho: None. R.J. Wakefield: None. P. Emery: None. M.H. Buch: None.

P214 Classifiable early RA shows heterogeneous imaging features and potential means of stratification

Abstract Background/Aims Synovitis and tenosynovitis (TS) are key findings seen in patients with rheumatoid arthritis (RA) on musculoskeletal ultrasound (MSUS). Both power Doppler TS (PDTS) and power Doppler synovitis (PDUS) have also been reported in preclinical-RA. We aimed to describe the prevalence of PDTS and PDUS in a treatment-naïve, new-onset RA trial cohort and their association with clinical and disease activity data. Methods The VEDERA (Very early Etanercept and Methotrexate versus Methotrexate with/without Delayed Etanercept in RA) trial recruited 120 treatment-naïve, new-onset RA patients fulfilling ACR-EULAR 2010 classification criteria with at least moderate DAS28-ESR to either first-line etanercept + methotrexate (ETN+MTX) or methotrexate treat-to-target (MTX-TT), and included clinical and dominant hand (MCP 1-5, wrist, flexor tendons 1-5 and extensor carpii ulnaris tendon) MSUS assessments. Joint Grey scale (GS), PDUS and PDTS were assessed semi-quantitatively and converted to dichotomous scale (absent = total score 0; present = total score ≥ 1). Patients were categorised in one of four groups based on presence of PDUS and/or PDTS: no PDUS or PDTS (PDUS-PDTS-), PDTS alone (PDUS-PDTS+), PDUS alone (PDUS+PDTS-) and both PDUS and PDTS (PDUS+PDTS+). Clinical and GS features are described for these groups. Results At time of diagnosis, 17% (20/120) were PDUS-PDTS-, 16% (19/120) PDUS-PDTS+, 15% (18/120) PDUS+PDTS- and 53% (63/120) PDUS+PDTS+. Table 1 details demographic, clinical and disease activity data. GS was present in 60% (12/20) and 74% (14/19) of PDUS-PDTS- and PDUS-PDTS+ groups respectively, while all patients with PDUS + (PDUS+PDTS- and PDUS+PDTS+ groups) demonstrated GS (table 1). The PDUS+PDTS+ group had the shortest symptom duration. PDUS-PDTS- group had lower DAS28ESR, tender/swollen joint counts (28 joints), CRP and pain. The two groups with PDTS + (with/without PDUS) had higher visual analogue pain scores. Conclusion Heterogeneous imaging features of classifiable, new-onset RA likely reflect different rates of progress along the continuum and presentation, and timing in capturing PDUS and PDTS. Severe clinical and imaging features associate with more rapid presentation. A minor proportion show no clear features of PDUS or GS yet are classifiable as RA indicating other contributors to diagnosis. MSUS features may offer a means of stratifying patients and tailoring treatment approaches. Disclosure R. Shukla: None. R. Levy: None. P. Ho: None. K. Mankia: None. R.J. Wakefield: None. P. Emery: None. M.H. Buch: None.

Fine particulate matter components and interstitial lung disease in rheumatoid arthritis.

Exposure to ambient fine particulate matter with an aerodynamic diameter <2.5 μg·m-3 (PM2.5) is a risk factor for pulmonary and systemic autoimmune diseases; however, evidence on which PM2.5 chemical components are more harmful is still scant. Our goal is to investigate potential associations between major PM2.5 components and interstitial lung disease (ILD) onset in rheumatoid arthritis (RA). New-onset RA subjects identified from a US healthcare insurance database (MarketScan) were followed for new onset of RA-associated ILD (RA-ILD) from 2011 to 2018. Annual concentrations of ambient PM2.5 chemical components (i.e. sulfate, nitrate, ammonium, organic matter, black carbon, mineral dust and sea salt) were estimated by combining satellite retrievals with chemical transport modelling and refined by geographically weighted regression. Exposures from 2006 up to 1 year before ILD onset or end of study were assigned to subjects based on their core-based statistical area or metropolitan division codes. A novel time-to-event quantile-based g (generalised)-computation approach was used to estimate potential associations between RA-ILD onset and the exposure mixture of all seven PM2.5 chemical components adjusting for age, sex and prior chronic obstructive pulmonary disease (as a proxy for smoking). We followed 280 516 new-onset RA patients and detected 2194 RA-ILD cases across 1 394 385 person-years. The adjusted hazard ratio for RA-ILD onset was 1.54 (95% CI 1.47-1.63) per every decile increase in all seven exposures. Ammonium, mineral dust and black carbon contributed more to ILD risk than the other PM2.5 components. Exposure to components of PM2.5, particularly ammonium, increases ILD risk in RA.

AB0668 THE ONSET OF RHEUMATOID ARTHRITIS AFTER COVID-19 – COINCIDENCE OR CONNECTED?

Background:COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been suggested to induce autoimmune phenomena. Multiple studies have reported the presence of autoantibodies in patients with COVID-19. Also the presence of anti-citrullinated protein antibodies (ACPA) and flaring of rheumatoid arthritis (RA) after COVID-19 has been described.[1, 2] Furthermore, in rheumatology clinics patients may present with polyarthritis compatible with RA shortly after SARS-CoV-2 infection. However, it is unclear how often ACPA occur after COVID-19 and whether preceding COVID-19 impacts on disease presentation of RA and phenotype of the ACPA response.Objectives:This study aims to determine the seroprevalence of ACPA after COVID-19 and to investigate the association between preceding COVID-19 infection and disease presentation of new-onset RA, including clinical phenotype and autoantibody response.Methods:To estimate the prevalence of ACPA after COVID-19 we measured ACPA IgG in samples from 61 patients visiting the specialized post-COVID outpatient clinic of the LUMC 5 weeks after hospitalization, using routine tests or in-house ELISA. Furthermore, we identified 5 patients presenting with polyarthritis compatible with RA after SARS-CoV-2 infection. To study the impact of COVID-19 on disease presentation, we examined clinical phenotype, autoantibody isotype positivity and ACPA IgG variable domain (V-domain) glycosylation of these patients and compared these features to regular RA patients. Autoantibody isotypes, including rheumatoid factor (RF) IgM/IgA, anti-CCP2 IgG/ IgM/IgA and anti-carbamylated protein antibodies (anti-CarP) IgG were measured using in-house ELISA’s. The percentage of V-domain glycosylation of purified ACPA IgG was measured with UHPLC.Results:None of the 61 post-COVID patients tested positive for ACPA 5 weeks after hospitalization, except two patients previously diagnosed with ACPA-positive RA. Thus, we could not observe an increase in ACPA-positivity shortly after COVID-19. Of the 5 patients who developed polyarthritis compatible with RA after SARS-CoV-2 infection, the average age was 63.6 years and 2/5 were female. 4/5 patients had been hospitalized due to severe COVID-19. On average, joint complaints started 6.6 weeks after infection, although two patients reported symptoms before infection. 4/5 patients fulfilled the ACR 2010 criteria for RA. Three patients (patient 1, 4, 5) were phenotypically very similar to regular new-onset RA patients. Patient 3 had a history of seronegative RA and had been in DMARD-free remission for 5 years. She flared 6 weeks after SARS-CoV-2 infection. Patient 2 had a remarkably different presentation. He was admitted with a suspected septic polyarthritis or pneumonia with reactive polyarthritis 6 weeks after COVID-19. ACPA level was low positive. The patient died unexpectedly after two days and autopsy revealed dilating myocarditis of unclear underlying cause. No causative pathogen could be identified.Previous studies have shown that RA-patients are most often either seronegative or triple-positive for RF, ACPA and anti-CarP antibodies. Autoantibody measurements on sera of the post-COVID polyarthritis patients revealed a similar pattern (Figure 1A) with two patients being completely seronegative, and three patients positive for a range of autoantibodies. In all post-COVID samples, the percentage of ACPA IgG V-domain glycosylation was significantly increased compared to total IgG (Figure 1B), similar as in regular RA.Conclusion:In conclusion, we found that the seroprevalence of ACPA is not increased post-COVID and that most patients presenting with polyarthritis after COVID-19 resemble regular RA patients, both regarding clinical phenotype and autoantibody characteristics. Although sample size and follow-up was limited, it appears that RA post-COVID may be coincidence rather than connected.

Circular RNAs hsa-circ0000175 and hsa-circ0044235 in plasma are novel biomarkers for new-onset rheumatoid arthritis

Circular RNAs (circRNAs) are a class of non-coding RNAs that could serve as potential molecular markers for disease diagnosis. However, the role of circRNAs in plasma from new-onset rheumatoid arthritis (RA) has not been extensively investigated. In this study, the expression of hsa-circ0000175 and hsa-circ0044235 in plasma from RA patients, healthy controls (HCs), systemic lupus erythematosus (SLE) patients, osteoarthritis (OA), and undiagnosed arthritis (UA) patients were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Correlation analysis was used to assess the correlation of the two circRNAs and clinical variables of RA. The receiver operating characteristic (ROC) curves were created to evaluate the diagnostic value and multivariate analysis (logistic regression) was performed to analyse the risk factors. We confirmed that hsa-circ0000175 was significantly elevated in plasma from patients with new-onset RA compared with HC and patients with new-onset SLE, but significantly was reduced when compared with OA + UA patients. Hsa-circ0044235 was found to be significantly decreased in plasma from patients with new-onset RA compared with HC and OA + UA patients, but was significantly increased compared with SLE patients. The expression of plasma hsa-circ0000175 in new-onset RA patients was associated with platelet count (PLT), plateletcrit (PCT), and platelet large cell ratio (PLR), the expression of plasma hsa-circ0044235 new-onset RA patients was associated with swollen joint count (SJC), painful joint count (PJC), and disease activity score 28 (DAS28). ROC curve analysis suggested that the combination of hsa-circ0000175 and hsa-circ0044235 has some value in the diagnosis of new-onset RA from HC, patients with SLE and patients with OA + UA. The logistic regression analysis revealed that the expression of hsa-circ0000175 and hsa-circ0044235 in plasma were risk factors for RA. This study suggests that the combination of plasma hsa-circ0000175 and hsa-circ0044235 improves the diagnostic accuracy for new-onset RA. Moreover, the expression levels of plasma hsa-circ0000175 and hsa-circ0044235 were associated with disease activity and severity of RA.

Circular RNAs Hsa_circ_0002715 and Hsa_circ_0035197 in Peripheral Blood Are Novel Potential Biomarkers for New-Onset Rheumatoid Arthritis.

This study is aimed at exploring the levels of peripheral blood circular RNAs (circRNAs) as biomarker candidates for the diagnosis of new-onset rheumatoid arthritis (RA). The selected twenty-two circRNAs in peripheral blood from new-onset RA patients and healthy controls (HC) were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The levels of hsa_circ_0002715, hsa_circ_0001947, hsa_circ_0000367, and hsa_circ_0035197 were significantly increased in the peripheral blood of new-onset RA patients than in the peripheral blood of HC. And, there were obvious differences in the above four peripheral blood circRNAs between new-onset RA patients and systemic lupus erythematosus (SLE) patients and ankylosing spondylitis (AS) patients. Moreover, there were obvious differences in hsa_circ_0001947 and hsa_circ_0035197 between new-onset RA patients and patients with undiagnosed arthritis (UA). Receiver operating characteristic (ROC) curve analysis suggested that the levels of hsa_circ_0002715 and hsa_circ_0000367 in peripheral blood could distinguish new-onset RA patients from the HC, AS patients, and SLE patients, and the levels of hsa_circ_0001947 and hsa_circ_0035197 in peripheral blood could distinguish new-onset RA patients from the HC, AS patients, SLE patients, and UA patients. The logistic regression model showed that the combination of hsa_circ_0002715 and hsa_circ_0035197 could provide the best diagnostic accuracy with an area under the curve (AUC) of 0.758 (sensitivity: 72.9%, specificity: 71.4%). Moreover, the levels of peripheral blood hsa_circ_0002715 were correlated with swollen joint count (SJC), tender joint count (TJC), disease duration, rheumatoid factor (RF), anticitrullinated protein antibodies (ACPA), and hematologic disorder. And, the levels of peripheral blood hsa_circ_0035197 were correlated with hematologic disorder. This study suggests that the combination of hsa_circ_0002715 and hsa_circ_0035197 in peripheral blood may be a potential biomarker of patients with new-onset RA and may be associated with disease activity.

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OBJECTIVES/SPECIFIC AIMS: Rheumatoid arthritis (RA) is one of the most prevalent systemic autoimmune diseases. It is caused by a combination of genetic and environmental factors. In humans, the intestinal microbe Prevotella copri strongly correlates with RA in previously untreated new-onset rheumatoid arthritis (NORA) patients. Metagenomic assembly of P. copri from NORA patients and healthy controls suggests genetic differences between P. copri from each group. In order to test the hypothesis that genetic differences in P. copri from arthritis patients promote arthritis, I am performing genomic comparison of primary P. copri isolates from NORA patients and healthy controls, and analysis of the immune response to P. copri in mice. Mice colonized with P. copri have increased susceptibility to DSS-induced weight loss and death compared with uncolonized controls. Future experiments will assess the local and systemic immune response in P. copri-colonized, DSS-treated mice. If this work is successful, then it may be possible to exploit genetic variation in P. copri. This could lead to new biomarkers for human disease or even insight into drug metabolism. METHODS/STUDY POPULATION: To validate a strategy to screen for the presence of P. copri in feces, qPCR primers were designed to amplify 8 regions across the 3.5 Mb P. copri reference genome using NCBI PrimerBlast. Primers were validated with DNA from feces for which P. copri abundance was previously determined by 16S rDNA sequencing. P. copri genome-specific primers were used to screen bacterial isolates from NORA patients and healthy controls. The 16S V3-V4 region was sequenced and compared with the P. copri reference 16S sequence to confirm &gt;97% similarity. Genomes of 2 NORA patient isolates were sequenced on Illumina MiSeq, and sequences were compared with the reference genome. A strategy was developed to colonize mice with P. copri: 3-week-old C57BL/6 mice were treated with antibiotics in drinking water for 2 weeks, then switched to water for 2 days before oral gavage with P. copri; 6–7 days after inoculation, P. copri colonization was assessed by plating feces from inoculated mice, and by qPCR of fecal DNA with P. copri-specific primers. A systemic immune response to P. copri was assessed by microbe-specific ELISA for IgG and IgA in the sera of colonized mice. RESULTS/ANTICIPATED RESULTS: P. copri was detected in the stool of 20% of healthy individuals and 50% of NORA patients. P. copri was isolated form 4 healthy individuals and 6 NORA patients. Whole genomes of 96 primary isolates from NORA patients and healthy controls will be sequenced on the Illumina HiSeq platform, and their genomes will be assembled and compared using Spades software. For 2 P. copri isolates for a NORA patient, 89% of 250 bp reads aligned &gt;95% to the P. copri reference genome. Mice can be colonized with P. copri gavaged at &gt;106 CFU. P. copri-specific IgG and IgA were detected in the sera of colonized mice. DISCUSSION/SIGNIFICANCE OF IMPACT: Several primary isolates of P. copri have been collected from healthy controls and NORA patients, which will enable whole genome comparison of these isolates. For the 2 P. copri isolates sequenced, 89% of 250 bp reads aligned &gt;95% to the P. copri reference genome, indicating variability between NORA patient P. copri strains and the P. copri reference genome. The establishment of colonization of mice with P. copri will allow further characterization of the immune response to P. copri at steady state and under pro-inflammatory conditions. Further, the systemic immune response to P. copri indicates that this microbe may have potential to play a role in systemic disease.

Evidence of the Immune Relevance of Prevotella copri, a Gut Microbe, in Patients With Rheumatoid Arthritis.

Prevotella copri, an intestinal microbe, may overexpand in stool samples from patients with new-onset rheumatoid arthritis (RA), but it is not yet clear whether the organism has immune relevance in RA pathogenesis. HLA-DR-presented peptides (T cell epitopes) from P copri were sought directly in the patients' synovial tissue or peripheral blood mononuclear cell (PBMC) samples using tandem mass spectrometry. The antigenicity of peptides or their source proteins was examined in samples from the RA patients or comparison groups. T cell reactivity was determined by enzyme-linked immunospot assay; antibody responses were measured by enzyme-linked immunosorbent assay, and cytokine/chemokine determinations were made by bead-based assays. Serum and synovial fluid samples were examined for 16S ribosomal DNA for P copri using nested polymerase chain reaction analysis. In PBMCs, we identified an HLA-DR-presented peptide from a 27-kd protein of P copri (Pc-p27), which stimulated Th1 responses in 42% of patients with new-onset RA. In both new-onset RA patients and chronic RA patients, 1 subgroup had IgA antibody responses to either Pc-p27 or the whole organism, which correlated with Th17 cytokine responses and frequent anti-citrullinated protein antibodies (ACPAs). The other subgroup had IgG P copri antibodies, which were associated with Prevotella DNA in synovial fluid, P copri-specific Th1 responses, and less frequent ACPAs. In contrast, P copri antibody responses were rarely found in patients with other rheumatic diseases or in healthy controls. Subgroups of RA patients have differential IgG or IgA immune reactivity with P copri, which appears to be specific for this disease. These observations provide evidence that P copri is immune-relevant in RA pathogenesis.

Glucocorticoid exposure and fracture risk in patients with new-onset rheumatoid arthritis.

We evaluated the impact of systemic glucocorticoid exposure on fracture risk among relatively young patients with new-onset rheumatoid arthritis (RA). Using administrative data, we identified 42,127 RA patients diagnosed January 1, 2005-December 31, 2012, age 18-64years, with benefits coverage for ≥12months before RA diagnosis. Follow-up extended to clinical fracture, cancer diagnosis, or December 31, 2012. Glucocorticoid users were new to therapy. Fracture incidence rates (IR) were stratified by glucocorticoid exposure expressed as prednisone equivalent doses. Cox's proportional hazards models estimated fracture risk adjusted for demographics and baseline clinical characteristics to assess dose-response relationships with current (daily) and prior (cumulative) dose, and by time since discontinuation. Most patients (85%) had glucocorticoid exposure. Exposed and unexposed patients were demographically similar (74% female; mean age 49.7 and 48.8years); 1% had prior fracture. Fracture IRs (95% confidence intervals) were 5 to 9 per 1000 person-years at doses <15mg/day, 16.0 (11.0, 22.6) at doses ≥15mg/day, and 13.4 (10.7, 16.7) at cumulative doses ≥5400mg. Adjusted fracture risk was approximately 2-fold higher at highest dose levels compared with 0mg/day current daily dose and <675mg cumulative dose, respectively. Fracture risk was 29% lower at 60-182days post-discontinuation compared with ongoing use and was similar to unexposed patients by 12months. Findings were similar among patients age <50years. Among younger, new-onset RA patients, fracture risk was significantly elevated at high levels of daily and cumulative dose, and was similar to unexposed patients by 12months post-discontinuation.

Early medication use in new-onset rheumatoid arthritis may delay joint replacement: results of a large population-based study.

IntroductionUse of disease-modifying anti-rheumatic drugs (DMARDs) in rheumatoid arthritis (RA) may prevent joint damage and potentially reduce joint replacement surgeries. We assessed the association between RA drug use and joint replacement in Quebec, Canada.MethodsA cohort of new-onset RA patients was identified from Quebec’s physician billing and hospitalization databases from 2002–2011. The outcome was defined using procedure codes submitted by orthopedic surgeons. Medication use was obtained from pharmacy databases. We used alternative Cox regression models with time-dependent variables measuring the cumulative effects of past use during different time windows (one model focussing on the first year after cohort entry) for methotrexate (MTX), and other DMARDs. Models were adjusted for baseline sociodemographics, co-morbidity and prior health service use, time-dependent cumulative use of other drugs (anti-tumor necrosis factor [anti-TNF] agents, other biologics, cyclooxygenase-2 inhibitors [COXIBs], nonselective nonsteroidal antiinflammatory drugs [NSAIDs], and systemic steroids), and markers of disease severity.ResultsDuring follow-up, 608 joint replacements occurred among 11,333 patients (median follow-up: 4.6 years). The best-fitting model relied on the cumulative early use (within the first year after cohort entry) of MTX and of other DMARDs, with an interaction between MTX and other DMARDs. In this model, greater exposure within the first year, to either MTX (adjusted hazard ratio, HR = 0.95 per 1 month, 95 % confidence interval, 95 % CI 0.93-0.97) or other DMARDs (HR = 0.97, 95 % CI 0.95-0.99) was associated with longer time to joint replacement.ConclusionsOur results suggest that longer exposure to either methotrexate (MTX) or other DMARDs within the first year after RA diagnosis is associated with longer time to joint replacement surgery.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0713-3) contains supplementary material, which is available to authorized users.