Muslima Chowdhury

Heightened immune response to autocitrullinated Porphyromonas gingivalis peptidylarginine deiminase: a potential mechanism for breaching immunologic tolerance in rheumatoid arthritis

Background Rheumatoid arthritis (RA) is characterised by autoimmunity to citrullinated proteins, and there is increasing epidemiologic evidence linking Porphyromonas gingivalis to RA. P gingivalis is apparently unique among periodontal pathogens in possessing a citrullinating enzyme, peptidylarginine deiminase (PPAD) with the potential to generate antigens driving the autoimmune response. Objectives To examine the immune response to PPAD in patients with RA, individuals with periodontitis (PD) and controls (without arthritis), confirm PPAD autocitrullination and identify the modified arginine residues. Methods PPAD and an inactivated mutant (C351A) were cloned and expressed and autocitrullination of both examined by immunoblotting and mass spectrometry. ELISAs using PPAD, C351A and another P gingivalis protein arginine gingipain (RgpB) were developed and antibody reactivities examined in patients with RA (n=80), individuals with PD (n=44) and controls (n=82). Results Recombinant PPAD was a potent citrullinating enzyme. Antibodies to PPAD, but not to Rgp, were elevated in the RA sera (median 122 U/ml) compared with controls (median 70 U/ml; p<0.05) and PD (median 60 U/ml; p<0.01). Specificity of the anti-peptidyl citrullinated PPAD response was confirmed by the reaction of RA sera with multiple epitopes tested with synthetic citrullinated peptides spanning the PPAD molecule. The elevated antibody response to PPAD was abolished in RA sera if the C351A mutant was used on ELISA. Conclusions The peptidyl citrulline-specific immune response to PPAD supports the hypothesis that, as a bacterial protein, it might break tolerance in RA, and could be a target for therapy.

Efficacy and safety of apremilast, an oral phosphodiesterase 4 inhibitor, in ankylosing spondylitis

Objectives To evaluate the efficacy and safety of an oral phosphodiesterase 4 inhibitor, apremilast, in treatment of ankylosing spondylitis (AS) by monitoring symptoms and signs in a pilot study including exploratory investigation of effects of PDE4 inhibition on blood biomarkers of bone biology. Methods In this double-blind, placebo-controlled, single-centre, Phase II study, patients with symptomatic AS with active disease on MRI were randomised to apremilast 30 mg BID or placebo over 12 weeks. Bath Indices were monitored serially. Patients were followed for 4 weeks after stopping medication. Bone biomarkers were assessed at baseline and day 85. Results 38 subjects were randomised and 36 subjects completed the study. Although the primary end-point (change in BASDAI at week 12) was not met, apremilast was associated with numerically greater improvement from baseline for all clinical assessments compared with placebo with mean change in BASDAI (−1.59±1.48 vs −0.77±1.47), BASFI (−1.74±1.91 vs −0.28±1.61) and BASMI (−0.51±1.02 vs −0.21±0.67); however, differences did not achieve statistical significance. The clinical indices returned to baseline values by 4 weeks after cessation of apremilast. Six apremilast patients (35.3%) vs 3 placebo (15.8%) achieved ASAS20 responses (p=0.25). There were statistically significant decreases in serum RANKL and RANKL:osteoprotegrin ratio and plasma sclerostin but no significant changes in serum DKK-1, bone alkaline phosphatase, TRAP5b, MMP3, osteoprotegrin, or osteocalcin. Conclusions Although a small pilot study, these results suggest that apremilast may be effective and well tolerated in AS and modulates biomarkers of bone biology. These data support further research of apremilast in axial inflammation.

The autoantibody repertoire in periodontitis: a role in the induction of autoimmunity to citrullinated proteins in rheumatoid arthritis?

Background Studies suggest that periodontitis may be a risk factor for rheumatoid arthritis (RA). The purpose of this study was to determine whether periodontitis is associated with autoantibodies characteristic of RA. Methods Serum samples were tested for anti-cyclic citrullinated peptide (CCP), anti-mutated citrullinated vimentin (MCV), anti-citrullinated α-enolase peptide-1 (CEP-1), anti-citrullinated vimentin (cit-vim), anti-citrullinated fibrinogen (cit-fib) and their uncitrullinated forms anti-CParg (negative control for anti-CCP), anti-arginine-containing α-enolase peptide-1 (REP-1), anti-vimentin and anti-fibrinogen antibodies in patients with and without periodontitis, none of whom had RA. Results Periodontitis, compared with non-periodontitis, was associated with a normal frequency of anti-CCP and anti-MCV (∼1%) but a higher frequency of positive anti-CEP-1 (12% vs 3%; p=0.02) and its uncitrullinated form anti-REP-1 (16% vs 2%; p<0.001). Positive antibodies against uncitrullinated fibrinogen and CParg were also more common among those with periodontitis compared to non-periodontitis patients (26% vs 3%; p<0.001, and 9% vs 3%; p=0.06). After adjusting for confounders, patients with periodontitis had 43% (p=0.03), 71% (p=0.002) and 114% (p<0.001) higher anti-CEP-1, anti-REP-1 and anti-fibrinogen titres, compared with non-periodontitis. Non-smokers with periodontitis, compared with non-periodontitis, had significantly higher titres of anti-CEP-1 (103%, p<0.001), anti-REP-1 (91%, p=0.001), anti-vimentin (87%, p=0.002), and anti-fibrinogen (124%, p<0.001), independent of confounders, confirming that the autoantibody response in periodontitis was not due to smoking. Conclusions We have shown that the antibody response in periodontitis is predominantly directed to the uncitrullinated peptides of the RA autoantigens examined in this study. We propose that this loss of tolerance could then lead to epitope spreading to citrullinated epitopes as the autoimmune response in periodontitis evolves into that of presymptomatic RA.

Smoking, the HLA-DRB1 shared epitope and ACPA fine-specificity in Koreans with rheumatoid arthritis: evidence for more than one pathogenic pathway linking smoking to disease

Objectives Data from North European rheumatoid arthritis (RA) populations has suggested a particularly strong association of gene-environment interaction between smoking and HLA-DRB1 shared epitope (SE) with antibodies to citrullinated α-enolase (CEP-1) and vimentin (cVim) peptides. We investigated this further by examining anticitrullinated peptide/protein antibody (ACPA) fine specificity in a Korean cohort, where there are notable differences in the RA-associated HLA-DRB1 alleles. Methods Antibodies to fibrinogen (cFib), α-enolase (CEP-1) and vimentin (cVim) peptides and cyclic citrullinated peptide (CCP) were measured in 513 cases. The Mann-Whitney U test was used to compare antibody levels. Logistic regression generated ORs for RA in a case-control analysis with 1101 controls. Association of ACPA status and erosion in patients with RA was examined by logistic regression. Results Anti-CCP, CEP-1, cVim and fibrinogen peptides were found in 86.7%, 63.9%, 45.5% and 74.7%, respectively. The number of ACPA and their levels were associated with SE, with evidence of a gene-dosage effect. There was a particular association of smoking with levels of anti-CEP-1. However, a gene-environment interaction was associated with all the ACPA positive subgroups, albeit the highest OR was seen with the anti-CCP+/cVim+ subset. In the absence of SE, smoking only conferred risk for anti-CCP negative subsets. The presence of erosions was not associated with the number of positive ACPA or specificity. Conclusions The SE governed the magnitude and diversity of the ACPA response, but its interaction with smoking did not exclusively segregate with any of the ACPA specificities studied here. Smoking was associated with RA by SE-dependent and independent effects.

Serotyping for an extended anti-citrullinated peptide autoantibody panel does not add value to CCP2 testing for diagnosing RA in an early undifferentiated arthritis cohort

The now widely employed CCP2 assay, which detects circulating autoantibodies to a panel of synthetic, cyclic citrullinated peptides, carries a positive predictive value of 0.93 for rheumatoid arthritis (RA) among early undifferentiated arthritis (UA) patients,1 thus providing a cornerstone of recently evolved scoring systems that predict and classify early RA.2 However, approximately 80% of newly presenting UA patients are anti-CCP2- negative 3 and a quarter of these evolve into RA within 3 years,1 thus experiencing diagnostic delay. The peptides used in the CCP2 assay do not necessarily correspond to in vivo generated proteins, yet citrullinated antigens of putative pathophysiological relevance have recently been described in association with specific circulating autoantibodies in RA.4 Therefore, we hypothesised that the detection of one or any such autoantibodies, as well as those against filaggrin components of the CCP1 assay,5 IgA or IgM rheumatoid factor (RF), might add to the diagnostic utility of CCP2 testing alone in predicting RA progression among early UA patients. Consecutive adult UA patients with …

Autodeimination of Porphyromonas gingivalis peptidylarginine deiminase: a novel antigen in rheumatoid arthritis

Backgroundand objectives Porphyromonas gingivalis, a major periodontal pathogen, has been implicated in the pathogenesis of rheumatoid arthritis (RA). It is the only pathogenic prokaryote known to possess a bacterial peptidylarginine deiminase (PPAD), an enzyme that catalyses the post-translational modification of arginine to citrulline. The enzyme is surface expressed and citrullinates c-terminal arginines of human fibrinogen and enolase peptides. Here, the authors show that PPAD is capable of autocitrullination and able to raise an immune response in RA. Materials and methods PPAD was expressed as a GST/His-tagged fusion protein using a bacterial expression system. Site-directed mutagenesis using a PCR based method was used to create a PPAD mutant (mPPAD) with a single residue mutation (cysteine 351 to alanine) and with no enzymatic activity. Citrullination was determined by immunoblotting with an antimodified citrullinated antibody kit (AMC, New York, NY, USA). Mass spectrophotometry was carried out to verify PPAD-GST citrullination. ELISAs using recombinant PPAD and recombinant mPPAD were developed to test sera from patients with periodontitis (PD) (n=44), RA (n=82) and healthy controls (C) (n=80). Reactivity was expressed as arbitrary units per ml (AU/ml) using a standard curve. Results Sera from patients with RA, PD and healthy controls were tested for IgG antibodies to PPAD and showed that RA sera have a significantly elevated antiPPAD IgG response (mean 182.4 AU/ml) compared to PD (mean 96.4AU/ml; p<0.01) and healthy control sera (mean 99.2AU/ml; p<0.05). An ELISA with the enzymatically non-active, C351A mutated PPAD (mPPAD), showed that the antibody response in the RA group was no longer significantly elevated compared to the antibody responses in the PD and healthy control sera. Antimodified citrulline immunoblotting demonstrated that recombinant PPAD was citrullinated whereas the mPPAD was not. Citrullination of PPAD was confirmed by mass spectrophotometry which showed that 7 of the 18 arginines in PPAD were citrullinated. All citrullines detected were internal and no c-terminal citrullination was observed. Conclusions The authors present evidence that PPAD is capable of autocitrullination and its target site is not restricted to c-terminal peptide arginines. The antibody response to autocitrullinated PPAD was significantly elevated in RA patients compared to PD patients and controls. This indicates that PPAD is a potential novel antigen in RA and may form part of the anticitrullinated protein antigen response seen in this disease, thus substantiating a possible link between PD and RA.

The anti-citrullinated antibody repertoire in periodontitis: a role in the induction of autoimmunity in rheumatoid arthritis?

Abstract Background Data suggest that periodontal disease (periodontitis) may be a risk factor for rheumatoid arthritis. Anti-citrullinated peptide/protein antibodies (ACPA) are highly specific for rheumatoid arthritis, and epitope spreading arises years before clinical onset of the disease. The purpose of this study was to determine the immune reactivity to ACPA and their uncitrullinated control peptides in patients with periodontitis. Methods ACPA serology was determined in patients with and without periodontitis, including anti-cyclic citrullinated peptide antibodies (anti-CCP), anti-mutated citrullinated vimentin antibodies (anti-MCV), and antibodies with specificity for different citrullinated peptides, including enolase (CEP-1), vimentin (cit-vim), fibrinogen (cit-fib), and their uncitrullinated forms (REP-1, vim, and fib, respectively). Findings We included 96 patients with and 98 without periodontitis, none of whom had rheumatoid arthritis at inclusion. Compared with patients without periodontitis, patients with the disease had a higher frequency of antibodies against CEP-1 (3% [3/98] vs 12% [12/96], p=0·02) and REP-1 (2% [2/98] vs 6% [6/96], p vs 3% [3/98], p vs 3% [3/98], p=0·06, respectively). 79 (49%) of 149 non-smokers with periodontitis had significantly higher titres of antibodies against CEP-1 (103% difference in titres, p Interpretation In patients without rheumatoid arthritis, periodontitis is associated with higher titres of antibodies to both citrullinated and uncitrullinated peptides in non-smokers suggesting that periodontitis may contribute to the development of autoimmunity related to rheumatoid arthritis. Funding Gums & Joints Collaborative EU Framework Programme 7 project and Birmingham and Black Country Comprehensive Local Research Network.

IS PERIODONTAL DISEASE A RISK FACTOR FOR RHEUMATOID ARTHRITIS? THE ANTI-CITRULLINATED ANTIBODY REPERTOIRE IN PERIODONTAL DISEASE

Background Anticitrullinated peptide antibodies (ACPA) epitope spreading has been shown to occur several years prior to RA clinical onset. Recent studies suggest an association between rheumatoid arthritis (RA) and periodontal disease (PD). Since P gingivalis is a common periodontal pathogen with a peptidylarginine deiminase that citrullinates proteins, the authors hypothesised that PD may have a causal role in the aetiology of RA. The purpose was to determine the immune reactivities to citrullinated antigens in subjects with and without PD. Methods Participants were recruited from the Birmingham Dental Hospital, Birmingham, UK. The presence of PD was based on a periodontal examination and/or radiographs. ACPA presence was determined on serum samples. In addition to anti-CCP2 and anti-MCV, the authors tested for antibodies with specificity for different citrullinated peptides, including α-enolase peptide 1 (CEP-1), fibrinogen (cit-fib) and vimentin (cit-vim). Antibodies against the uncitrullinated form of the same peptides were determined as negative controls. Comparisons between groups were done using χ2 or Mann–Whitney as appropriate. Non-normally distributed variables were log transformed for analyses using linear regression models. Results The total sample included 194 subjects of whom 81 had PD and 113 did not (non-PD). None of the participants had RA at the time of the study. As expected, there was a low frequency of ACPA, including anti-CCP2, anti-MCV, anti-CEP-1, anti-cit-vim and anti-cit-fib. Anti-CCP2 antibodies were present in 1.3% of the PD group and none of the controls. Anti-MCV was present in one subject in each of the groups. Anti-CEP-1 antibodies and anti-REP1 (arginine control peptide for CEP-1) were present in 8.6% and 1.8% (p=0.02) and 38.3% and 14.2% (p<0.001) of the cases and controls, respectively. Among those with PD, those who were anti-CEP-1 positive were non-smokers and among those who were anti-REP-1 positive only 13% were smokers. Anti-fib antibodies (arginine control peptide for cit-fib) were also more common among those with PD compared with non-PD (45.7% vs 13.3%, p<0.001). There was no statistical difference between groups for anti-cit-fib, anti-cit-vim or anti-vim positivity. Compared with non-PD, presence of PD was associated with both the citrullinated peptides (anti-CEP-1 (p<0.001), anti-cit-vim (p=0.007) and anti-cit-fib p=0.004)) and their uncitrullinated controls (anti-vim (p<0.001), anti-REP-1 (p<0.001) and anti-fib (p<0.001)) in linear regression models. These differences were independent of smoking. Conclusions Distinct ACPA reactivity was observed between individuals with and without PD. Antibodies against CEP-1 and cit-fib and their uncitrullinated forms (REP-1 and fib, respectively) were more common in PD, and higher levels of antibodies were observed among those with PD compared with non-PD, independent of smoking, suggesting that the uncitrullinated peptide may break tolerance in PD, with epitope spreading to citrullinated epitopes in the small proportion of patients which may evolve into RA.