Nathalie Balandraud

Safety of TNF-blocking agents in rheumatic patients with serology suggesting past hepatitis B state: results from a cohort of 21 patients

IntroductionReactivation of hepatitis B virus (HBV) infection in patients with past infection has been described in 5% to 10% of individuals undergoing immunosuppressive therapies. No data are available to date on the outcome of patients treated by tumour necrosis factor-alpha (TNFα) inhibitors for chronic arthritis with a serological pattern of past HBV infection. The aim of our study was to monitor HBV markers in HBV surface antigen (HBsAg)-negative/anti-HBcAb-positive patients treated with a TNFα inhibitor for inflammatory arthritides.MethodsTwenty-one HBsAg-negative/anti-HBcAb-positive patients were included. HBV serological patterns were compared with those determined before starting TNFα inhibitors. Serum HBV DNA testing by polymerase chain reaction was additionally performed. Spearman correlation analysis was used and P < 0.05 was chosen as the significance threshold.ResultsBefore starting therapy, mean anti-HBsAb titre was 725 IU/L, no patient had an anti-HBsAb titre <10 IU/L, and 18 patients had an anti-HBsAb >100 IU/L. At a mean time of 27.2 months following therapy introduction, mean anti-HBsAb titre was 675 IU/L and anti-HBsAb titre remained >100 IU/L in 17 patients. There was a strong correlation between the first and second anti-HBsAb titres (r = 0.98, P = 0.013). Moreover, no patient had an anti-HBsAb titre below 10 IU/L or HBV reactivation (HBsAg seroreversion or positive HBV DNA detection). However, the anti-HBsAb titre decreased by more than 30% in 6 patients. The mean anti-HBsAb titre at baseline was significantly lower (P = 0.006) and the mean duration of anti-TNFα therapy, although non-significant (P = 0.09), was longer in these six patients as compared to patients without a decrease in anti-HBsAb titre.ConclusionsAnti-TNFα treatments are likely to be safe in patients with past hepatitis B serological pattern. However, the significant decrease of anti-HBsAb titre observed in a proportion of patients deserves HBV virological follow-up in these patients, especially in those with a low anti-HBsAb titre at baseline.

New autoantigens in rheumatoid arthritis (RA): screening 8268 protein arrays with sera from patients with RA

Objective: To identify new IgG autoantibodies in sera from patients with rheumatoid arthritis (RA). Methods: We tested serum samples from 19 patients with RA with given human leukocyte antigen (HLA)-DR genotypes, from 7 patients with spondylarthropathy, 2 patients with lupus, 4 patients with systemic sclerosis and 10 healthy individuals on 8268 human protein arrays. Results: We identified four antigens (peptidyl arginine deiminase 4 (PAD4), protein kinase Cβ1 (PKCβ1), phosphatylinositol 4 phosphate 5 kinase type II γ (PIP4K2C) and v raf murine sarcoma viral oncogene homologue B1 catalytic domain (BRAF)) that were recognised almost uniquely by sera from patients with RA on protein arrays. Using purified proteins, we confirmed that PAD4 and BRAF are recognised almost uniquely by patients with RA. Conclusion: We identified PAD4 and BRAF as RA specific autoantigens.

HLA-DRB1 genotypes and the risk of developing anti citrullinated protein antibody (ACPA) positive rheumatoid arthritis.

Objective To provide a table indicating the risk for developing anti citrullinated protein antibody (ACPA) positive rheumatoid arthritis (RA) according to one’s HLA-DRB1 genotype. Methods We HLA-DRB1 genotyped 857 patients with ACPA positive RA and 2178 controls from South Eastern and Eastern France and calculated Odds Ratios (OR) for developing RA for 106 of 132 possible genotypes accounting for 97% of subjects. Results HLA-DRB1 genotypic ORs for developing ACPA positive RA range from 28 to 0.19. HLA-DRB1 genotypes with HLA-DRB1*04SE (HLA-DRB1*0404, HLA-DRB1*0405, HLA-DRB1*0408), HLA-DRB1*04∶01, HLA-DRB1*01 are usually associated with high risk for developing RA. The second HLA-DRB1 allele in genotype somewhat modulates shared epitope associated risk. We did not identify any absolutely protective allele. Neither the Reviron, nor the du Montcel models accurately explains our data which are compatible with the shared epitope hypothesis and suggest a dosage effect among shared epitope positive HLA-DRB1 alleles, double dose genotypes carrying higher ORs than single dose genotypes. Conclusion HLA-DRB1 genotypic risk for developing ACPA positive RA is influenced by both HLA-DRB1 alleles in genotype. We provide an HLA-DRB1 genotypic risk table for ACPA positive RA.

Autoantibodies to PAD4 and BRAF in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that causes cartilage and bone destruction. The mechanisms leading to RA are unknown. There is currently no reliable cure for RA. Early treatment can reduce inflammation, joint damage and bone destruction. Thus, early diagnosis of RA is critical. However, definitive diagnosis of RA can be difficult. Immunologic tests that can be performed for the diagnosis of RA include detection of anti citrullinated protein antibodies (ACPAs). However, one third of RA patients have no ACPAs. To identify new autoantibodies in RA, we used the sera of RA patients to screen protein arrays containing 8000 human proteins. We found and validated two major autoantigens: PAD4 (peptidyl arginine deiminase 4) and BRAF (v raf murine sarcoma viral oncogene homolog B1) catalytic domain. We identified peptide targets of anti PAD4 and BRAF autoantibodies. We observed that anti PAD4 are inhibitory whereas anti BRAF stimulate BRAF activity. Anti PAD4 and anti BRAF antibodies may be used to diagnose RA, particularly in the absence of anti citrullinated protein antibodies.

Patients with ankylosing spondylitis have been breast fed less often than healthy controls: a case–control retrospective study

Objective Ankylosing spondylitis (AS) is a chronic inflammatory disease affecting the spine and pelvis of young adults. On the HLA-B27 genetic background, the occurrence of AS is influenced by the intestinal microbiota. The goal of our study was to test whether breast feeding, which influences microbiota, can prevent the development of AS. Methods First, 203 patients with HLA-B27-positive AS fulfilling the modified New York criteria were recruited in the Department of Rheumatology, Ste Marguerite hospital in Marseilles. A total of 293 healthy siblings were also recruited to make up a control group within the same families. Second, 280 healthy controls, and 100 patients with rheumatoid arthritis and their siblings were recruited. The data collected were age, gender, number of brothers and sisters, age at disease onset, type and duration of feeding (breast or bottle). Results Patients with AS had been breast fed less often than healthy controls. In families where children were breast fed, the patients with AS were less often breast fed than their healthy siblings (57% vs 72%), giving an OR for AS onset of 0.53 (95% CI (0.36 to 0.77), p value=0.0009). Breast feeding reduced familial prevalence of AS. The frequency of breast feeding was similar in the AS siblings and in the 280 unrelated controls. However, patients with AS were less often breast fed compared with the 280 unrelated controls (OR 0.6, 95% CI (0.42 to 0.89), p<0.01). Conclusions Our study suggests a breastfeeding-induced protective effect on the occurrence of AS. To our knowledge, this is the first study of breastfeeding history in patients with AS.

HLA-DRB1*0404 is strongly associated with anticalpastatin antibodies in rheumatoid arthritis

Objective: To test whether HLA-DR alleles influence the production of particular autoantibodies in rheumatoid arthritis (RA) patients, we screened synovial proteins with sera of RA patients homozygous for different HLA-DR alleles by using 2D blots. We found that sera of RA patients homozygous for HLA-DRB1*0404 recognised a 100-kDa synovial protein identified as calpastatin. We studied B and T cell epitopes on calpastatin and their association with HLA-DRB1*0404. Methods: The frequency of positive sera in patients expressing different RA-associated HLA-DR allele combinations was calculated by inhouse ELISA using purified synovial calpastatin or calpastatin peptides encompassing the entire calpastatin protein as immunosorbent. Interaction between calpastatin peptides and HLA-DR alleles was tested by a direct binding assay. T cell responses to calpastatin were measured in RA patients and controls. Results: We found that RA-associated HLA-DR alleles are associated with presence of autoantibodies to synovial calpastatin in RA patients’ sera. HLA-DRB1*0404 is strongly associated with antisynovial calpastatin in RA sera. One linear B cell epitope is preferentially associated with HLA-DRB1*0404. Multiple peptides from calpastatin bind every tested HLA-DR allele associated or not with RA. Peptides from domain 1 and 4 of calpastatin are the best HLA-DR allele binders. The T cell response to calpastatin is frequent in RA patients and independent of the HLA-DR background. Conclusions: HLA-DRB1*0404 is strongly associated with anticalpastatin antibodies in rheumatoid arthritis.

Autoantibodies to BRAF, a new family of autoantibodies associated with rheumatoid arthritis

IntroductionBRAF (v raf murine sarcoma viral oncogene homologue B1) is a serine-threonine kinase involved in the mitogen-activated protein kinase (MAPK) signalling pathway, known to be implicated in the production of pro-inflammatory cytokines.We have observed that sera from rheumatoid arthritis (RA) patients recognize the BRAFs catalytic domain, which encompasses amino acids 416 to 766. Here, we identify peptide targets of anti-BRAF autoantibodies and test whether anti-BRAF autoantibodies may interfere with BRAF kinase activity.MethodsAnti-BRAF autoantibodies were detected by ELISA (enzyme-linked immunosorbent assay) in the serum of RA patients and controls, using 40 overlapping 20mer peptides encompassing the catalytic domain of BRAF as immunosorbents. To test whether autoantibodies to BRAF influence BRAF kinase activity, we developed an in vitro phosphorylation assay of MEK1 (mitogen extracellular regulated kinase), a major BRAF substrate. MEK1 phosphorylation by BRAF was tested in the presence of purified anti-BRAF autoantibodies from RA patients or control antibody.ResultsWe found that one BRAF peptide, P25 (656 to 675), is specifically recognized by autoantibodies from RA patients. Of interest, anti-P25 autoantibodies are detected in 21% of anti-CCP (cyclic citrullinated peptides) negative RA patients. Anti-BRAF autoantibodies activate the in vitro phosphorylation of MEK1 mediated by BRAF.ConclusionsAnti-BRAF autoantibodies from RA patients preferentially recognize one BRAF peptide: P25. Autoantibody responses to P25 are detected in 21% of anti-CCP negative RA patients. Most anti-BRAF autoantibodies activate BRAF kinase activity.

New autoantibodies in early rheumatoid arthritis

IntroductionRheumatoid arthritis (RA) is a chronic inflammatory joint disease causing articular cartilage and bone destruction. Since irreversible joint destruction can be prevented by intervention at the early stages of disease, early diagnosis of RA is important. In this study, we identified new autoantibodies in the sera of patients with early (less than one year) RA.MethodsWe screened the sera of 20 RA patients with disease duration less than one year, 19 RA patients with disease duration more than five years and 23 controls on 8,268 human protein arrays. We confirmed the validity of protein array detection by ELISA assays. We then performed epitope mapping with overlapping 15-mers to analyze RA sera reactivity.ResultsWIBG (within BGCN homolog (Drosophila)), GABARAPL2 (GABA(A) receptor associated protein like 2) and ZNF706 (zinc finger protein 706) proteins are preferentially recognized by autoantibodies from early RA patients. Of interest, autoantibodies to WIBG are very specific for early RA. Indeed, 33% of early RA patients sera recognize WIBG versus 5% of RA patients with disease duration more than 5 years and 2% of controls. We identified three linear peptides on WIBG GABARAPL2 and ZNF706 that are preferentially recognized by sera of early RA patients.ConclusionsWe identified new autoantibodies associated with RA with disease duration less than one year. These autoantibodies could be used as diagnosis markers in RA patients.

Improving agreement in assessment of synovitis in rheumatoid arthritis.

OBJECTIVEnSynovitis assessment through evaluation of swollen joints is integral in steering treatment decisions in rheumatoid arthritis (RA). However, there is high inter-observer variation. The objective was to assess if a short collegiate consensus would improve swollen joint agreement between rheumatologists and whether this was affected by experience.nnnMETHODSnEighteen rheumatologists from French university rheumatology units participated in three 30 minutes rounds over a half day meeting evaluating joint counts of RA patients in small groups, followed by short consensus discussions. Agreement was evaluated at the end of each round as follows: (i) global agreement of swollen joints (ii) swollen joint agreement according to level of experience of the rheumatologist (iii) swollen joint count and (iv) agreement of disease activity state according to the Disease Activity Score (DAS28). Agreement was calculated using percentage agreement and kappa.nnnRESULTSnGlobal agreement of swollen joints failed to improve (kappa 0.50 to 0.52) at the joint level. Agreement between seniors did not improve but agreement between newly qualified rheumatologists and their senior peer, which was initially poor (kappa 0.28), improved significantly (to 0.54) at the end of the consensus exercises. Concordance of DAS28 activity states improved from 71% to 87%.nnnCONCLUSIONnConsensus exercises for swollen joint assessment is worthwhile and may potentially improve agreement between clinicians in clinical synovitis and disease activity state, benefit was mostly observed in newly qualified rheumatologists.

Antinuclear Antibodies in Patients with Psoriatic Arthritis Treated or Not with Biologics

Background With the emergence of biotherapies, accurate diagnosis in early arthritis is needed. At this time, there is no biological marker of psoriatic arthritis. Objective To test whether antinuclear antibodies (ANA) can be used as a diagnostic tool in psoriatic arthritis (PsA), we evaluated the prevalence of ANA in biologic-naïve PsA patients and in healthy blood donors. Methods 232 patients from the Rheumatology department, St Marguerites Hospital, Marseilles, who fulfilled the CASPAR criteria for PsA, underwent clinical and laboratory investigations. Antinuclear antibodies (ANA), anti-extractable nuclear antigen antibodies (ENA), rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA) were assayed. Ninety-one healthy blood donors were also tested. Results Detection of ANA by indirect immunofluorescence was significantly more frequent in sera from PsA patients than those from controls at serum dilution of 1:100 (57% compared with 40%, Odds Ratio (OR) 1.98 (1.2-3.4) p<0.02) and 1:160 (52% compared with 24%, OR 3,7 (1.9-7.2) p<0.001). No patients had lupus specific autoantibodies, 15 % had RF (34/232), and 1.7 % had ACPA (4/232). Conclusions Detection of ANA was more frequent in sera from PsA patients than in those from healthy controls. This suggests that ANA could be a diagnosis orientation tool in PsA. Nevertheless, the specificity of these antibodies still remains to be investigated.