Rheumatoid arthritis is the most common inflammatory arthritis and is a major cause of disability. It existed in early Native American populations several thousand years ago but might not have appeared in Europe until the 17th century. Early theories on the pathogenesis of rheumatoid arthritis focused on autoantibodies and immune complexes. T-cell-mediated antigen-specific responses, T-cell-independent cytokine networks, and aggressive tumour-like behaviour of rheumatoid synovium have also been implicated. More recently, the contribution of autoantibodies has returned to the forefront. Based on the pathogenic mechanisms, specific therapeutic interventions can be designed to suppress synovial inflammation and joint destruction in rheumatoid arthritis.

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Evolving concepts of rheumatoid arthritis

Rheumatoid factor (RF) and autoantibodies against cyclic citrullinated peptide (CCP) are markers that might help in the diagnosis of rheumatoid arthritis. The investigators examined whether anti-CC...

Meta-analysis: Diagnostic Accuracy of Anti–Cyclic Citrullinated Peptide Antibody and Rheumatoid Factor for Rheumatoid Arthritis

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1-13), anti-double stranded DNA antibodies (14-18), specific antibodies (19-23) and validation of methods (24-25) were created. Significant differences between experts were observed regarding recommendations 24-25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

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International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies

Background: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein antibodies can be detected in rheumatoid arthritis (RA) sera. Objective: To determine the diagnostic values of RF, anticitrullinated protein antibodies, and the shared epitope (SE), and their associations with radiological progression rates and extra-articular manifestations. Methods: Population 1 consisted of sera from 315 patients, consecutively sent for detection of anticitrullinated protein antibodies, of which 264 were used to determine the sensitivity and specificity of RF and of antibodies against three synthetic citrullinated peptides: peptide A (pepA), peptide B (pepB), and CCP2. Population 2 consisted of sera from 180 longstanding RA patients and was used to determine associations of RA associated antibodies and the SE with radiological progression rates and extra-articular manifestations. Antibodies to pepA and pepB were detected by line immunoassay, and antibodies to CCP2 by ELISA. HLA Class II typing was performed by LiPA. Results: In population 1, we defined adapted cut offs corresponding to a specificity of ⩾98.5%. This yielded the following sensitivities: RF 12.8%; anti-pepA antibodies 63.6%; anti-pepB antibodies 54.2%; and anti-CCP2 antibodies 73.7%. In population 2, significant differences in radiological progression rates were found between positive and negative patients for different RA antibodies and the SE. RF, but not anticitrullinated protein antibodies or the SE, were more frequent in patients with extra-articular manifestations. Conclusion: A valid comparison of RA associated antibodies shows superior sensitivity of the anticitrullinated protein antibodies compared with RF. The presence of RA associated antibodies and the SE are indicative for poorer radiological outcome, and presence of extra-articular manifestations is associated with RF but not with anticitrullinated protein antibodies.

https://ard.bmj.com/content/annrheumdis/63/12/1587.full.pdf

Rheumatoid factor and anticitrullinated protein antibodies in rheumatoid arthritis: diagnostic value, associations with radiological progression rate, and extra-articular manifestations

Objective To identify features of antinuclear antibody (ANA)-HEp-2 test results that discriminate ANA-positive healthy individuals and patients with autoimmune rheumatic diseases (ARDs). Methods We sequentially retrieved data on 918 healthy individuals and 153 patients with ARDs after clinical assessment. ANA-positive healthy individuals for whom data were available were reevaluated after 3.6-5.0 years. An ANA-HEp-2 test result was considered positive when a clear ANA pattern was observed at 1:80 dilution in 2 distinct commercial HEp-2 slides by 2 blinded independent observers. Results ANAs were present in 118 healthy individuals (12.9%) and 138 patients with ARDs (90.2%). The ANA titer was higher in patients with ARDs than in healthy individuals (P Conclusion Our findings suggest that the titer, and especially the pattern, on the ANA-HEp-2 test strongly enhances our ability to discriminate ANA-positive healthy individuals and patients with ARDs.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.30084

Pattern on the antinuclear antibody–HEp-2 test is a critical parameter for discriminating antinuclear antibody–positive healthy individuals and patients with autoimmune rheumatic diseases

Objective

To identify immunodominant epitopes in natural filaggrin that are reactive with antifilaggrin autoantibodies (AFA) in the sera of patients with rheumatoid arthritis (RA) and to explore their use in a diagnostic assay format.



Methods

Based on the results of epitope mapping of human natural filaggrin as well as molecular modeling and computational chemistry, synthetic peptides together with recombinant citrullinated filaggrin were evaluated by a line immunoassay (LIA) for AFA detection. Diagnostic performance was assessed using 336 RA and 253 disease control sera and was compared with that of reference methods.



Results

Several immunoreactive epitopes were identified in natural filaggrin, all of which contained at least 1 citrulline residue. Three antigenic substrates, including 2 synthetic peptides and recombinant citrullinated filaggrin showing maximal reactivity on LIA, were finally selected. Using the 3-antigen LIA3, overall sensitivity, specificity, and positive predictive value for RA were 65.2%, 98.0%, and 89.1%, respectively, compared with 61.9%, 98.8%, and 92.8% using the 2-antigen LIA2 (without recombinant protein). Thirty-seven percent of the rheumatoid factor (RF)-negative RA samples (30 of 81) were AFA-positive by LIA2, and 52 of 54 RF-positive control samples had no AFA detected on LIA2. Higher specificity and sensitivity were obtained by LIA2 versus anti-RA33 immunoblot, whereas good agreement was observed with antikeratin antibody testing. LIA performed significantly better than AFA immunoblotting using natural filaggrin, at a specificity level of 99% (P = 0.0047).



Conclusion

Citrullinated residues are present in immunoreactive epitopes of natural human filaggrin. AFA can be readily detected by citrullinated peptides in an LIA-based test, resulting in high specificity and positive predictive value for RA. The LIA could serve as a user-friendly alternative to existing immunofluorescence tests and AFA immunoblot techniques. Given its complementarity to RF, this test can be a valuable tool in the differential diagnosis of arthritis.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/art.10229

Identification of citrullinated rheumatoid arthritis-specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection by line immunoassay.

Objectif : Etudier les caracteristiques cliniques de patients qui ont des auto-anticorps de la proteine CENP-F du centromere et la frequence des auto-anticorps CENP-F chez des patients qui ont diverses maladies. Conception : Etude clinique et serologique retrospective. Methodes : On a identifie 36 patients atteints d'anti-CENP-F au moyen d'un trace caracteristique d'immunofluorescence indirecte (IFI) sur les cellules HEp-2. On a etudie aussi 50 patients atteints d'un melanome, 50 d'un cancer du sein, 10, d'un cancer du poumon, 354 souffrait de sclerodermie generalisee, 120, de lupus erythemateux dissemine et 50, de polyarthrite rhumatoide. On a produit des proteines recombinantes a partir de clones de 5 CENP-F de l'ADNc representant les acides amines 2192-3317 (p-F1), 5561-7126 (p-F2), 5892-6883 (p-F3), 7538-10116 (p-F4) et 9242-10096 (p-F5). On a etudie la presence de l'antigene CENP-F dans une souche de cellules de cancer du sein, des cryosections de cancer du sein, des tissus normaux du sein et des amygdales. Resultats : Vingt-deux des 36 patients qui avaient des anticorps CENP-F avaient des neoplasmes; on a diagnostique surtout des cancers du sein (9/22) et du poumon (5/22). Trente-trois serums etaient disponibles pour une etude plus poussee. Lorsqu'on en a analyse la reactivite aux peptides recombinants, les serums de 21 patients sur 21 qui avaient des neoplasmes et de 5 patients sur 12 qui avaient d'autres maladies ont lie le peptide p-F4 C-terminal. Lorsqu'on a etudie le tiers terminal du peptide p-F4 (p-F5), on n'a pas detecte de difference significative dans la tendance a la reactivite. Comme comparaison, la frequence de la reactivite avec des peptides representant d'autres domaines du CNP-F a ete inferieure a celle de la reactivite avec le p-F4 (p-F2 > p-F3 > p-F1). On n'a pas trouve d'auto-anticorps CENP-F dans aucun des serums temoins provenant de patients atteints de lupus erythemateux dissemine, de polyarthrite rhumatoide ou de sclerodermie generalisee, ou dans des serums non selectionnes provenant de diverses tumeurs malignes. On a identifie des antigenes CENP-F dans des tissus de cancer du sein, mais rarement dans des tissus normaux. Conclusions: Une proportion elevee d'individus qui ont des anticorps CENP-F ont des neoplasmes et ces serums presentent un biais en faveur de la reactivite avec des determinants dans le domaine carboxyle terminal du CENP-F. Les antigenes CENP-F semblent tres frequents dans les tumeurs malignes.

High frequency of neoplasia in patients with autoantibodies to centromere protein CENP-F.

Objective: To determine whether anti-Ro52 is an independent serum marker in connective tissue disease. Methods: Over a two year period, 1727 consecutive antinuclear antibody (ANA) positive serum samples were analysed in parallel by double immunodiffusion with thymus/spleen nuclear extract and by line immunoassay with recombinant Ro52, recombinant La/SSB, and natural Ro60. Sera that were only reactive towards Ro52 were further analysed by a variety of additional anti-SSA/Ro detection methods and by specific anti-Ro52 and anti-Ro60 assays. Natural purified SSA/Ro was analysed by immunoblot and protein sequencing. Results: Analysis of natural purified SSA/Ro (Immunovision, Springdale, AR) showed only Ro60 and no immunoreactive Ro52. Consequently, assays based on this substrate only identify sera with anti-Ro60 reactivity. Twenty serum samples showed anti-Ro52 without anti-Ro60 and anti-SSB/La on line immunoassay. By additional testing, 2/20 sera were found positive for anti-Ro60 reactivity. The remaining 18 sera were not identified by any of the classical anti-SSA/Ro assays and were considered to be reactive only with Ro52 and not with Ro60. This anti-Ro52 reactivity was confirmed by natural and recombinant Ro52 in 16/18 cases. 12/18 sera corresponded to connective tissue diseases. Conclusion: Anti-Ro52 positive sera without any evidence of anti-Ro60 and anti-La/SSB reactivity can be considered as an independent group that is systematically missed by classical anti-SSA/Ro detection methods owing to a bias towards anti-Ro60 reactivity. The anti-Ro52 sera are precipitin negative, not retrieved by SSA/Ro enzyme linked immunosorbent assays (ELISAs) based on natural SSA/Ro, and show no specific ANA fluorescence staining pattern. These findings together with the clinical data indicate that anti-Ro52 should be considered as an additional and independent serum marker.

https://ard.bmj.com/content/annrheumdis/61/10/929.full.pdf

Anti-Ro52 reactivity is an independent and additional serum marker in connective tissue disease.

Autoimmunity to the cell cycle-dependent centromere protein p330d/CENP-F in disorders associated with cell proliferation

Primary biliary cirrhosis (PBC) is a chronic disease characterized by portal inflammation and necrosis of small intrahepatic bile ductules (1). PBC is an irreversible condition, and destruction of the bile ductules leads to progressive cholestasis and fibrosis, and may eventually lead to the development of cirrhosis. PBC is most likely an autoimmune disorder (2), and anti-mitochondrial antibody (anti-M2) has been a diagnostically very useful marker: several studies have reported a positivity rate for anti-M2 of >95% in biopsy-confirmed PBC patients (3). The major mitochondrial antigen was identified as the 74-kDa E2 subunit of the pyruvate dehydrogenase complex (PDC), a member of the 2-oxoacid dehydrogenase complex family (4). The traditional technique for the detection of anti-M2 is immunofluorescence (5), but recently new serological assays, such as ELISA, immunoblotting, and enzyme inhibition (EI), have been developed (6)(7). A miniaturized EI assay for performance on microtiter plates has already been described (8)(9). In this study we evaluated a new commercially available and completely automated EI assay manufactured by Trace Scientific (Victoria, Australia) and compared it to the immunofluorescence and ELISA techniques performed in our laboratory.

The TRACE enzymatic procedure is a unique method based on the PDC inhibitory properties of the principal anti-74-kDa antibody. PDC catalyzes the following reaction: \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[\mathrm{Pyruvate}{+}\mathrm{NAD}^{\mathrm{{+}}}{+}\mathrm{CoA}\ \begin{array}{l}\mathrm{PDC}\\{\rightarrow}\end{array}\ \mathrm{Acetyl-CoA}{+}\mathrm{NADH}{+}\mathrm{H}^{{+}}{+}\mathrm{CO}_{2}\] \end{document} In the absence of anti-M2, this reaction will proceed at the maximal rate after the addition of a serum sample. If a sample contains anti-M2, the PDC activity will be inhibited, greatly reducing the rate of reaction, typically to <70%. The rate of reaction can be simply monitored by measuring the rate of increase in absorbance at 340 …

/pdf/evaluation-of-an-automated-enzyme-inhibition-assay-for-the-4y1qm9g6ve.pdf

René Louis Humbel

Papers

Identification of citrullinated rheumatoid arthritis-specific epitopes in natural filaggrin relevant for antifilaggrin autoantibody detection by line immunoassay.

High frequency of neoplasia in patients with autoantibodies to centromere protein CENP-F.

Anti-Ro52 reactivity is an independent and additional serum marker in connective tissue disease.

Autoimmunity to the cell cycle-dependent centromere protein p330d/CENP-F in disorders associated with cell proliferation

Evaluation of an Automated Enzyme Inhibition Assay for the Detection of Anti-Mitochondrial M2 Autoantibodies