Ger J. M. Pruijn

Anti‐cN1A autoantibodies are helpful for diagnosing inclusion body myositis (IBM). We have developed an immunofluorescence assay based on the perinuclear accumulation of GFP‐cN1A. The analysis of patient sera indicated that this assay shows a higher sensitivity for anti‐cN1A in IBM compared to alternative assays, without loss of specificity.

Objectives: ACPAs play an important role in the classification of RA and can be detected by serological tests. Several ACPA detection assays are available for clinical use, which are all based on ELISA(-like) tests with citrullinated peptides (CCP2) or alternative citrullinated proteins/peptides. We aimed to facilitate ACPA detection in low-volume laboratories and resource-poor environments by developing a rapid and easy-to-perform test. Methods: We designed and generated an agglutination mediator by protein engineering. A single-chain antibody fragment that binds to glycophorin A, one of the major surface proteins of erythrocytes, was conjugated to a CCP2-like peptide. This agglutination mediator was used to assess ACPA in RA and PsA patients and in healthy individuals. Results: The agglutination mediator bound to erythrocytes was reactive with ACPA and induced haemagglutination in an ACPA-dependent fashion, which can be detected by the naked eye. The applicability was assessed by the analysis of fresh blood samples from 204 RA patients, 77 PsA patients and 100 healthy individuals. Agglutination was observed in up to 61% of the RA samples, which correlated well with the results obtained with a standardized anti-CCP2 ELISA (63–67%). Depending on the minimal agglutination score, agglutination was observed in only 3–21% of the PsA samples and in 1% of the healthy controls. Conclusion: We conclude that the agglutination mediator allows rapid and efficient detection of ACPA by haemagglutination in human blood samples and offers a low-threshold method for ACPA detection.

One of the main strategies of neutrophils in responding to microbial infections is the formation of neutrophil extracellular traps (NETs). NETs are web-like structures of decondensed chromatin associated with antimicrobial proteins. Citrullination plays an important role during NET formation and a substantial fraction of NET-associated proteins appeared to be citrullinated. The release of citrullinated intracellular proteins from netting neutrophils led to the hypothesis that the production of anti-citrullinated protein autoantibodies by autoimmune patients, in particular patients with rheumatoid arthritis, might be initiated when citrullinated NET components are not properly cleared and are exposed to the immune system. Here, we discuss the processes that lead to NET formation, including the role of peptidylarginine deiminase activation and our current knowledge on citrullinated NET-associated proteins. Citrulline-dependent epitopes do not appear to play a major role in the recognition of NETs by autoantibodies from rheumatoid arthritis and systemic lupus erythematosus patients, even though anti-NET autoantibodies are frequently observed in sera from these patients. The neutrophil proteases associated with NETs have a major impact on the integrity of NET-associated proteins when NET formation is induced by activating isolated human neutrophils. Cleavage/degradation of these proteins also resulted in a strong reduction of the reactivity with autoantibodies. This article is part of the Theo Murphy meeting issue ‘The virtues and vices of protein citrullination’.

Background Autoantibodies are found in up to 80% of patients with idiopathic inflammatory myopathies (IIM) and are associated with distinct clinical phenotypes [1]. Autoantibodies targeting cytosolic 5´-nucleotidase 1A (anti-cN1A) are currently the only known serum biomarker for the subgroup inclusion body myositis (IBM) (2), although detected even in other autoimmune diseases. Objectives To identify new autoimmune targets in IIM by antigen bead array assay. Methods In a first cross-sectional exploratory study, 357 antigens representing 268 proteins were incubated with plasma samples from 219 IIM (108 Polymyositis (PM), 80 Dermatomyositis (DM) and 31 IBM) patients, 349 Systemic Lupus Erythematosus (SLE) patients and 306 population controls for screening of IgG reactivity by antigen bead array. All samples were identified in the local biobank of the Rheumatology clinic, Karolinska University Hospital. Interesting results obtained for the IBM subgroup were then validated in an independent larger cohort of 287 patients with IBM followed at nine European rheumatological or neurological centers. IBM serum samples were explored by antigen bead array and results validated by western blot. As controls, serum samples from 30 patients with PM and 30 with DM, HLA-matched with the IBM Swedish cohort, were included. Demographics, laboratory, clinical, and muscle biopsy data of the IBM cohort was retrieved. Results In the exploratory study IgG reactivity towards NADH dehydrogenase 1 α subcomplex 11 (NDUFA11), a subunit of the membrane-bound mitochondrial respiratory chain complex I, was discovered with higher frequency in the IBM (9,7%) than PM (2,8%) and DM samples (2,5%), although the difference was not statistically significant. Anti-NDUFA11 IgG was also found in 2,3% of SLE and 2,6% of population control samples. In the validation study anti-NDUFA11 autoantibodies were detected in 11/287 IBM patients (3,8%), 0/30 PM and 0/30 DM patients. Reactivity against NDUFA11 could be confirmed by western blot (Table 1, Figure 1). The eleven anti-NDUFA11 positive patients showed a trend of lower frequency of wheelchair/walker ever use and higher creatine kinase levels at time of IBM diagnosis compared to the anti-NDUFA11 negative group. Ragged red fibers were significantly more prevalent in anti-NDUFA11 positive than negative patients (p=0.04). Anti-cN1A autoantibodies were detected in 98/287 (34,1%) of IBM, 3/30 (10%) DM and 9/29 (31%) PM patients, p=0.03. Coexistence of anti NDUFA11 and anti-cN1A antibodies was observed in 3 IBM patients. Conclusion Our results reveal a new autoimmune target in the mitochondrial respiratory chain complex I that might be specifically associated with IBM. This is of particular interest as mitochondrial abnormalities are known histological findings in muscle biopsies of IBM patients. References [1]Galindo-Feria AS, Wang G, Lundberg IE. Autoantibodies: Pathogenic or epiphenomenon. Best Pract Res Clin Rheumatol. 2022;36(2):101767. [2]Herbert MK,et al. Disease specificity of autoantibodies to cytosolic 5’-nucleotidase 1A in sporadic inclusion body myositis versus known autoimmune diseases. Ann Rheum Dis. 2016;75(4):696-701. NDUFA11 protein fragments (PrESTs) Legend Amino acid sequence and molecular weight of the protein fragments loaded on the western blot gel. NDUFA11, NADH dehydrogenase 1 α subcomplex 11; His6ABP, six histidine and albumin binding protein. Validation of bead array assay results with western blot Legend Western blot showing the reactivity of two IBM patients against the NDUFA11 protein fragments (PrEST1,2,3) expressed with a His6ABP tag. Patient#1 was reactive to PrEST1 and not to PrEST2 and 3 in the bead array assay while patient#2 did not display any reactivity. NDUFA11, NADH dehydrogenase 1 α subcomplex 11; His6ABP, six histidine and albumin binding protein. Disclosure of Interests Antonella Notarnicola: None declared, Cecilia Hellstrom: None declared, Begum Horuluoglu: None declared, Charlotta Preger: None declared, Francesco Bonomi: None declared, Boel De Paepe: None declared, Jan De Bleecker: None declared, Anneke J. van der Kooi: None declared, Marianne de Visser: None declared, Sabrina Sacconi: None declared, Pedro Machado: None declared, Umesh A. Badrising: None declared, Anke Rietveld: None declared, Ger Pruijn: None declared, Simon Rothwell: None declared, James B. Lilleker: None declared, Hector Chinoy: None declared, Olivier Benveniste: None declared, Elisabet Svenungsson: None declared, Helena Idborg: None declared, Per-Johan Jakobsson: None declared, Peter Nilsson: None declared, Ingrid E. Lundberg Shareholder of: stock shares in Roche and Novartis, Consultant of: Consulting fees from Corbus Pharmaceuticals, Inc and research grants from Astra Zeneca and has been serving on the advisory board for Astra Zeneca, Bristol Myers Squibb, EMD Serono Research & Development Institute, Argenx, Octapharma, Kezaar, Orphazyme, Pfizer and Janssen.

Background Anti-citrullinated protein antibodies (ACPA) are the most specific serological marker of rheumatoid arthritis (RA) and are produced by about 75% of RA patients. The presence of ACPA early in the disease, even before onset of clinical symptoms, facilitates early diagnosis and ACPA is among the most prominent classification criteria for RA.^[1] Importantly, early diagnosis and immediate start of treatment is strongly correlated with improved outcomes. Several ACPA-detection assays are available for clinical use, which nearly all are based on the same principle: ELISA or related assays with cyclic citrullinated peptides or citrullinated proteins.^[2] While these methods can be automated in modern diagnostic laboratories, they are ill-suited for low volume laboratories or resource-poor environments. Objectives The aim of this study was to develop a rapid and easy to perform assay for ACPA detection. The assay is based on ACPA-dependent agglutination of erythrocytes and can be executed with whole blood. Methods An agglutination mediator was developed by protein engineering. Addition of this mediator to (diluted) blood samples results in hemagglutination when ACPA are present. After optimization of the assay with RA serum-spiked blood samples, the applicability was assessed by the analysis of fresh blood samples from 100 RA patients and from 100 psoriatic arthritis (PsA) patients as a control group. Anti-CCP2 and RF levels in these patient samples were determined by standardized ELISAs. Results The agglutination mediator that was generated is based on a single-chain antibody fragment that binds to glycophorin A,^[3] one of the major surface proteins of erythrocytes. It is conjugated to a citrullinated peptide that is efficiently recognized by ACPA. In the presence of erythrocytes and ACPA the mediator induces agglutination of the erythrocytes, which can be detected by the naked eye. The addition of the mediator resulted in detectable agglutination in 64% percent of the RA patient samples. Agglutination correlated well with the results obtained with a commercial anti-CCP2 ELISA for ACPA detection. Efficient agglutination was observed with only 7% of the PsA samples. No correlation with rheumatoid factor levels was observed. Conclusion An ACPA-dependent hemagglutination mediator was generated. This agglutination mediator allows the rapid and efficient detection of ACPA by hemagglutination in human blood samples. References [1]Aletaha, D. et al. 2010 Rheumatoid arthritis classification criteria. Arthritis Rheum. 2010; 62:2569-81. [2]Pruijn, G.J.M. et al. The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis. Arthritis Res Ther. 2010; 12:203. [3]Bigbee, W.L. et al. Binding specificities of eight monoclonal antibodies to human glycophorin A – studies with M^cM, and M^kEn(UK) variant human erythrocytes and M and MN^V-type chimpanzee erythrocytes. J Immunol. 1984: 133:3149-55. Acknowledgements: NIL. Disclosure of Interests Ilmar Kruis: None declared, Annemarie Van der Heijden: None declared, Martin Salden Employee of: CEO/ R&D director Novio Catalpa BV, Ger Pruijn Grant/research support from: Research grant from Novio Catalpa B.V.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

The hallmark autoantibodies in rheumatoid arthritis are characterized by variable domain glycans (VDGs). Their abundant occurrence results from the selective introduction of N-linked glycosylation sites during somatic hypermutation, and their presence is predictive for disease development. However, the functional consequences of VDGs on autoreactive B cells remain elusive. Combining crystallography, glycobiology, and functional B cell assays allowed us to dissect key characteristics of VDGs on human B cell biology. Crystal structures showed that VDGs are positioned in the vicinity of the antigen-binding pocket, and dynamic modeling combined with binding assays elucidated their impact on binding. We found that VDG-expressing B cell receptors stay longer on the B cell surface and that VDGs enhance B cell activation. These results provide a rationale on how the acquisition of VDGs might contribute to the breach of tolerance of autoreactive B cells in a major human autoimmune disease.

RNase MRP is a ribonucleoprotein complex involved in the endoribonucleolytic cleavage of different RNAs. Mutations in the RNA component of the RNP are the cause of cartilage hair hypoplasia. Patients with cartilage hair hypoplasia are characterized by skeletal dysplasia. Biochemical purification of RNase MRP is desired to be able to study its biochemical function, composition and activity in both healthy and disease situations. Due to the high similarity with RNase P, a method to specifically isolate the RNase MRP complex is currently lacking. By fusing a streptavidin-binding RNA aptamer, the S1m-aptamer, to the RNase MRP RNA we have been able to compare the relative expression levels of wildtype and mutant MRP RNAs. Moreover, we were able to isolate active RNase MRP complexes. We observed that mutant MRP RNAs are expressed at lower levels and have lower catalytic activity compared to the wildtype RNA. The observation that a single nucleotide substitution at position 40 in the P3 domain but not in other domains of RNase MRP RNA severely reduced the binding of the Rpp25 protein subunit confirmed that the P3 region harbours the main binding site for this protein. Altogether, this study shows that the RNA aptamer tagging approach can be used to identify RNase MRP substrates, but also to study the effect of mutations on MRP RNA expression levels and RNase MRP composition and endoribonuclease activity.

Objective: To assess anti–cytosolic 5′‐nucleotidase 1A (anti–cN‐1A) autoantibodies in children with juvenile dermatomyositis (DM) and healthy controls, using 3 different methods of antibody detection, as well as verification of the results in an independent cohort.Methods: Anti–cN‐1A reactivity was assessed in 34 Dutch juvenile DM patients and 20 healthy juvenile controls using the following methods: a commercially available full‐length cN‐1A enzyme‐linked immunosorbent assay (ELISA), a synthetic peptide ELISA, and immunoblotting with a lysate from cN‐1A–expressing HEK 293 cells. Sera from juvenile DM patients with active disease and those with disease in remission were analyzed. An independent British cohort of 110 juvenile DM patients and 43 healthy juvenile controls was assessed using an in‐house full‐length cN‐1A ELISA.Results: Anti–cN‐1A reactivity was not present in sera from juvenile DM patients or healthy controls when tested with the commercially available full‐length cN‐1A ELISA or by immunoblotting, in either active disease or disease in remission. Additionally, in the British juvenile DM cohort, anti–cN‐1A reactivity was not detected. Three Dutch juvenile DM patients had weakly positive results for 1 of 3 synthetic cN‐1A peptides measured by ELISA.Conclusion: Juvenile DM patients and young healthy individuals did not show anti–cN‐1A reactivity as assessed by different antibody detection techniques.