ObjectivesAnti-centromere antibodies (ACAs) are detected in patients with various autoimmune diseases such as Sjögren’s syndrome (SS), systemic sclerosis (SSc) and primary biliary cholangitis (PBC). However, the targeted antigens of ACAs are not fully elucidated despite the accumulating understanding of the molecular structure of the centromere. The aim of this study was to comprehensively reveal the autoantigenicity of centromere proteins.MethodsA centromere antigen library including 16 principal subcomplexes composed of 41 centromere proteins was constructed. Centromere protein/complex binding beads were used to detect serum ACAs in patients with SS, SSc and PBC. ACA-secreting cells in salivary glands obtained from patients with SS were detected with green fluorescent protein-fusion centromere antigens and semiquantified with confocal microscopy.ResultsA total of 241 individuals with SS, SSc or PBC and healthy controls were recruited for serum ACA profiling. A broad spectrum of serum autoantibodies was observed, and some of them had comparative frequency as anti-CENP-B antibody, which is the known major ACA. The prevalence of each antibody was shared across the three diseases. Immunostaining of SS salivary glands showed the accumulation of antibody-secreting cells (ASCs) specific for kinetochore, which is a part of the centromere, whereas little reactivity against CENP-B was seen.ConclusionsWe demonstrated that serum autoantibodies target the centromere–kinetochore macrocomplex in patients with SS, SSc and PBC. The specificity of ASCs in SS salivary glands suggests kinetochore complex-driven autoantibody selection, providing insight into the underlying mechanism of ACA acquisition.

Rationale:Hypertrophic pachymeningitis (HP) is an uncommon, life-threatening disease that is seen in elderly patients with antineutrophil cytoplasmic antibody (ANCA) positivity. Proteinase-3 (PR3)-ANCA-positive HP has not been reported in adolescents. Here, we report the first case of adolescent PR3-ANCA-positive HP successfully treated with immunosuppressive therapy.Patient concerns:A 14-year-old female presented with fullness and pain in her right ear unresponsive to antibiotics. Laboratory tests showed an elevated C-reactive protein and PR3-ANCA positivity. Computed tomography and magnetic resonance imaging revealed mastoiditis in the right temporal bone. Surgical biopsy revealed severe fibrosis and prominent inflammatory-cell infiltration. She received prednisolone and methotrexate therapy, and then underwent a right mastoidectomy. Five months later, she developed headache, dysarthria, and multiple cranial nerve palsies. Further imaging revealed enhancement and thickening of the right hemispheric dura.Diagnosis:PR3-ANCA-positive HP.Interventions:She was successfully treated with steroid pulse therapy for 3 days, followed by high doses of prednisolone and intravenous cyclophosphamide.Outcome:The treatment resulted in significant improvement of her symptoms, laboratory data, and radiologic findings.Lessons:PR3-ANCA-positive HP can present not only in the elderly, but also in adolescence, and prompt diagnosis and treatment with immunosuppressive therapy is vital.

Background:Anti-centromere antibodies (ACA) are detected in the serum of patients with various autoimmune diseases including Sjögren’s syndrome (SjS), systemic sclerosis (SSc) and primary biliary cholangitis (PBC). ACA positivity is correlated with clinical manifestations such as Raynaud’s phenomenon and sclerodactyly and these features are commonly seen across diseases. Although CENPB is thought to be the major antigen against ACA, autoimmune features of other centromere proteins have not been fully evaluated.Objectives:The aim of this study is to elucidate centromere autoantigens comprehensively and clarify their association with pathogenesis of SjS, SSc and PBC.Methods:A centromere protein library was created by cloning 6 single proteins and 10 complexes consisting of 35 proteins belonging to human centromere region. The centromere antigens were immobilized on beads and incubated in the serum of patients with SjS (n = 86), SSc (n = 35), PBC (n = 10), patients with two or more diseases above (n = 44), and healthy volunteers (n = 68). Autoantibodies to each centromere protein were analyzed by flow cytometry.Results:Patients had a wide variety of antibodies against most of centromere antigens including 4 newly identified autoantigens. The hierarchical clustering of each antigen distinguished 2 antigen clusters. The reactivity of autoantibodies against a centromere protein of one cluster was mutually correlated regardless of disease types, indicating that these proteins/protein complexes might be the target of ACA. In addition, our method enabled us to detect sera reacted against multiple centromere antigens in some of the ACA-negative patients with existing methods.Conclusion:We identified 4 novel centromere autoantigens and our data suggested that the main target of ACA was the protein complex rather than a single specific antigen in SjS, SSc and PBC patients. Using the combination of centromere proteins may be useful to detect ACA with higher sensitivity.References:[1]Fritzler MJ, Rattner JB, Luft LM, Edworthy SM, Casiano CA, Peebles C, Mahler M. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F. Autoimmun Rev. 2011;10:194-200.Disclosure of Interests:Nobuhiko Kajio: None declared, Masaru Takeshita: None declared, Katsuya Suzuki: None declared, Tsutomu Takeuchi Grant/research support from: Eisai Co., Ltd, Astellas Pharma Inc., AbbVie GK, Asahi Kasei Pharma Corporation, Nippon Kayaku Co., Ltd, Takeda Pharmaceutical Company Ltd, UCB Pharma, Shionogi & Co., Ltd., Mitsubishi-Tanabe Pharma Corp., Daiichi Sankyo Co., Ltd., Chugai Pharmaceutical Co. Ltd., Consultant of: Chugai Pharmaceutical Co Ltd, Astellas Pharma Inc., Eli Lilly Japan KK, Speakers bureau: AbbVie GK, Eisai Co., Ltd, Mitsubishi-Tanabe Pharma Corporation, Chugai Pharmaceutical Co Ltd, Bristol-Myers Squibb Company, AYUMI Pharmaceutical Corp., Eisai Co., Ltd, Daiichi Sankyo Co., Ltd., Gilead Sciences, Inc., Novartis Pharma K.K., Pfizer Japan Inc., Sanofi K.K., Dainippon Sumitomo Co., Ltd.

BackgroundClinical remission is a clinical goal in the treatment of rheumatoid arthritis (RA). Sustained, biologics-free and true remission is an unachieved goal of the “treat-to-target” approach in most patients, and the determinants for achievement are still unclear. In our recent prospective study using multiomics analysis, we proposed that a molecular signature in peripheral whole blood can be a predictor for subsequent disease activity or activities of daily living.1 We also showed that tocilizumab (TCZ) induced deep clinical remission associated with gene expression in peripheral CD4+ T cells.2ObjectivesTo consolidate and expand our hypothesis, we investigated the significance of molecular signatures in sustained remission in a larger scale cohort.MethodsTo build and validate the diagnostic model, we collected 73 peripheral blood samples from 30 patients with active RA, 30 patients in clinical remission induced by TCZ and 13 healthy controls. We then collected another 23 samples at a point before TCZ was halted due to sustained clinical remission. In total, 96 samples were analyzed by a multiomics platform, which included RNA sequencing and comprehensive proteomics.ResultsWe first developed an optimized partial least-squares regression (PLSR) model using data from 5,436 genes and 255 proteins extracted in our previous model.1 The odds ratio in the model clearly reflected the clinical state with high fidelity (Figure 1). In that study, TCZ induced nearly half of the patients with clinical remission into molecular remission, with an odds ratio of less than zero. To clarify the characteristics of the molecular signature at sustained clinical remission under TCZ continuation, 23 samples were applied to the model. The odds ratio was largely the same as that for clinical remission. Next, we investigated the association with disease flare after cessation of TCZ. At some points before cessation, the median odds ratio in patients who experienced disease flare after stopping TCZ tended to be higher than that in patients with sustained remission after stopping TCZ in the transcriptomics model but not in the proteomics model. Thirty-five differentially expressed genes were identified between the two groups under the conditions of a >1.5-fold change and P-value<0.05.Figure 1.Odds ratio in the partial least-squares regression model using transcriptomics (A) and proteomics (B) data from rheumatoid arthritis and healthy control groupsConclusionOur larger scale study validated the idea in our previous study that TCZ induces molecular remission. A certain substantial gap associated with prognosis after quitting TCZ may exist as a molecular signature of sustained clinical remission induced by TCZ. These multiomics data sets enable us to understand sustained clinical remission at a molecular level.References[1]Nat Commun. 9(1):2775, 2018, 2) Sci Rep.11(1):16691, 2021Graphs:AcknowledgementsWe acknowledge funding by Chugai Pharmaceutical Co., Ltd.Disclosure of InterestsNobuhiko Kajio: None declared, Katsuya Suzuki Speakers bureau: AbbVie, AsahiKasei, Astellas, Ayumi, Bristol-Myers Squibb, Chugai, Eisai, Eli Lilly, Gilead, Janssen, Mitsubishi Tanabe, Pfizer, Sanofi, Viatris, Consultant of: AbbVie, Asahi Kasei, Janssen, Pfizer, Grant/research support from: Chugai, Daiichi-Sankyo, Eli Lilly, Mitsubishi Tanabe, Ono, Takeda, Kotaro Matsumoto: None declared, Hiroshi Iijima: None declared, Seiji Nakamura: None declared, Yohei Ishizawa: None declared, Jun Inamo: None declared, Masaru Takeshita: None declared, Keiko Yoshimoto: None declared, Yuko Kaneko Speakers bureau: Chugai, Consultant of: Chugai, Grant/research support from: Chugai, Tsutomu Takeuchi Speakers bureau: Chugai, Consultant of: Chugai, Grant/research support from: Chugai.

Background: Artificial intelligence (AI) techniques including deep learning have been rapidly evolving and have yielded appreciable benefits in many fields in recent years. In rheumatology field, however, these techniques have not been used often. Objectives: In an early phase of development of an AI-based automatic radiographic evaluating system for bone destruction, we aimed to develop learning-based models to automatically detect hand joint region, ankylosis and subluxation in radiographic images. Methods: A total of 130 radiographic image sets of both hands were randomly obtained from rheumatoid arthritis patients who had visited our division at Keio University Hospital in 2015. Well-trained rheumatologists determined the boundaries of regions of MP and PIP/IP joints and evaluated the presence of ankylosis and subluxation of each joint in radiographs. These evaluations of hand joints were performed using our developed annotation software tool [1]. In learning phase, joint images were randomly divided into five sets for 5-fold cross validation. As deep learning models, we utilized Single Shot Multibox Detector (SSD) method [2] with ensemble learning for detecting ankylosis and subluxation of MP and PIP/IP joint regions. Results: Our model showed 100% detection rate of MP and PIP/IP joint regions. As a performance of detecting hand joint ankylosis and subluxation, our model presented precision values of 0.85 and 0.73, recall values of 0.94 and 0.79, and F-measure values of 0.90 and 0.76, respectively. Conclusion: Deep learning-based models to automatically detect hand joint region, ankylosis and subluxation in radiographic images were developed with relatively small samples, which suggests that the predictive performance may increase by collecting more training dataset. Next, we are elaborating a plan for a deep learning-based evaluating system for erosion and joint space narrowing.