1. ANA and Assays (1)First Author / Presenter Affiliation Title

Manfred HeroldMedical University of Innsbruck, Department ofInternal Medicine VI, Innsbruck, Austria,EASIAustria

ANA testing: the clinicians’ point of view

Kai Fechner Institute for Experimental Immunology,EUROIMMUN AG Automated evaluation of ANA patterns with EUROPattern meets the ICAP guideline

Kai Fechner Institute for Experimental Immunology,EUROIMMUN AG Evaluation of Crithidia luciliae IFT can be reliably automated with EUROPattern

Kai Fechner Institute for Experimental Immunology,EUROIMMUN AG Evaluation of modified Crithidia luciliae IIFT for detection of anti-dsDNA antibodies

Werner Klotz Medical University of Innsbruck, Department ofInternal Med. VI ANA screening on DFS70-knockout HEp-2 cells in a routine setting

Rico Hiemann Brandenburg University of Technology Cottbus-Senftenberg ANA testing on HEp-2 cells and antigen-coated microparticles including DFS70

Rico Hiemann Brandenburg University of Technology Cottbus-Senftenberg Automated digital classification of HEp-2 pattern according to ICAP classification tree

Kishore Malyavantham Immco Diagnostics Novel Engineered HEp-2 Substrate and its Impact on DFS70 and ANA ScreeningAlgorithm

Alessandra Dellavance

Research and Development Division, FleuryLaboratoriesRheumatology Division, Escola Paulista de Medicina,Universidade Federal de São Paulo

Proposal of an International Standard for Anti-DFS70/LEDGFp75 Antibodies

2. ANA and Assays (2)First Author / Presenter Affiliation Title

Nadja Roeber Institute of Immunology, Medical Faculty Carl GustavCarus, Technical University Dresden Harmonization of HEp-2 cell assays using anti-cell pattern description proposed by ICAP

Nadja Roeber Institute of Immunology, Medical Faculty Carl GustavCarus, Technical University Dresden Differentiation of immunofluorescence pattern of anti-Ro/SS-A antibody positive sera

Torsten Matthias AESKU. Diagnostics GmbH & Co. KG Performance comparison of the HELMED® Blot Module and HELIA® usingAESKUBLOTS® ANA-17 Pro

Torsten Matthias AESKU. Diagnostics GmbH & Co. KG MULTICENTER STUDY OF ANA VALIDATION BY IFA IN THE AUTOMATEDHELIOS® AND HELMED®

Torsten Matthias AESKU. Diagnostics GmbH & Co. KG IMMUNOFLUORESCENCE STUDIES FOR THE SPECIFIC DIAGNOSTICS OFMYASTHENIA GRAVIS BY IFA IN THE AUTOMATED HELIOS®

Tsuyoshi Shirai Tohoku University School of Medicine An effective method to identify autoantigens expressed on the cell surface: SARF

[ Poster Session 1 ]15:15-16:15 11th October (Tue), 2016

3. Rheumatoid Arthritis - BasicFirst Author / Presenter Affiliation Title

Yuichi Maeda

Department of Respiratory Medicine, Allergy andRheumatic Diseases, Osaka University GraduateSchool of Medicine Department of Microbiology andImmunology, Osaka University Graduate School ofMedicine

Gut microbiota from rheumatoid arthritis patients induce severe arthritis in SKG mice

Namiko OkabeDepartment of Rheumatology and ClinicalImmunology, Kyoto University Graduate School ofMedicine

STS-2 (Suppressor of TCR signaling-2) suppresses collagen-induced arthritis in mice.

Yuichi Yokoyama Division of Rheumatology, Department of internalmedicine, Hyogo College of Medicine

INVOLVEMENT OF REGULATORY T CELLS AND MICRORNAS IN REGULATIONOF COLLAGEN-INDUCED ARTHRITIS IN MICE TREATED WITH IL-2/ANTI-IL-2IMMUNE COMPLEXES

Hoshimi Kawaguchi Department of Internal Medicine, Faculty ofMedicine, University of Tsukuba Crucial involvement of citrullinated proteins and ACPA in peptide GPI-induced arthritis

Hui Jin

Laboratory of Immunochemistry, World PremierInternational (WPI) Immunology Frontier ResearchCenter, Osaka UniversityDepartment of Immunochemistry, Research Institutefor Microbial Diseases, Osaka University

Neo-self IgG/HLA-DR complexes are involved in RA susceptibility as autoantibody targets

Takayoshi Morita

Department of Respiratory Medicine, Allergy andRheumatic diseases, Osaka University GraduateSchool of MedicineDepartment of Experimental Immunology,Immunology Frontier Research Center, OsakaUniversity

The proportion of regulatory T cells in rheumatoid arthritis; comparison of the results fromdifferent definition of regulatory T cells

Masato MoriDepartment of Rheumatology and ClinicalImmunology, Graduate School of Medicine, KyotoUniversity

Adhesion molecules on synovial fibloblast promote cytokine production from CD4+T cells

Naoto UmedaDepartment of Internal Medicine, Faculty ofMedicine, University of Tsukuba,Department ofRheumatology, Tsuchiura Kyodo General Hospital

Co-localization of citrullinated proteins, PAD4 and ACPAs in CD68+ cells in RAsynovium

4. Rheumatoid Arthritis - ClinicalFirst Author / Presenter Affiliation Title

Nozomi IshigookaDepartment of Rheumatology and ClinicalImmunology,Kyoto University Graduate School ofMedicine

Clinical significance of anti-mutated citrullinated vimentin antibodies in RA patients

MASATO OKADA Immuno-Rheumatology Center, St. Luke'sInternational University Hospital

Prediction of Onset of Rheumatoid Arthritis with ACPA Screening in 11758 AsymptomaticMiddle-Aged Women

Tetsuji Sawada Rheumatology, Tokyo Medical University Anti-CCP titers in patients with elderly onset rheumatoid arthritis (RA): Analysis using anationwide RA database in Japan (NinJa)

Okinori Murata Division of Rheumatology, Department of InternalMedicine, Keio University School of Medicine Radiographic thymus alteration associates with clinical feature in autoimmune diseases

Shuichiro NakaboDepartment of Rheumatology and ClinicalImmunology, Graduate School of Medicine, KyotoUniversity

Anti-carbamylated protein antibody is detectable in various collagen vascular diseases

Tatsuhiro Yamamoto Division of Rheumatology, Depertment of InternalMedicine, School of Medicine, Toho University

Intracellular methotrexate polyglutamates is regulated by gene polymorphisms of a folate-related enzyme.

Torsten Matthias AESKU.Diagnostics GmbH & Co. KG Autoantibodies against CD74 - A new diagnostic marker forSpondyloarthritis (SpA)

Torsten Matthias AESKU.KIPP Institute, Germany Elevated MMP-3 levels in early RA patients sera with Borrelia Antibodies

Hiroshi Ishida Division of Rheumatology, Ishida RheumatologyClinic

Analysis of bio-free markers in Japanese rheumatoid arthritis(RA) patients from cytokinelevels and autoantibodies

5. Intestinal Imunity and DiseasesFirst Author / Presenter Affiliation Title

Kosuke Fujimoto

Department of Microbiology and Immunology,Graduate School of Medicine, Osaka UniversityDepartment of Respiratory Medicine, Allergy andRheumatic Diseases, Graduate School of Medicine,Osaka UniversityWPI Immunology Frontier Research Center,Osaka University

The ulcerative colitis susceptibility gene RNF186 regulates intestinal homeostasis

Takashi Mishima Laboratory of Immune Signal, National Institute ofBiomedical Innovation, Health and Nutrition

Leucine-rich α2-glycoprotein (LRG) enhances colonic inflammation by increasingleukocyte adhesion to TGF-β-stimulated endothelium

Torsten Matthias AESKU.KIPP Institute New markers for celiac disease: anti-neo-epitope human and microbial transglutaminases

Torsten Matthias AESKU.KIPP Institute COMPARISON OF THE RELIABILITY OF CELIAC DISEASE SEROLOGY TOREFLECT INTESTINAL DAMAGE.

Torsten Matthias AESKU.Diagnostics GmbH & Co. KG,AESKU.KIPPInstitute

TISSUE/MICROBIAL TRANSGLUTAMINASE NEO-EPITOPES SHOW SIMILARIMMUNO-EPITOPES IN CELIACS

Torsten Matthias AESKU.KIPP Institute 3-D STRUCTURES OF HUMAN AND MICROBIAL TRANSGLUTAMINASESCOMPLEXED TO GLIADIN ARE SIMILAR

Torsten Matthias AESKU.KIPP Institute ANTI ENTEROCYTE AUTOANTIBODIES IN CELIAC DISEASE.

Torsten Matthias AESKU.KIPP Institute GLUTEN IMMUNOLOGICAL SIDE EFFECTS ARE DETRIMENTAL TO HUMANHEALTH.

Torsten Matthias AESKU.KIPP Institute THE INTESTINE AS PRIMARY SITE FOR POST-TRANS MODIFICATION INSYSTEMIC AUTOIMMUNE DISEASE

Torsten Matthias AESKU.KIPP Institute Peripheral organs’ autoimmunity is highly connected to the intestinal ecosystem’s events

Kai Fechner EUROIMMUN AG Labormedizinische DiagnostikaAG Dassow Evaluation of liver tissue as substrate for detection of endomysium antibodies

6. Skin and Liver DiseasesFirst Author / Presenter Affiliation Title

Noriko Kubota Department of Dermatology, Faculty of Medicine,University of Tsukuba Autoimmune CD8 T cell-mediated interface dermatitis is regulated by Langerhans cells

Akimasa Saito Department of Dermatology, University of Tsukuba Perforin is essential for induction of keratinocytes death in a murine model of autoimmunemucocutaneous disease with interface dermatitis

Mende Miriam Institute for Experimental Immunology, affiliated toEUROIMMUN AG A multivariant profile ELISA for one-step diagnostics of autoimmune bullous dermatoses

Takaharu Ikeda Department of Dermatology, Wakayama MedicalUniversity IgG4-positive cells in the affected skin of alopecia areata

Takashi Himoto Department of Medical Technology, KagawaPrefectural University of Health Sciences Diversity of humoral responses to the centromere proteins in with chronic liver diseases

Joris Vanderlocht

Tissue Typing Laboratory, Department ofTransplantation Immunology, GROW - School forOncology and Developmental Biology, MaastrichtUniversity Medical Center

Multiplex autoantibody detection for autoimmune liver diseases and autoimmune gastritis

S. John Calise Department of Oral Biology, University of Florida Jade-2 is a novel component of autoantigenic 'rod and ring' intracellular structures

7. Myopathies and AutoantibodiesFirst Author / Presenter Affiliation Title

Shusaku NakashimaDivision of Hematology, Rheumatology andRespiratory Medicine, Department of Internalmedicine, Faculty of Medicine, Kagawa University

Myositis-specific and myositis-associated autoantibodies in patients with myositis.

Shinji Sato Division of Rheumatology, Department of InternalMedicine, Tokai University School of Medicine

Comparison of Prognosis in Interstitial Lung Disease Between Polymositis/Dermatomyosis Patients with Anti-Jo-1 Antibody and Anti-EJ Antibody

Yumiko SugiyamaDepartment of Stem Cell and Immune Regulation,Yokohama City University Graduate School ofMedicine Y-CURD Study Group

The analysis of inflammatory myopathies complicated with interstitial lung disease withanti-Jo-1 antibody

Ayako Kawano Department of Rheumatology, Infectious Diseasesand Laboratory Medicine, University of Miyazaki

Anti-aminoacyl-tRNA synthetases (ARS) antibody seems to be associated with the severityof muscular manifestation and alveolar-endothelial damages.

Yoshihiro Hishitani Department of Rheumatology and Allergy, HyogoPrefectural Kobe Children's Hospital

Anti-MDA5 Antibody Positive Juvenile Dermatomyositis with Hemophagocytic Syndrome(a case report and literature review)

Kazuhiro Komura Tsuruga Hospital Nailfold vascular findings of anti-melanoma differentiation-associated gene 5 antibody-positive patients with dermatomyositis

Mirei ShirakashiDepartment of Rheumatology and ClinicalImmunology, Kyoto University Graduate School ofMedicine

Predictive factors of poor prognosis and utility of plasmaexchange in anti-MDA5(+)dermatomyositis with interstitial lung disease under immunosuppressive therapy

Hiroko Kadota Department of Allergy and Rheumatology, NipponMedical School

In situ expression of transcriptional intermediary factor-1γ (TIF-1γ) in patients with cancer-associated myositis (CAM)

Shigeaki Suzuki Department of Neurology, Keio University School ofMedicine Clinical features and prognosis in anti-SRP and anti-HMGCR necrotizing myopathy

Keiichiro Kadoba Department of Rheumatology and ClinicalImmunology, Kyoto University Hospital Interstitial Lung Disease in Patients with Anti-Signal Recognition Particle Antibodies

Mende Miriam EUROIMMUN AG Presence of anti-cN-1A (Mup44, NT5c1A) IgG is specific for Sporadic Inclusion BodyMyositis

Shinji Sato Division of Rheumatology, Department of InternalMedicine, Tokai University School of Medicine

Clinical Significance of Cyclic Citrullinated Peptide-specific Antibody and RheumatoidFactor in Patients with Polymyositis/Dermatomyositis

Mariko Ogawa-Momohara Department of Dermatology, Nagoya UniversityGraduate School of Medicine Autoantibodies to Su/Argonaute 2 in Japanese patients with inflammatory myopathy

[ Poster Session 2 ]15:10-16:10 12th October (Wed), 2016

8. Systemic Lupus ErythematosusFirst Author / Presenter Affiliation Title

Kazuhisa Nozawa Department of Rheumatology, Juntendo University Antibody to Ribonuclease-H2 is a novel autoantibody specifically recognized in SLE;Importance of intramolecular epitope spreading for autoantibody production

Shuzo Sato Department of Gastroenterology and Rheumatology,Fukushima Medical University School of Medicine Association of anti-TPI antibodies with aseptic meningitis in patients with NPSLE

Kazuhisa Nozawa Department of Rheumatology, Juntendo University Antibodies to Microtubule Associated Protein-2 in the Cerebrospinal Fluid are a UsefulDiagnostic Biomarker for Neuropsychiatric Systemic Lupus Erythematosus

Sachiko ArakiDepartment of Hematology, Clinical Immunology andInfectious Diseases, Ehime University GraduateSchool of Medicine

Novel autoantigens associated with lupus nephritis

Koji Kitagori

Department of Rheumatology and ClinicalImmunology, Graduate School of Medicine, KyotoUniversityCenter for Innovation in ImmunoregulativeTechnology and Therapeutics, Graduate School ofMedicine, Kyoto University

Cleaved Form of Osteopontin in Urine as a Clinical Marker of Lupus Nephritis

Goh Murayama

Department of Internal Medicine and Rheumatology,Juntendo University School of Medicine,Departmentof Immunology, Juntendo University School ofMedicine

Role of Mucosal-associated Invariant T (MAIT) Cells In Lupus Dermatitis.

Mikiko Iguchi Division of Rheumatology, National HospitalOrganization Kyoto Medical Center

Angioimmunoblastic T-cell lymphoma with autoimmune hemolytic anemia andthrombocytopenia mimicking SLE

Kenji OkuDivision of Rheumatology, Endocrinology andNephrology Hokkaido University Graduate School ofMedicine

A patient-derived autoimmune IgG monoclonal anticardiolipin antibody that binds to beta2 glycoprotein domain I but not to total beta 2 glycoprotein I molecule

Hongmei Song Department of Pediatrics,Peking Union MedicalCollege Hospital

Clinical Analysis of 12 Pediatric Patients of Antiphospholipid Syndrome with PulmonaryEmbolism

9. Systemic Sclerosis and Sjögren's SyndromeFirst Author / Presenter Affiliation Title

Shuji AkizukiDepartment of Rheumatology and ClinicalImmunology, Kyoto University Graduate School ofMedicine

Genetic mutation of phospholipase D4 disturbs the homeostasis and immunologicaltolerance of B cells in mice.

Jiucun Wang School of Life Sciences, Fudan University Clinical and serological features of anti-centromere antibody-positive systemic sclerosis inChinese patients

Michihito KatayamaDept. of Respiratory Medicine, Allergy andRheumatic Disease, Graduate School of Medicine,Osaka University

Anti-activin type IIB receptor intracellular domain (ACVRIIB-in) Ab may relate the causeof Systemic sclerosis (SSc) with anti-topoisomerase-I antibody (ATA).

Gary Norman Dept Research and Development, Inova Diagnostics Serum Calprotectin levels in Japanese patients with diffuse and limited systemic sclerosis

Tetsuya Furukawa Division of Rheumatology, Department of InternalMedicine, Hyogo college of Medicine

Biomaker of YKL-40 for the Presence of Interstitial Pneumonia and Pulmonary ArterialHypertension in Systemic Sclerosis

Toshio OdaniDivision of Rheumatology, Endocrinology andNephrology, Hokkaido University Graduate School ofMedicine

Efficacy of autologous stem cell transplantation in the treatment of systemic sclerosis

Takashi Fujimoto The Center for Rheumatic Diseases,Nara Medical University

Regenerating Gene Protein as a Novel Autoantigen in the Pathogenesis of Sjögren’sSyndrome

Akihiro Mukaino Department of Neurology,Kumamoto University Hospital Serum ganglionic acetylcholine receptor antibodies in patients with Sjögren’s syndrome

Yasuhiko Itoh Department of Pediatrics, Nippon Medical School Alteration of clinical course by Immunosuppressants in children with positive anti-Roantibodies and with chronic nonspecific complaints

10. ANCA and VasculitisFirst Author / Presenter Affiliation Title

Jan Damoiseaux Central Diagnostic Laboratory, Maastricht UniversityMedical Center, The Netherlands

Detection of ANCA: a multicenter EUVAS evaluation of the value of indirectimmunofluorescence versus antigen-specific immuno-assays

Estela Motta Immunology Department, Hospital Dr. Oscar Alende,Mar del Plata Effects of urea treatment on the antineutrophil anticytoplasmic antibodies (ANCA)

Ryosuke Hiwa

Department of Rheumatology and ClinicalImmunology, Graduate School of Medicine, KyotoUniversityLaboratory of Immunochemistry, World PremierInternational (WPI) Immunology Frontier ResearchCenterDepartment of Immunochemistry, Research Institutefor Microbial Diseases, Graduate School of Medicine,Osaka University

Myeloperoxidase/HLA class II complexes as targets for autoantibodies in microscopicpolyangiitis

Nadja Roeber Institute of Immunology, Medical Faculty Carl GustavCarus, Technical University Dresden PR3-ANCA in children and adolescents with inflammatory bowel disease

Kai Fechner EUROIMMUN AG, Institute for ExperimentalImmunology

DNA-BOUND LACTOFERRIN IS THE MAJOR TARGET FOR P-ANCA INULCERATIVE COLITIS

Toshiki Nakajima Department of clinical immunology andrheumatology,Kyoto University IL-12p40 in Pathophysiology of Takayasu Arteritis

Michihiro KonoDivision of Rheumatology, Endocrinology andNephrology, Hokkaido University Graduate School ofMedicine

Three cases of varied size vessel vasculitis; Large vessel vasculitis with small or mediumvessel involvement.

11. Innate Immunity and DiseasesFirst Author / Presenter Affiliation Title

Tomonori Kimura Department of Nephrology, Osaka University Regulation of autoimmune responses through TRIM-regulated precision autophagy andsecretion.

Toshiaki Nakano

Graduate Institute of Clinical Medical Sciences,Chang Gung University College of Medicine,LiverTransplantation Center and Department of Surgery,Kaohsiung Chang Gung Memorial Hospital

Histone H1: a novel alarmin-like mediator and molecular target for immune regulation

Takashi Fujimura

Hiroshima Research Center for Healthy Aging(HiHA), Department of Molecular Biotechnology,Graduate School of Advanced Sciences of Matter,Hiroshima University

Extracellular linker histone H1 deteriorates IgE-mediated allergic hypersensitivity

Miharu IzumikawaDepartment of Internal Medicine, Hematology,Rheumatology and Respiratory Medicine, Faculty ofMedicine, Kagawa University

Anti-nuclear antibody positive rate in familial Mediterranean fever patients in Kagawauniversity hospital

Tokutaro Tsuda Immuno-Rheumatology Center, St. Luke'sInternational Hospital A case of drug hypersensitivity mimicking adult-onset still's disease

12. Miscellaneous DiseasesFirst Author / Presenter Affiliation Title

Hiromi Honda Laboratory of Immune Signal, National Institutes ofBiomedical Innovation, Health and Nutrition Leucine rich α-2 glycoprotein promotes lung fibrosis by modulating TGF-β signaling

Yoshimasa HamanoDept. of Respiratory Medicine, Allergy andRheumatic Disease, Graduate School of Medicine,Osaka University

Anti-GM-CSF/MHC class II complex antibody in autoimmune pulmonary alveolarproteinosis

Syuichi Koarada Department of Rheumatology, Saga University The clinical significance of autoantibody-producing CD180-lacking plasmablasts insystemic autoimmune and immune-based inflammatory disorders

Isao MurakamiDepartment of Rheumatology and Clinicalimmunology,Kyoto University Graduate School ofMedicine

The role of novel lipid mediators in connective tissue diseases

MASATO OKADA Immuno-Rheumatology Center, St. Luke'sInternational University Hospital The Effect of Quality Indicator Monitoring for Glucocorticoid-Induced Osteoporosis

Kiyomitsu Miyachi Rheumatology and Women's Health, KeiguClinic,Health Sciences Research Institute

Is hormone replacement therapy an effective treatment for arthralgia in post-/ peri-menopausal women? Two case reports from an ongoing trial.

a90128 [2. Poster or Oral]ANA testing: the clinicians’ point of view

1Medical University of Innsbruck, Department of Internal Medicine VI, Innsbruck,Austria,2Medical University of Graz, Deptartment of Internal Medicine Rheumatology &Immunology, Graz, Austria,3EASI Austria, Vienna, Austria,4Thermo Fisher Scientific - PhadiaAustria GmbH, Vienna, AustriaManfred Herold1, 3,Ulrike Demel2,Werner Klotz1,Thomas Horn3, 4,Members of EASI Austria3

The European Autoantibody Standardization Initiative (EASI) evaluated the current approach ofANA knowledge among clinicians using a questionnaire adapted from a Dutch questionnaire (1). InAustria the original English version of the adapted EASI questionnaire was distributed to 51physicians. The doctors’ speciality was stated as internal medicine (30), rheumatology (28),family doctor (10) and others.If ANA is positive, doctors are not always confident with the interpretation of results (9 always,18 frequently, 16 sometimes). Nevertheless, a discussion of the results with laboratoryspecialists is not common (11 never, 14 rarely, 24 sometimes).The questionnaire included one section asking for clinical conditions, resulting in doctor’srequest for ANAs. The answers were quite diverse, and ranged in all named clinical conditionsbetween never, rarely, sometimes and frequently.One chapter was dealing with the connection of ANA request and the conclusion of the results.Again, a high variability was found in all answers.Most clinicians are not familiar with the various laboratory techniques used for detection ofANA and do not consider the laboratory technique when interpreting the ANA result.Laboratory specialists agree, that the method used for ANA detection should be reported to theclinicians (2) but do not recognize clinicians’ missing knowledge about laboratory methods.

1. EASI questionnaire adapted from Marie Jose Sousa, Portugal.2. Agmon-Levin N. et al. Ann Rheum Dis 2013

a90140 [1. Poster only]Automated evaluation of ANA patterns with EUROPattern meets the ICAP guideline

1Institute for Experimental Immunology, EUROIMMUN AG, Seekamp 31, 23560 Lübeck, Germany

Fechner Kai1,Rohwäder Edda1,Krause Christopher1,Becker Claudia1,Ockenga Wymke1,JahnkeAnnika1,Voigt Jörn1,Fraune Johanna1,Stöcker Winfried1

Recently, an international consensus on the nomenclature of anti-nuclear antibody (ANA)patterns (ICAP) has been published (Chan et al, Frontiers Immunol, 2015). The consensus aims atstandardizing evaluation and reporting of ANA patterns and titer information. Accordingly,competent ANA patterns, encompassing nuclear but also cytoplasmic patterns need to bereported on non-transgenic HEp-2 cells, mixed patterns should be identified and titers ought tobe titrated to end-point. We aligned the performance of the EUROPattern-Suite (EuroimmunAG), an automation platform for computer-aided immunofluorescence microscopy, with the ICAPspecifications to check whether the system fulfils the essential criteria.

The classification software of the EUROPattern-Suite is able to distinguish several nuclearpatterns (homogeneous, speckled, nucleolar, centromere, nuclear envelope, nuclear dots) as wellas cytoplasmic pattern on native HEp-2 cells. Furthermore, mixed patterns in variouscombinations can be reported in ANA positive samples. The titer is automatically calculated onthe basis of several dilutions. All results are finally synthesized into one report per samplewhich is presented to the physician.

While other automation systems lack the capability of cytoplasmic or mixed patternidentification or provide titer calculation derived from single well estimation, the EUROPattern-Suite meets the essential goals laid down by the ICAP. Thus, the system is able to support thephysician via automated evaluation of ANA immunofluorescence in accordance with currentinternational guidelines.

a90136 [1. Poster only]Evaluation of Crithidia luciliae IFT can be reliably automated with EUROPattern

1Institute for Experimental Immunology, EUROIMMUN AG, Seekamp 31, 23560 Lübeck, Germany

Fechner Kai1,Gerlach Stefan1,Affeldt Kai1,Pototzki Lena1,Krause Christopher1,OckengaWymke1,Jahnke Annika1,Voigt Jörn1,Fraune Johanna1,Stöcker Winfried1

Automation of immunofluorescence microscopy resolves general limitations of IIFT inautoimmune diagnostics, such as high work load and subjectivity of interpretation. An additionalsoftware concept of “EUROPattern-Suite” - a combination of automated microscope andclassification software - now enables automated evaluation of anti-dsDNA in Crithidia luciliaebased IIFT (CLIFT). Anti-dsDNA are primarily present in systemic lupus erythematosus (SLE) witha varying prevalence of 20% to 90%.

60 samples of clinically confirmed SLE patients and 117 samples of patients with otherautoimmune or infectious diseases were tested for anti-dsDNA using CLIFT. Results,automatically retrieved by the classification software, were compared to visual fluorescenceinterpretation performed by two independent experts on the computer screen.

Visual evaluation of CLIFT revealed 27/60 anti-dsDNA positive SLE patients (prevalence 45%).26 of them were also classified positive by the “EUROPattern-Suite”, in total the systemidentified 28 positive patients. Of 33 SLE patients and 115 control patients determinednegative by the experts, the software classified 31 and 112 patients likewise negative.

With respect to visual IIFT interpretation, the “EUROPattern-Suite” was 96.3% sensitive and96.6% specific and performed in major agreement with the experts, reaching an accordance of96.6%. This high agreement is a precondition for reliable application of the software in routinediagnostics, so it can support reduction of human workload and standardization of fluorescenceread-out without losing quality.

a90137 [1. Poster only]Evaluation of modified Crithidia luciliae IIFT for detection of anti-dsDNA antibodies

1Institute for Experimental Immunology, EUROIMMUN AG, Seekamp 31, 23560 Lübeck, Germany

Fechner Kai1,Affeldt Kai1,Pototzki Lena1,Ockenga Wymke1,Jahnke Annika1,FrauneJohanna1,Stöcker Winfried1

The presence of autoantibodies against double-stranded DNA (anti-dsDNA) is considered asimportant criterion for the diagnosis of systemic lupus erythematosus (SLE), though theprevalence is reported to range between 20% and 90%. Standard methods for the detection ofanti-dsDNA are enzyme or radio immunoassays and indirect immunofluorescence using Crithidialucilae (CLIFT) – a protist exhibiting tightly packed dsDNA within its kinetoplast. CLIFT is knownto be particular disease specific at, however, moderate sensitivity. Here, we report on theperformance of a novel substrate intended to enhance sensitivity of CLIFT.

A patient panel comprising 60 samples of clinically confirmed SLE patients as well as a controlpanel with 117 samples of individuals having other autoimmune or infectious diseases wereanalysed regarding anti-dsDNA using the standard CLIFT (Euroimmun, Germany) and the Crithidialuciliae sensitive IIFT (Euroimmun, Germany).

Among the patient panel, 27 SLE patients were tested positive for anti-dsDNA by the standardCLIFT (45% sensitivity) whereas 39 SLE patient samples were reactive in the Crithidia luciliaesensitive IIFT (65% sensitivity). With respect to the control panel, the two tests revealedsimilar specificities of 98.3% (standard CLIFT) and 97.4% (Crithidia luciliae sensitive IIFT).

Thus, sensitivity for the detection of anti-dsDNA may be increased by use of the Crithidialuciliae sensitive IIFT without significant loss of specificity.

a90126 [2. Poster or Oral]ANA screening on DFS70-knockout HEp-2 cells in a routine setting

1Medical University of Innsbruck, Department of Internal Med. VI, Innsbruck, Austria,2Tirol-Kliniken, Landeskrankenhaus Innsbruck, Innsbruck, AustriaWerner Klotz1,Silvia Blunder2,Helene Naschberger2,Manfred Herold1, 2

Immunofluorescence on HEp-2 cells is the gold standard for ANA screening with high sensitivity.However specifity for SARD is low due to antibodies not associated with SARD. For easyconfirmation of DFS70 Abs during ANA screening a new HEp-2 cell preparation (10% traditionalcells, 90% HEp-2 cells knocked out for psip1 gene producing the antigen LEDGF responsible forthe DFS70 pattern).The study was aimed to evaluate the efficiency of ANA HEp-2 DFS70 KO cells in a routinesetting.842 consecutive serum samples sent to our laboratory for ANA screening were tested withtraditional HEp-2 slides(INOVA Diagnostics, San Diego, CA, USA) and HEp-2 ELITE DFS70 KOslides (IMMCO Diagnostics, Buffalo NY, USA) using a screening dilution of 1:40. Out of 842samples 283 (33.6%) were classified as 1:40 positive using DSF70 KO cells, 281 samples (33.4%)were positive on traditional HEp-2 cells. Overall agreement was 96.9 %. Samples positive forcytoplasmic or mitotic patterns without staining of the nucleus (true ANA) were classified asANA negative.23 out of 28 AC-pattens (Chan EKL et al. Front Immunol 2015;6:412) according to ICAP could befound within the routine samples. All patterns seen on traditional HEp-2 cells were also found onDSF70 KO cells. „Rare“ pattens like nuclear dots (AC-6,7), nuclear envelope (AC-11,12) orpolar/Golgi like (AC-22) were found with both HEp-2 cell preparations.HEp-2 DFS70 KO cells revealed reliable results within routine ANA-testing and simultaneousverification of anti-DFS70 antibodies.

IMMCO Diagnostics supported the studies by supplying reagents.

a90158 [2. Poster or Oral]ANA testing on HEp-2 cells and antigen-coated microparticles including DFS70

1Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany,2Researchand Development Department, GA Generic Assays GmbH, Dahlewitz/Berlin, Germany ,3Instituteof Immunology, Technical University of Dresden, Dresden, GermanyRico Hiemann1,Juliane Scholz2,Kai Grossmann2,Mandy Sowa2,Nadja Röber3,PeterSchierack1,Karsten Conrad3,Dirk Roggenbuck1, 2

Standard anti-nuclear antibody (ANA) detection starts with indirect immunofluorescence (IIF)reading on HEp-2 cells followed by confirmation of ANA positives using different immunoassaytechniques. This recommended two-step strategy is time- and cost-intensive. Hence, wedeveloped a multiplex one-step CytoBead assay, which enables ANA screening on HEp-2 cells withsimultaneous analysis of clinically relevant ANA specificities. HEp-2 cells and autoantigen-coated microparticles were fixed on standard glass slides with a novel well layout. Antigen-coated microparticles comprise populations coated with dsDNA, DFS70, Ro52, Ro60, La/SS-B,RNP-Sm, Sm, and Scl-70. Sera of patients with systemic autoimmune rheumatic disease (SARD)(n=403), various control groups (n=304) and anti-DFS70 positive sera (n=10) wereinvestigated. ANA findings by the standard two-step approach were compared with results ofthe novel combination assay. Compared to the standard two-step approach, the novelcombination assay showed very good agreement of ANA and antibody testing to dsDNA, DFS-70,La/SS-B, RNP-Sm, Scl-70, Ro52, and Ro60 (Cohen’s kappa>0.8). All patient sera with anti-DFS70antibodies scored also positive using the DFS70-microparticle population in this combinationassay. Thus, the novel one-step CytoBead assay may replace the standard two-step ANA analysis.Furthermore, anti-DFS70 antibody testing may help physicians to exclude patients with non-SARD and to establish a more specific algorithm to diagnose SARD.

a90159 [2. Poster or Oral]Automated digital classification of HEp-2 pattern according to ICAP classification tree

1Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany,2Instituteof Immunology, Technical University of Dresden, Dresden, GermanyRico Hiemann1,Nadja Röber2,Peter Schierack1,Karsten Conrad2

The aim of this study was the development of algorithms for automated classification of HEp-2cell pattern according to ICAP classification tree. Digital images of positive HEp-2 cell tests(n=1000) from routine samples were acquired with the Aklides system (Medipan GmbH,Dahlewitz, Germany) and classified by two experts according to ICAP classification scheme.These images were re-analyzed with a modified version of the Aklides software allowingdetection of all cell cycle phases and extraction of more than 2000 morphological features foreach nucleus. A first digital model for description of intensities and structures of patternrelevant features was developed. Model information is still incomplete because some patternwere rarely found in routine diagnostics (e.g., AC-17, AC-22). Deeper digital analysis of pre-classified samples revealed that implemented textural and structural features allow recognitionof differences between samples and further classification into ICAP tree. In general,classification algorithms were able to classify typical representative nomenclature samples,such as sera showing AC-1, AC-3 and AC-4 pattern in 100 %, 100%, 96% of cases, respectively.However, majority of samples (57%) showed a mixed or often slightly deviant pattern where itwas impossible to place it into one specific expert-level pattern group. Further extended studiesincluding more samples and adapted image acquisition strategies with multiple focus points arerequired to provide more accurate structure details especially for metaphase and cytoplasmicstaining.

a90087 [2. Poster or Oral]Novel Engineered HEp-2 Substrate and its Impact on DFS70 and ANA Screening Algorithm

1Immco Diagnostics, Buffalo, NY, USA,2Laboratory of Clinical Pathology, San Antonio Hospital,Tolmezzo, ItalyKishore Malyavantham1,Lakshmanan Suresh1,Nicola Bizzaro2

The recommended screening method for ANAs is indirect immunofluorescence (IIF) using HEp-2.Interpretation of a widely reported dense fine speckled (DFS70) pattern on HEp-2 can bechallenging on a conventional substrate and therefore needs a confirmation step. Solid phaseassays (EIA/CLIA/Immuno-Blots (IB)) for DFS70 require a two-phase diagnostic step andadditional costs. Here we report the impact of a novel IIF HEp2 substrate composed ofconventional HEp2 along with cells that lack LEDGF antigen responsible for DFS70 pattern onANA screening-confirmation algorithm.398 sera (62 DFS70 suspect by IIF, 118 infectious disease controls, 138 autoimmune controlsand 80 healthy donors) were evaluated on the novel HEp-2 substrate at the Laboratory ofClinical Pathology, San Antonio Hospital.92.6% (25) of the 27 IIF+/CLIA+/IB+ samples for DFS70 were confirmed as DFS70 patternpositive using the novel HEp-2 substrate in one step. All 27 of the DFS70 suspect samples thatwere CLIA-/IB- for DFS70 were found to be negative for DFS70 pattern by novel HEp-2. NovelHEp-2 substrate did not interfere with the expected classic ANA patterns for 336 controls.Based on these results, the new engineered HEp-2 substrate can substitute the traditional HEp-2 for ANA screening, achieve all the objectives of ANA screening step, simultaneously provideconfirmation for DFS70 pattern and greatly reduce the need for a DFS70 specific confirmationstep. The new method also improves the overall specificity of ANA screening by reducing thefalse positive rates associated with DFS70 pattern.

A90182 [2. Poster or Oral]

Proposal of an International Standard for Anti-DFS70/LEDGFp75 Antibodies

Alessandra Dellavance1,2, Danielle C Baldo1,2, Andressa Mathias2,3, Andressa

Rodrigues Maldonado3, Camila Motta Batista3, Herika Santiago Okamoto

Shiguedomi3, Juliana Caceres Lopes3, Louise Seselgis Tendler3, Marcelle

Grecco2,3, Marcia Scarpa, Raquel Suelen de Souza3, Thais Aguilar de Alcantra3,

Thiago Katsuhiko Fukuda3, Luis Eduardo Coelho Andrade2,3

1- Research and Development Division, Fleury Laboratories, São Paulo, Brazil. 2 – Rheumatology Division, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil. 3- Immunology Division, Fleury Laboratories, São Paulo, Brazil.

Antibodies to 70/75kD LEDGF yield nuclear dense fine speckled (DFS) pattern on ANA-HEp-

2 test. Isolate occurrence of anti-DFS70/LEDGFp75 antibodies virtually rules out diagnosis

of systemic autoimmune rheumatic diseases. An international anti-DFS70 standard would

help worldwide recognition of DFS pattern. We built such standard by pooling up samples

from 760 individuals with ≥1/640 DFS pattern, typical 75kDa band on western blot (WB)

and anti-DFS70 reactivity in ELISA and CLIA. Samples with bona fide DFS pattern were

retrieved within a 2-year period. A progressive pooling strategy included PENTA-pools (5

individual samples), ICOSA-pools (4 PENTA), SEMIFINAL pools (3-5 ICOSA) and the FINAL

POOL (8 SEMIFINAL). Intermediate pools and FINAL were validated in ANA-HEp-2, WB,

ELISA (cutoff 0.9U) and CLIA (cutoff 20CU). Anti-DFS70 CLIA for 760 individual samples had

median of 337.7CU (26.1 to 3,547), 152 PENTA had median of 420.6CU (60.4-3,518), and

38 ICOSA had median of 407.2U (77.5-1,823). Anti-DFS70 ELISA for the 8 SEMIFINAL had

median of 5.2U (3.8-6.2) and 5.1U for the FINAL pool. Intermediate pools and FINAL

yielded the characteristic DFS pattern and 75kDa WB band. In conclusion, pooling samples

from hundreds of individuals with anti-DFS70 preserves the original reactivity of individual

samples encompassed, as judged by different assays. The present anti-DFS70 standard will

integrate the panel of ANA standards of the Autoantibody Standardization Committee

affiliated with the International Union of Immunology Societies, to be distributed by the

Centers for Disease Control.

a90057 [1. Poster only]Harmonization of HEp-2 cell assays using anti-cell pattern description proposed by ICAP

1Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden,Dresden, GermanyNadja Roeber1,Benjamin Glauche1,Christine Gräfe1,Uta Kiessling1,Andrea Brandis1,ManuelaRejzek1,Annett Skupin1,Karsten Conrad1

The ICAP (International Consensus on ANA Patterns) is an initiative started to promoteconsensus on description and classification of ANA patterns. Such a consensus is a majorprerequisite to promote harmonization and understanding of autoantibody test nomenclature, aswell as interpretation guidelines for ANA testing. However, the harmonization of the HEp-2 cellassays may be hampered by the use of different HEp-2 cell assays in routine practice. Wecompared 8 different commercially available HEp-2 cell assays regarding the applicability ofanti-cell (AC) pattern description and differentiation proposed by the ICAP. For this purpose weused monospecific in-house reference sera that correspond to certain consensus patterns. Apoint score was used to evaluate and compare the results obtained with the analyzed HEp-2 cellkits. Nuclear patterns mostly show minor variations between different kits whereas in mitoticand especially cytoplasmic patterns major differences are observed. The diagnostic performanceis more or less different between the tested kits. The applicability of some test systems forcertain diagnostic issues (e.g. diseases associated with cytoplasmic patterns) has to bediscussed and requires further investigation. Different influences in autoantigenic epitopeexpression (especially different fixation procedures) as well as different autoantibody finespecificities have to be considered a major challenge regarding harmonization of autoantibodyscreening using HEp-2 cells.

a90058 [1. Poster only]Differentiation of immunofluorescence pattern of anti-Ro/SS-A antibody positive sera

1Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden,Dresden, GermanyNadja Roeber1,Marie Luise Reimer1,Uta Kiessling1,Christine Gräfe1,Karsten Conrad1

The aim of this study was the detailed analysis of the fluorescence patterns of anti-Ro/SS-Aantibody (Ro-Ab) positive sera on HEp-2-cells as a basis for optimizing the patterndifferentiation by automated image analysis. The testing of 381 in ELISA Ro-Ab positive serawith different autoantibody (AAb) profiles (monospecific Ro-Ab, Ro-Ab in combination withother AAb) showed a strong association with a first in 2013 described nuclear dot pattern. ThisRo/SS-A-Ab associated nuclear dot (RND) pattern is characterized by 10-30 small nuclear dotsper cell, which are predominantly located in the center of the nucleoplasm and irregularly, partlyroad shape arranged around and between the nucleoli. Compared with 165 in ELISA Ro-Abnegative patients and blood donors, the specificity of the RND pattern reaches 98% with anoverall sensitivity of 65% (including sera with AAb that mask the RND pattern) and a sensitivityof 85% in sera with combination of Ro52- and Ro60-Ab. Furthermore, a Ro-Ab associatedcytoplasmic dot (RCD) pattern was identified in 9.2% of the Ro-Ab positive sera with aspecificity of 99%. The simultaneous detection of RND and RCD indicates a sensitivity of 6.8%and a specificity of 100 % for the presence of Ro-Ab. From the results of this study it can beconcluded that the RND and RCD in the IIF on HEp-2-cells point with a very high specificity, but astill insufficient sensitivity to the presence of Ro-Ab and may be suitable for the developmentof new software algorithms for pattern recognition in the context of an optimized automatedimage analysis.

a90083 [1. Poster only]Performance comparison of the HELMED® Blot Module and HELIA® using AESKUBLOTS® ANA-17Pro1AESKU. Diagnostics GmbH & Co. KG,2AESKU.Systems, Wendelsheim Germany

Torsten Matthias1,Karola Krausse1,Christian Klein1,Carsten Jung2

Introduction: Immunoblotting is a common method for efficient profile testing of autoimmuneand infectious diseases. Automation offers higher throughput testing, thereforeAESKU.SYSTEMS developed two solutions to facilitate automated immunoblot testing.Objective:To compare the performance of the HELMED® Blot Module that is a fully automated Blotprocessor, and the HELIA®, an automated analyzer for line immunoassays.Methods: 39 routinesamples were tested on the AESKUBLOTS® ANA-17 Pro (AESKU.DIAGNOSTICS) utilizing in parallelthe HELMED® Blot Module and the HELIA® system (both AESKU.SYSTEMS, Wendelsheim). Byperforming samples with the HELMED® Blot Module the AESKUBLOTS® were analyzed by theAESKU.SCAN® software.Results: 28 samples were found positive for one or more parameters. 2samples showed equivocal results and 9 were completely negative for all ANA antigens. Overallagreement (concordance correlation coefficient) between HELMED® Blot Module and the HELIA®

system was 0.9476 (95% CI: 0.9216 to 0.9652). Notably, all discordant samples werecharacterized by very borderline signal. Comparing the level of immunoreactivity of thedifferent coated antigens and sample diversity the Pearson precision (ρ) was 0.9718 (95% CI:0.9547 to 0.9825; p<0.0001).Conclusions: The HELMED® Blot Module and the HELIA® system areable to identify the ANA positive samples with the same level of band intensity of the coatedantigens. Both approaches are able to reduce inter-laboratory variability and time required toperform ANA testing, especially in high throughput laboratories.

a90084 [1. Poster only]MULTICENTER STUDY OF ANA VALIDATION BY IFA IN THE AUTOMATED HELIOS® AND HELMED®

1AESKU. Diagnostics GmbH & Co. KG,2Dinámica IPS, Calle 27 No. 45 – 109, Medellín,Colombia,3Laboratorio Clínico Compensar, Calle 63 No. 28 – 42, Bogotá, Colombia,4AnalizarLaboratorio Clínico Automatizado, Calle 103 No. 14A-76, Bogotá, Colombia,5Quimiolab, Carrera38 No 55-40, Bogotá, ColombiaTorsten Matthias1,Adriana Londoño2,Victoria Estrada2,Claudia Ruiz Castaño3,Francy GuiovannaMora Serna3,Diana Milena Pedraza Sánchez3,Constanza Sánchez4,Patrcicia Tarazona5

Objective: To verify the performance of the AESKUSLIDES® ANA HEp-2, AESKUSLIDES® ANCA,AESKUSLIDES® nDNA and AESKUSLIDES® rLKS from AESKU.Diagnostics in automated systems forindirect immunofluorescence HELMED® and HELIOS® (AESKU.Systems).Methods: In total 436samples were tested at three different reference laboratories in Colombia, comprising thefollowing samples: 131 DNA, 177 ANA, 64 ANCA and 64 LKS. All were tested on the automatedsystems HELMED® (DNA, ANCA and LKS) and HELIOS® (ANA). In addition, reading of ANA slideswas performed by HELIOS® and manually by microscope. The results were compared to the daily-routine-method used in which samples were run on the PHD system with Bio-Radreagents.Results: ANA samples had a correlation between 91.9% and 98.3%. A comparative titerdiscrepancy of 1 dilution was observed in 1.69% to 3.57% of the samples. Pattern differenceswere focused on non-observation of patterns on both sides and were found in 7.14% to 18.64%of the samples. 1.78% to 5.08% of the differences were expressed in patterns belonging tocytoplasm. A concordance of 97.56% to 100% was obtained for DNA, 100% for ANCA, 90.0% to96.15% for LKS.Following Kappa indexes were calculated: ANA 0.84-0.96, DNA 1.0, ANCA 1.0,and LKS 0.90-0.94. Interpretation of the kappa index is very good for all tests.Conclusions:Performance of HELIOS® and HELMED® showed a very good correlation to standard method in alllaboratories for all tests. Thereby, both systems able to simplify the daily routine workflowregarding process speed, precision, reliability and efficiency.

a90085 [1. Poster only]IMMUNOFLUORESCENCE STUDIES FOR THE SPECIFIC DIAGNOSTICS OF MYASTHENIA GRAVIS BYIFA IN THE AUTOMATED HELIOS®1AESKU. Diagnostics GmbH & Co. KG

Torsten Matthias1,Eva Schweikhard1,Kathrin Eichler1,Saskia Backes1

Objective: Myasthenia gravis is an autoimmune disease that is characterized by muscle weaknessand fatigue. It is associated with antibodies directed against e.g. the acetylcholine receptor(AchR) and muscle-specific kinase (MusK) in the postsynaptic membrane at the neuromuscularjunction. AchR antibodies (AchRAb) are highly specific and present in 80-90 % of patients,whereas autoantibodies against MusK (MusKAb) are found in 30-40 % of seronegative patients.Immunofluorescence studies were performed to underline the specificity of AchRAbs whilecomparing with other autoimmunological syndromes by verifying the performance of theAESKUSLIDES_AChR® from AESKU.DIAGNOSTICS in the automated systems for indirectimmunofluorescence HELIOS® (AESKU.SYSTEMS).

Methods: In total 80 sera were tested, comprising the following samples: 20 AchRAb, 10 ANA, 10ENA, 10 SLE, 10 MusK and 20 blood donors. All were tested on the automated system HELIOS®.

Results: Our data show that 19 out of 20 AchRAb sera showed AchR autoantibody binding withAESKUSLIDES_AChR®. Interestingly, all MusK positive sera and healthy blood donors werenegative. ANA, ENA and SLE sera showed different fluorescent patterns.

Conclusions: AESKUSLIDES_AChR® showed a very good distinction of AchRAbs among MusK, ANA,ENA, SLE and healthy blood donors’ sera. Thereby, differences were expressed in patternsbelonging to the cytoplasm, nuclear and plasma membrane.

a90037 [2. Poster or Oral]An effective method to identify autoantigens expressed on the cell surface: SARF

1Tohoku University School of Medicine

Tsuyoshi Shirai1,Hiroshi Fujii1,Tomoyuki Muto1,Tomonori Ishii1,Hideo Harigae1

Background: Autoantibodies against integral membrane proteins are generally accepted aspathogenic, but identification of such molecules was difficult by using conventional methodsincluding proteomics. Anti-endothelial cell antibodies (AECAs) are autoantibodies against cellsurface molecules on the endothelium, and play important roles on promoting vascularinflammation. To identify cell-surface autoantigens, we constructed a Serological identificationsystem for Autoantigens using a Retroviral vector and Flow cytometry (SARF).Methods and Results: cDNA library of ECs were generated and inserted into the retroviralvector, then rat myeloma cells were infected with these retroviruses. Distinct AECA-positiveclones were isolated by flow cytometry by using different serum IgG from patients with collagendiseases. By DNA sequencing and microarray, we identified cDNA inserted into each of the clones.These molecules included FLRT2, EphB2, and Pk for systemic lupus erythematosus, and ICAM-1for rheumatoid arthritis. We further identified autoantigens in Takayasu arteritis. Expressionsof these molecules on ECs were confirmed and AECA IgGs bound specifically to the cells whichwere transfected with each of the molecules. Importantly, anti-FLRT2 antibody inducedcomplement-dependent cytotoxicity.Conclusions: We successfully identified membrane proteins as autoantigens against AECAs byusing SARF. Using this system, it is possible to achieve a comprehensive understanding ofautoantibody-mediated injury in inflammatory diseases and develop more specific interventions.

a90054 [2. Poster or Oral]Gut microbiota from rheumatoid arthritis patients induce severe arthritis in SKG mice

1Department of Respiratory Medicine, Allergy and Rheumatic Diseases, Osaka UniversityGraduate School of Medicine,2Department of Microbiology and Immunology, Osaka UniversityGraduate School of MedicineYuichi Maeda1, 2,Atsushi Kumanogoh1,Kiyoshi Takeda2

Manifestation of rheumatoid arthritis (RA) can be attributed to both genetic and environmentalfactors. Some researchers have been focusing on gut microbiota which is thought to be one ofthe environmental factors that enhance the development of RA. The advancement of highthroughput microbial DNA sequencing had enabled us to investigate the correlation between gutmicrobiota and host immune systems. In this study, we showed that altered composition ofmicrobiota (dysbiosis) was observed in early RA patients. Dysbiosis was also detected in the RApatients from United States, China and Finland. However, it remains unclear how dysbiosiscontributes to arthritis development. We inoculated fecal samples from RA patients whoharbored relatively high abundance of Prevotella and healthy controls into germ-free SKG miceand evaluated severity of arthritis and immune responses. We also analyzed whetherlymphocytes of SKG mice harboring microbiota from RA patients react with arthritis-relatedautoantigen, RPL23A.SKG mice harboring microbiota from RA patients contained an increased number of Th17 cells inlarge intestine and developed severe arthritis with zymosan injection. Lymphocytes fromregional lymph nodes and large intestine of these mice showed increased IL-17 responses toRPL23A. Naïve SKG CD4+ T cells, co-cultured with P. copri-stimulated dendritic cells, producedIL-17 in response to RPL23A and rapidly induced arthritis. These results support the idea thatautoreactive T cells were activated in the large intestine, and possibly migrated to the joint toinduce arthritis.

a90034 [2. Poster or Oral]STS-2 (Suppressor of TCR signaling-2) suppresses collagen-induced arthritis in mice.

1Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School ofMedicine,2Osaka Red Cross Hospital, collagen diseases internal medicine,3Center for Rheumaticdisease, Kyoto University HospitalNamiko Okabe1,Masaki Katayama2,Shuji Akizuki1,Kosaku Murakami1,Ran Nakashima1,MotomuHashimoto3,Yoshitaka Imura1,Hajime Yoshifuji1,Masao Tanaka3,Koichiro Ohmura1,TsuneyoMimori1

Objective: STS-2 has been identified as a susceptibility gene for rheumatoid arthritis (RA) bygenome-wide association study. STS-2 is known to negatively regulate downstream of TCRsignaling, but the role in autoimmune disease is not clear. In this study, we examined STS-2function in collagen-induced arthritis (CIA) model. Methods: We induced CIA to 46 STS-2 knock-out (KO) mice (C57BL/6 background) and 41 wild-type (WT) mice, then evaluated CIA incidenceand clinical score over time, and measured anti-type II collagen (CII) antibody (Ab) titer. We alsoexamined peripheral lymphocyte fraction and cytokine production from CD4+T cells in arthriticmice by flowcytemetry and analyzed regulatory T cell (Treg) function by in vitro suppressionassay with non-immunized STS-2 KO and WT mice. Results: Compared with WT mice, STS-2 KOmice showed significantly higher CIA incidence (50 % vs 25 %, p<0.05) at 6 week from CIIimmunization. But, there was no significant difference in clinical score and anti-CII Ab titerbetween STS-2 KO and WT mice. At the point of 8 week from immunization, STS-2 and WT miceshowed similar numbers of T cells, B cell, and also IFN-γ-, IL-17-, IL-4- and IL-10-producing CD4+

T cells, whereas IL-2 producing CD4+ T cells were significantly increased in STS-2 KO mice. STS-2KO Treg revealed similar suppressive function to that of WT. Conclusion: STS-2 suppressed CIAonset but not clinical severity. STS-2 KO CD4+ T cells produced more IL-2, but did not alter Tregfunction. It is suggested that IL-2 overproduction affects effector T cell activity in CIA.

a90117 [1. Poster only]INVOLVEMENT OF REGULATORY T CELLS AND MICRORNAS IN REGULATION OF COLLAGEN-INDUCED ARTHRITIS IN MICE TREATED WITH IL-2/ANTI-IL-2 IMMUNE COMPLEXES1Division of Rheumatology, Department of internal medicine, Hyogo College of Medicine ,2HyogoUniversity of Health Sciences,31st Department of Internal Medicine, Kansai Medical UniversityYuichi Yokoyama1,Tsuyoshi Iwasaki2,Sachie Kitano1,Tetsuya Furukawa1,Atsushi Satake3,NozomuMoriya2,Kiyoshi Matsui1,Hajime Sano1

Objectives: To clarify complex regulatory network of IL-2IC in autoimmune arthritis weexamined the effects of IL-2IC on Th1, Th17, Treg inductions and microRNAs (miRNAs)expressions which regulate T cell inductions.Methods: IL-2ICs were prepared by mixing 5μg ofanti-IL-2 antibody (clone JES6-1D) with 1μg of mouse IL-2 for 15 minutes. The mice wereinjected with either PBS as a control or IL-2IC intraperitoneally for 3 days. Peripheral blood cellswere analyzed by flow cytometry. Th1 and Th17 cells infiltrating in the synovium were examineby immunohistochemistry stained with anti-IFN-g and IL-17 mAb. MiRNAs expressions in theserum from CIA mice were analyzed using panel real-time PCR analysis. Results: We observed asignificant decrease in both the incidence and severity of arthritis in both established CIA andearly stage of disease. Injection of IL-2IC effectively elicited more than 2-fold expansion ofTregs in peripheral blood cells than control mice. Th1 and Th17 cells infiltrations in the synoviumwere inhibited by IL-2IC. We identified 24 differentially expressed miRNAs (fold change >2.0)and 72 differentially expressed miRNAs (fold change <0.5) comparing untreated mice. Of note,we observed miR-17-5p and miR-19b-3p which inhibits iTreg induction, and miR-326-3p whichinduces Th17 were significantly augmented in CIA mice but decreased in CIA mice treated withIL-2IC. Conclusions: These observations indicate that IL-2IC might play a complex regulatory rolein autoimmune arthritis by regulating Th1, Th17, Tregs and miRNAs which regulate these T cellinductions.

a90052 [2. Poster or Oral]Crucial involvement of citrullinated proteins and ACPA in peptide GPI-induced arthritis

1Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Ibaraki, Japan

Hoshimi Kawaguchi1,Isao Matsumoto1,Hiroshi Ebe1,Yuko Kurashima1,Naoto Umeda1,YuyaKondo1,Hiroto Tsuboi1,Takayuki Sumida1

[Objectives]To explore the pathogenic relevance of citrullinated proteins (Cit-P) and anti-citrullinatedprotein antibodies (ACPA) in peptide glucose-6-phosphate isomerase (pGPI)-induced arthritis(pGIA).

[Methods]1) The titers of anti-pGPI antibodies and ACPA in sera from pGIA mice were analyzed by ELISA.2) Cit-P expressions in joints and skins were examined by immunohistochemistry, those in serawere examined by Western blot analysis.3) Cl-amidine (PAD inhibitor) was injected intraperitoneally to pGIA mice. Clinical score, ACPAtiters, Cit-P expressions, T cell response and the level of proinflammatory cytokines in sera wereassessed.

[Results]1) The titers of anti-pGPI antibodies and ACPA in sera from pGIA mice were elevated from day14, and were significantly higher than those from control mice.2) In immunohistochemistry, Cit-P was detected in joints on day 14 and in skins on day 7 frompGIA mice, whereas not detected from control mice. In sera, Cit-P was detected from pGIA mice,and significantly increased than control mice.3) By Cl-amidine treatment, clinical score was significantly decreased, and ACPA titers tended tolower. Cit-P in joints, skins and sera from treated mice were clearly decreased. The level of IL-6in sera was significantly decreased, but pGPI-specific CD4+ T cell response was not changed.

[Conclusion]Cit-P and ACPA were increased in pGIA mice, and the inhibition of PAD suppressed arthritis andCit-P expressions. These results suggested that PAD was involved in the pathogenesis andmaintenance of autoimmune arthritis.

a90175 [2. Poster or Oral]Neo-self IgG/HLA-DR complexes are involved in RA susceptibility as autoantibody targets

1Laboratory of Immunochemistry, World Premier International (WPI) Immunology FrontierResearch Center, Osaka University,2Department of Immunochemistry, Research Institute forMicrobial Diseases, Osaka University,3Department of Dermatology, Graduate School of Medicine,Osaka University,4Department of Orthopaedic Surgery, Graduate School of Medicine, OsakaUniversity,5Division of Rheumatology, Endocrinology and Nephrology, Graduate School ofMedicine, Hokkaido University,6Department of Rheumatology and Clinical Immunology, GraduateSchool of Medicine, Kyoto University,7Laboratory of Cell Signaling, RIKEN Center for IntegrativeMedical Sciences (IMS-RCAI),8Laboratory of Cell Signaling, WPI Immunology Frontier ResearchCenter, Osaka University,9Institute for Advanced Study, Kyushu University,10Department ofMicrobiology and Immunology and the Cancer Research Institute, UCSFHui Jin1, 2,Noriko Arase1, 3,Kouyuki Hirayasu1,Masako Kohyama1, 2,Tadahiro Suenaga1, 2,FumijiSaito2,Kenji Tanimura2,Sumiko Matsuoka2,Kosuke Ebina4,Kenrin Shi4,Shinsuke Yasuda5,TetsuyaHorita5,Ryosuke Hiwa1, 2, 6,Koichiro Ohmura6,Hideki Yoshikawa4,Takashi Saito7, 8,TatsuyaAtsumi5,Takehiko Sasazuki9,Ichiro Katayama3,Lewis L Lanier10,Hisashi Arase1, 2

Specific HLA class II alleles are strongly associated with susceptibility to rheumatoid arthritis(RA); however, how HLA class II regulates susceptibility to RA has remained unclear. Recently, wefound a novel function of HLA class II molecules; their ability to aberrantly transport cellularmisfolded proteins to the cell surface without processing to peptides. Rheumatoid factor (RF) isan autoantibody that binds to denatured IgG or Fc fragments of IgG and is detected in 70-80%of RA patients, but also in patients with other diseases. Here, we report that intact IgG heavychain (IgGH) is transported to the cell surface by HLA class II via association with the peptide-binding groove and that IgGH/HLA-DR complexes are specifically recognized by autoantibodies inRF-positive sera from RA patients. In contrast, autoantibodies in RF-positive sera from non-RAindividuals did not bind to IgGH/HLA-DR complexes. Of note, a strong correlation betweenautoantibody binding to IgG complexed with certain HLA-DR alleles and the odds ratio for thatallele's association with RA was observed (r=0.81, P=4.6x10-5). Our findings suggest that IgGHcomplexed with certain HLA class II alleles is a target for autoantibodies in RA, which mightexplain why these HLA class II alleles confer susceptibility to RA. Because antigenicity of IgGHcomplexed with HLA class II molecules may be different from that of normal IgG, IgGH/HLA-DRcomplex might have induced autoantibody production in RA as a neo-self antigen.

a90120 [2. Poster or Oral]The proportion of regulatory T cells in rheumatoid arthritis; comparison of the results fromdifferent definition of regulatory T cells

1Department of Respiratory Medicine, Allergy and Rheumatic diseases, Osaka UniversityGraduate School of Medicine,2Department of Experimental Immunology, Immunology FrontierResearch Center, Osaka University, Osaka, Japan.,3Department of Experimental Pathology,Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.,4Division of Allergy,Rheumatology and Connective tissue disease, NTT west Osaka Hospital, Osaka, Japan.Takayoshi Morita1, 2,Yoshihito Shima1,James Badger Wing2,Shimon Sakaguchi2, 3,Atsushi Ogata1,

4,Atsushi Kumanogoh1

Background. Regulatory T cells (Tregs) have important functions in peripheral immune toleranceand failure of their functions is considered to cause autoimmune diseases. However the roles ofTregs in the pathogenesis of rheumatoid arthritis (RA) are controversial. The reason ofconfusion is that no Tregs definition with consensus. To clarify the status of Tregs in RA, weinvestigated the proportion of Tregs in the blood of RA patients.Methods. We performed asystematic review and meta-analysis of peripheral blood (PB) Tregs among CD4+ T cells in RApatients and control subjects. Studies were identified using PubMed and Google Scholar. Weevaluated the proportion of Tregs in RA patients relative to control subjects by combining thestandardized mean differences from different studies for meta-analysis.Results. A total of 31studies were selected. When we evaluated the proportion of PB Tregs regardless of definition,there was no difference of Tregs proportion between RA patients and control subjects. Theresults have extreme heterogeneity. The proportion of PB Tregs decreased in RA patients thanthat in control subjects in sub-analysis of selected definitions, CD25+FOXP3+ or CD25-high.Accurate definition reduced dispersion of results.Conclusion. The proportion of Tregs defined bymore accurate markers decreased in RA patients than that in control subjects. A low proportionof Tregs is not sufficient to maintain peripheral immune tolerance; therefore, the lowproportion of Tregs in RA might contribute to the failure of peripheral immune tolerance in thisdisease.

a90050 [1. Poster only]Adhesion molecules on synovial fibloblast promote cytokine production from CD4+T cells

1Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, KyotoUniversity,2Department of the Control for Rheumatic Diseases, Graduate School of Medicine,Kyoto University,3Center for Innovation in Immunoregulative Technology and Therapeutics,Kyoto University Graduate School of Medicine,4 Department of Experimental Pathology, Institutefor Frontier Medical Sciences, Kyoto UniversityMasato Mori1,Motomu Hashimoto2,Takashi Matsuo1,Moritoshi Furu2,Hiromu Ito2,TakaoFujii2,Hiromu Yoshitomi3,Yoshinaga Ito4,Shuji Akizuki1,Ran Nakashima1,YoshitakaImura1,Naoichiro Yukawa1,Hajime Yoshifuji1,Koichiro Ohmura1,Tsuneyo Mimori1, 2

[Objectives]To study the role of adhesion molecules on synovial fibloblast (FLS) in regulatingcytokine production from CD4+T cells. [Methods]We cultured naive CD4+T cells with or withoutFLS or skin fibroblast(SF), stimulated them with anti-CD3/28 in the presence or absence oftranswell plate or anti-ICAM-1 or VCAM-1 antibody. Cytokine production from CD4+T cells wasassessed by ELISA and cytokine beads array(CBA). In some experiments, CD4+CD161+ orCD4+CD161-T cell alone were stimulated by anti-CD3/28 and plate-bound recombinant ICAM-1or VCAM-1. [Results] Both FLS and SF enhanced cytokine production(IFN-γ, IL-17) from anti-CD3/28-stimulated CD4+T cells which was inhibited by transwell plate or anti-ICAM-1 or VCAM-1 antibody. Interestingly, IL-17 production was observed with FLS but not SF. FLS expressedboth ICAM-1 and VCAM-1, while SF expressed ICAM-1 alone. Plate-bound recombinant ICAM-1enhanced the production of IFN-γ from anti-CD3/28- stimulated CD4+CD161+T cells, whileplate-bound VCAM-1 enhanced the production of both IFN-γ and IL-17 from anti-CD3/28-stimulated CD4+CD161+T cells. [Conclusion] ICAM-1 expressed on FLS promoted IFN-γ but notIL-17 production, while VCAM-1 on FLS promoted both IFN-γand IL-17 production from naiveCD4+CD161+T cells.

a90036 [1. Poster only]Co-localization of citrullinated proteins, PAD4 and ACPAs in CD68+ cells in RA synovium

1Department of Internal Medicine, Faculty of Medicine, University of Tsukuba,2Department ofRheumatology, Tsuchiura Kyodo General HospitalNaoto Umeda1, 2,Isao Matsumoto1,Hoshimi Kawaguchi1,Hiroshi Ebe1,Takayuki Sumida1

Objective: To clarify the localization of citrullinated proteins and peptidylarginine deiminase(PAD) 4 in the synovium of RA, and to investigate the deposition sites of affinity purifiedACPAs.Methods: 1) The expression of citrullinated proteins in RA and OA synovium was identified byimmunochemical staining using anti-modified citrulline (AMC) antibody. 2) Expressing cells ofcitrullinated proteins in RA synovium were identified by immunofluorescence multiple stainingwith CD3, CD20 and CD68. 3) The PAD4 expression in RA synovium was identified using an anti-PAD4 antibody. Further, the PAD4 expression cells are examined by immunofluorescence staining.4) Three ACPAs, anti-cyclic citrullinated glucose-6-phosphate isomerase (CCG)-2, anti-CCG-7and anti-citrullinated enolase peptide (CEP)-1 antibody, were affinity purified from RA serum,respectively. These ACPAs were incubated with RA synovium to identify the deposition cells byimmunofluorescence staining.Results: 1) Citrullinated proteins were observed in the surface layer of the RA synovium. It wasnot observed in the OA synovium. 2) Citrullinated proteins in RA synovium were observed in mostof CD68 positive cells and part of CD3 positive cells. 3) The expression of PAD4 was observedstrongly in CD68 positive cells. 4) Anti CCG-2, CCG-7 and CEP-1 antibody were depositedrespectively to CD68 positive cells in the same way.Conclusion: These findings suggested that PAD4 induced citrullinated proteins were mainlyexpressed on CD68 positive cells in the RA synovium, resulting in ACPAs mediated arthritis.

a90023 [2. Poster or Oral]Clinical significance of anti-mutated citrullinated vimentin antibodies in RA patients

1Department of Rheumatology and Clinical Immunology,Kyoto University Graduate School ofMedicine,2Department of Rheumatology and Clinical Immunology,Wakayama Medical University,3Department of the Control for Rheumatic Disease,Kyoto University Graduate School ofMedicine,4Department of Orthopedic Surgery, Kyoto University Graduate School of MedicineNozomi Ishigooka1,Takao Fuji2,Seiko Kondo-Ishikawa1,Motomu Hashimoto3,Moritoshi Furu3,MasaoTanaka3,Hiromu Ito4,Ran Nakashima1,Kosaku Murakami1,Yoshitaka Imura1,HajimeYoshifuji1,Koichiro Ohmura1,Tsuneyo Mimori1

Mutated citrullinated vimentin (MCV) is one of the important targets of anti-citrullinatedprotein/peptide antibodies (ACPA) in patients with rheumatoid arthritis (RA). In the presentstudy, we tried to determine clinical significance of anti-MCV antibodies.Both anti-MCV (ORGENTEC Diagnostika) and anti-cyclic citrullinated peptide antibodies(MESACUPTM-2 test CCP, MBL) (anti-CCP) in sera from 280 RA patients in Kyoto University RAManagement Alliance cohort 2013 and 2014 were measured by ELISA. In 2013 cohort, anti-MCV+/CCP+, +/-, -/+, and -/- patients were 63.2, 1.5, 21.4, and 13.9 %. While anti-CCP-positivity was not changed, anti-MCV in 41positive patients (22.7%) disappeared during 2013and 2014. Patient’s age, gender, disease duration, Steinbrocker’s stage and DAS28 levels in 2013were not different between anti-MCV disappeared and continued (n=140, 77.3%) groups. During2013 and 2014, however, ΔDAS28 in disappeared group was more significant than that incontinued group (-0.58±1.1 vs 0.02±0.34, P = 0.028), and use of biologics in disappeared groupwas higher than that in continued group (64.7 vs 25.2%, P< 0.0001). Anti-MCV disappearancerate was equal among TNF inhibitors (25.5%), tocilizumab (22.2%) and abatacept (26.8%) users.The present results suggest that anti-MCV, but not anti-CCP, may be a useful marker for diseaseactivity and effectiveness in RA patients receiving biological agents, although the immunologicalmechanisms explaining the anti-MCV disappearance should be elucidated.

a90011 [1. Poster only]Prediction of Onset of Rheumatoid Arthritis with ACPA Screening in 11758 AsymptomaticMiddle-Aged Women1Immuno-Rheumatology Center, St. Luke's International University Hospital

MASATO OKADA1,Ryo Rokutanda1

Background/Purpose: Early diagnosis and intervention are associated with better long-termoutcome in rheumatoid arthritis (RA)Methods: From April 2014 to November 2014, ACPA and RF were simultaneously measured in11758 women who presented for their annual routine health examination. Multivariate analysiswas performed to identify the factors affecting the positivity of ACPA and RF. Cost-effectiveness was calculated for RA screening.Results: RF was positive in 7876 persons among the 105778 (mean age, ± years), and thepositive rate was increased from % in age 30-39 to % in age over 50. ACPA was positive in 74 outof the 629 persons who visited our Rheumatology clinic for follow-up from April 2006 to March2012. In the simultaneous screening of 11758 women from April 2014 to November 2014, ACPApositivity was identified in 1.3% of women, and the positive rate of ACPA increased from 0.5%in age 30-39, 0.90% in age 40-49 to 1.5% in age over 50. RF was positive in 10.8% women, andthe positive rate of RF increased from 6.2% in age 30-39, 8.9% in age 40-49 to 13.2% in ageover 50. ACPA positivity was also higher in older patients, but remained less than 2% even at age60-69. Positivity of both ACPA and RF (ACPA+RF+) was seen in 0.83% and 36.4% of women withpositive ACPA was RF negative. The multivariate analysis revealed that age, positivity of RF andhistory of malignancy were associated factors for ACPA positivity.Conclusion: ACPA is much less frequently positive than RF in women of general population, andsubstantial portion of ACPA positive women is RF negative.

a90169 [1. Poster only]Anti-CCP titers in patients with elderly onset rheumatoid arthritis (RA): Analysis using anationwide RA database in Japan (NinJa)

1Rheumatology, Tokyo Medical University,2Lifetime Clinical Immunology, Tokyo Medical andDental University,3Clinical Research Center for Allergy and Rheumatology, NHO SagamiharaHospitalTetsuji Sawada1,Eri Kato1,Toshihiro Matsui2,Koichiro Tahara1,Haeru Hayashi1,JinjyuNishino3,Shigeto Tohma3

Background: Previous studies demonstrated the lower prevalence of anti-CCP antibody (Ab) inelderly-onset RA (EORA). The aim of the present study was to investigate anti-CCP titers inpatients with EORA, using a nationwide RA database in Japan (NinJa).Methods: We analyzed 4,445 RA patients, whose anti-CCP titers were available. The cutoff agefor defining EORA was 65 years of age. Student unpaired t-test with Bonferroni correction wasused to compare the mean values.Results: Frequency of anti-CCP Ab in EORA (62.1%) was significantly lower than that inyounger-onset RA (YORA) (77.9%), as consistent with previous studies. The average anti-CCPtiter increased, as the age at RA onset increased. There was a significant difference in anti-CCPAb levels between 20-29 age group (170 U/mL) and 60-69 age group (284 U/mL). When anti-CCPAb levels were divided into quartiles (Q1: 4.5-40 Q2: 40-124 Q3: 124-434 Q4: >434 U/mL), theproportion of anti-CCP negative RA patients was higher in the groups with age at RA onsetabove 50 years. Furthermore, there was a difference in the pattern of anti-CCP distribution,depending on the ages at RA onset. Thus, the higher titer quartiles (Q3 and Q4) were morefrequently observed in RA patients with their age at disease onset above 50 years than in thosebelow 50 years.Conclusion: We have demonstrated the relationship of the lower positivity and higher titers ofanti-CCP Ab with increasing age at RA onset. It is suggested that a different pathogenic factorscould contribute to anti-CCP production in elderly onset RA.

a90063 [2. Poster or Oral]Radiographic thymus alteration associates with clinical feature in autoimmune diseases

1Division of Rheumatology, Department of Internal Medicine, Keio University School ofMedicine,2Division of Diagnostic Radiology, Department of Radiology, Keio University School ofOkinori Murata1,Katsuya Suzuki1,Hiroaki Sugiura2,Yasushi Kondo1,Hidekata Yasuoka1,KunihiroYamaoka1,Tsutomu Takeuchi1

Background/Purpose: Only a few small-scale studies for radiographic thymus alteration havebeen reported in systemic autoimmune diseases. We conducted a large-scale cross-sectionalanalysis on prevalence of radiographic thymus alteration and its clinical association in varioussystemic autoimmune diseases.Methods: Consecutive and unbiased 500 patients who had visited at our service had beenevaluated by chest CT scan between January 2013 and December 2015 were enrolled. Theassociation of their radiographic thymus alteration to clinical information was statisticallyanalyzed.Results: Patients with thymoma or thymic cyst, and less than 30 year-old were excluded, 488were served for following analysis. Thymic hyperplasia was found in 90 (18%) overall.Remarkably, patients with granulated pattern was more frequent (42%) as compared toundiagnosed controls. In these, RA patients was the most frequent (24% and 46%). Regardingclinical association, when RA patients is classified by hyperplasia and granulated pattern,proportion of serum anti-cyclic citrullinated peptide antibody (ACPA)-positivity weresignificantly higher in alteration group compared to normal group (93% vs. 70% and 89% vs.63%; P=0.004 and 0.0005, odds ratio=6.1 and 5.0). In addition, titer of ACPA was alsopositively correlated to both hyperplasia and granulated pattern (P=0.048 and 0.009).Conclusion: In RA patients, radiographic thymus alteration, especially granulated patternsignificantly correlates to ACPA status and may reflect activation of germinal centers in thethymic medulla.

a90029 [2. Poster or Oral]Anti-carbamylated protein antibody is detectable in various collagen vascular diseases

1Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, KyotoUniversity, Kyoto, JapanShuichiro Nakabo1,Koichiro Ohmura1,Tsuneyo Mimori1

[Background and Objective]Anti-carbamylated protein antibody (anti-CarP Ab) was reported to be detected in around 40%of rheumatoid arthritis (RA), and remarkably, 16% of anti-citrullinated protein antibody(ACPA)-negative RA. Since it is sometimes difficult to distinguish ACPA-negative RA from non-RA collagen vascular diseases (CVDs), anti-CarP Ab is expected to be a new helpful diagnosticbiomarker. However, its prevalence in non-RA CVDs has not been elucidated well. Therefore, wetried to elucidate the prevalence of anti-CarP Ab in non-RA CVDs as well as RA.[Method]The sera from 266 RA and 616 various CVD patients and 80 healthy controls were examined byin-house ELISA system of anti-CarP Ab which was established following the previous report.[Result]Anti-CarP Ab was positive in 54% of ACPA-positive RA and 21% of ACPA-negative RA. On theother hand, this was also seen in 38% of mixed connective tissue disease, 36% of primarySjögren’s syndrome, 23 % of systemic sclerosis, 22% of systemic lupus erythematosus, 22% ofvasculitis, 15% of adult onset Still’s disease, 8% of Behçet's disease, 8% of polymyalgiarheumatica, 7% of inflammatory myositis and 46% of others (relapsing polychondritis,sarcoidosis, primary antiphospholipid syndrome, and osteoarthritis). Anti-CarP Ab titer in ACPA-negative RA was not significantly higher than that in non-RA CVDs, the titer is not informativefor diagnosing ACPA-negative RA.[Conclusion]Anti-CarP Ab is detected in various non-RA CVDs and not useful for discriminating ACPA-negative RA from non-RA CVDs.

a90062 [1. Poster only]Intracellular methotrexate polyglutamates is regulated by gene polymorphisms of a folate-related enzyme.1Division of Rheumatology, Depertment of Internal Medicine, School of Medicine, Toho University

Tatsuhiro Yamamoto1,Kotaro Shikano1,Toshihiro Nanki1,Shinichi Kawai1

Objectives: To investigate major determinants of the intracellular concentrations ofmethotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA).Patients and Methods: In 271 RA patients on stable oral low dose weekly pulse MTX therapy, theconcentrations of MTXPGs in red blood cells (RBCs) were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry. Polymerase chain reaction-restrictionfragment length polymorphism analysis was performed to determine the genotypes of solutecarrier family 19 member 1 (SLC19A1), folylpolyglutamate synthase (FPGS), and gamma-glutamyl hydrolase (GGH).Results: The mean total MTXPG concentration and the concentrations of individual MTXPGsincreased dose-dependently, but reached a plateau at MTX doses >10 mg weekly. The MTXPG3-5/1-2 ratio was lower in patients with adverse events related to MTX than in patients withoutadverse events. Three polymorphisms of FPGS significantly influenced the MTXPG3-5/1-2 ratioin RBCs, while polymorphisms of SLC19A1 and GGH had no impact. The minor allele frequencies of2 FPGS genotypes were significantly increased in our patients compared with a Caucasianpopulation.Conclusion: FPGS may have a major role in regulating intracellular polyglutamation of MTX in RApatients receiving low-dose weekly MTX therapy.

a90082 [1. Poster only]Autoantibodies against CD74 – A new diagnostic marker forSpondyloarthritis (SpA)

1AESKU.Diagnostics GmbH & Co. KG,2AESKU.KIPP Institute, Germany,3Department of Internalmedicine and Rheumatology, Helios Ostseeklinik Damp, Germany ,4Department of Immunology andRheumatology, Medical University Hannover, GermanyTorsten Matthias1,Eva Schweikhard1,Sandra Reuter2,Maria Köhler3,Joachim Georgi3,NiklasBaerlecken4,Torsten Witte4

Objective: Spondyloarthritis (SpA) is a common debilitating inflammatory disorder. Pathogenesisof axial SpA (axSpA) including ankylosing spondylitis (AS) is still largely unclear. Diagnosis isdifficult, since abnormalities in conventional X-rays develop with a latency of several years andonly HLA-B27 is used as laboratory marker. The presence of radiographic sacroiliitis is essentialfor SpA diagnosis. To prevent destructive effects early diagnosis and intervention in SpApatients may be important.

Aim: To evaluate antibodies to the human leukocyte antigen class II-associated invariant chainpeptide (anti-CD74) as a diagnostic marker of SpA.

Methods: Sera of 117 patients with axial SpA and 38 non-SpA patients were analyzed for IgA andIgG antibodies against CD74 by ELISA. All donors provided informed consent for the studyapproved by the local ethics committee (project number 4928).

Results: Anti-CD74 antibodies were detected in 85.1% of SpA patients but only in 5% of non-SpA patients (p≤0.0001). Detection of IgG and IgA anti-CD74 antibodies for diagnosing SpArevealed a sensitivity of 77% and a specificity of 90%. Remarkably, IgA autoantibodies againstCD74 alone had a sensitivity of 67% and a specificity of 95%. IgA anti-CD74 antibodies wereeven more frequent in SpA patients with short disease duration and significantly correlate withadvanced radiological sacroiliitis and reduced spinal mobility.

Conclusion: Anti-CD74 IgA antibodies were strongly associated with SpA. Antibodies againstCD74 could provide an important additional tool for diagnosis of SpA.

a90081 [1. Poster only]Elevated MMP-3 levels in early RA patients sera with Borrelia Antibodies

1AESKU.KIPP Institute, Germany,2Department of Internal Medicine, Division of Rheumatology andClinical Immunology, Johannes Gutenberg-University, Mainz, GermanyTorsten Matthias1,Peter Trinder1,Patricia Jeremias1,Sandra Reuter1,Andreas Schwarting2

Aim: Investigate the influence of an infectious disease on Rheumatoid Arthritis (RA), patientsfrom the ADAPTHERA study cohort were screened for antibodies to Borrelia antigens.

Methods: 204 sera from early RA patients with disease duration < 6 months were screened usingthe following ELISAs: AESKULISA® Rf-AGM, CCP-IgG and -IgM, Borrelia-IgG and –IgM, DF MMP-3and AESKUBLOT® Borrelia-IgG and -IgM. Statistical analysis was performed using MedCalcV.15.6.1.

Results: 10.8% of the patients tested at their 1st visit were positive for Borrelia-specificantibodies. 18/204 patients were positive for IgG or IgM, or both, and 4/204 patients hadequivocal results. 5/22 Borrelia positive patients were CCP positive but negative for Rf-AGM.Interestingly, 10/13 patients that were negative for RA parameters had high MMP-3 levels,while only 1/9 of CCP positive patients showed elevated MMP-3 titres. Measurements werecontinued in 3 serologically RA negative up to visit 4. Anti-Borrelia IgM remained highly positiveand correlated with coexisting high MMP-3 levels. 2/3 patients remained positive in IgG BorreliaWestern blot and negative in RA parameters. 1/3 patients remained positive in CCP and showednegative Western blot results in Borrelia. Classical RA parameters remained negative in mostpatients which showed high MMP-3 and positive Borrelia outcomes.Co-measurement of MMP-3and correlation with further serological markers may assist in improved differential diagnosisand helps to eliminate unnecessary patient therapies, thus saving time, money and improvingoverall patient care.

a90041 [2. Poster or Oral]Analysis of bio-free markers in Japanese rheumatoid arthritis(RA) patients from cytokine levelsand autoantibodies1Division of Rheumatology, Ishida Rheumatology Clinic, Kyoto, Japan

Hiroshi Ishida1

Background: In Japan, many RA patients are received bio-DMARDs treatment based on the flatnational medical insurance system. However, this current leads to heavy budget for govermentalmedical burden.Purpose: In order to decrease the heavy burden, many factors for bio-free markers were analysedfrom diverse aspects not only extra-articular complications but also autoantibodies and serumcytokine levels.Methods: Bio-free group was selected 20 RA patients without bio-DMARDs including TNFblockers, interleukin(IL-6) receptor blocker and co-stimulation blocker. Control group wascomposed of the close characters and backgrounds RA patients with bio-DMARDs treatment.Both groups are compared with each other.Serum cytokine levels were measured by sandwichenzyme-linked immunoabsorbant assay (ELISA) for specific cytokines such as IL-6, IL-10, IL-17,IL-22, IL-23, IL-33 and TNF. Autoantibodies are assayed by radio immunoassay (RIA).Results:Bio-free group had high frequency eosinophilic allergic sinusitis (EOAS), higher titer ofanti-CCP antibody, IL-10, and IL-33, lower titer of IL-22, comparing to the control group. Othercytokines such as IL-6, IL-17, and TNF were not significant differences between the bio-freegroup and the control group.Conclusion: TNF and IL-6 are typical inflammatory cytokines in RA, but these cytokine levels inRA patients sera are NOT predict marker for bio-free.

a90046 [2. Poster or Oral]The ulcerative colitis susceptibility gene RNF186 regulates intestinal homeostasis

1Department of Microbiology and Immunology, Graduate School of Medicine, OsakaUniversity,2Department of Respiratory Medicine, Allergy and Rheumatic Diseases, GraduateSchool of Medicine, Osaka University,3WPI Immunology Frontier Research Center, OsakaKosuke Fujimoto1, 2, 3,Atsushi Kumanogoh2, 3,Kiyoshi Takeda1, 3

As well as other autoimmune diseases, both genetic and environmental factors play an importantrole in the pathogenesis of inflammatory bowel diseases (IBDs). However, genetic etiology ofIBDs, such as Crohn’s disease and ulcerative colitis (UC), is not fully understood. In this study,we analyzed the physiological function of RING finger protein 186 (RNF186), a gene variation ofwhich was identified to be associated with UC in genome-wide association studies andsubsequent deep sequencing analysis of the susceptible locus. RNF186, which was highlyexpressed in colonic epithelial cells, acted as an E3 ligase and polyubiquitinated its substratesincluding occludin. Permeability of small organic molecules was increased in the intestine ofRnf186−/− mice. In colonic epithelial cells of Rnf186−/− mice, expression pattern of occludin wasaltered and expression of other substrates was increased. Accordingly, Rnf186−/− mice showedincreased endoplasmic reticulum (ER) stress in colonic epithelial cells and high sensitivity tointestinal inflammation after dextran sulfate sodium (DSS) administration. RNF186 protein withthe UC-associated mutation had an impaired E3 ligase activity. Furthermore, introduction of theUC-associated Rnf186 mutation in mice led to increased ER stress in colonic epithelial cells andhigh sensitivity to DSS-induced intestinal inflammation. These findings demonstrate thatRNF186 controls protein homeostasis in colonic epithelial cells and thereby regulates intestinalhomeostasis.

a90116 [1. Poster only]Leucine-rich α2-glycoprotein (LRG) enhances colonic inflammation by increasing leukocyteadhesion to TGF-β-stimulated endothelium

1Laboratory of Immune Signal, National Institute of Biomedical Innovation, Health and Nutrition

Takashi Mishima1,Hayato Urushima1,Minoru Fujimoto1,Hiromi Honda1,Lee Hyun1,TomoharuOhkawara1,Satoshi Serada1,Tetsuji Naka1

Leucine-rich α2-glycoprotein (LRG) has been identified as a disease activity marker ofinflammatory diseases including inflammatory bowel disease (IBD). Whereas LRG was reported tomodulate TGF-β signaling, the role of LRG in these diseases has not yet been clarified. Here weinvestigated the function of LRG in IBD.The IBD-like condition, dextran sodium sulfate (DSS)-induced colitis, was induced in wild-type(WT) mice and LRG knockout (LRG-/-) mice. Weight loss in LRG-/- mice was less severe than thatin WT mice. Histological examination disclosed that neutrophil and macrophage invasion incolonic submucosal layer was much reduced in LRG-/- mice compared with WT mice at day 3 postDSS treatment. Interestingly, the expression of endoglin, one of adhesion molecules in vascularendothelial cells, was markedly reduced in LRG-/- mice compared with WT mice.To examine the contribution of LRG in cell adhesion, in vitro cell adhesion assay was performed.THP1 monocytes could adhere to TGF-β-treated endothelial cells better than to untreated cells,in accordance with the finding that TGF-β induced Smad1 phosphorylation in endothelial cellsand stimulated the expression of endoglin. Importantly, the addition of recombinant LRG couldenhance TGF-β-induced endoglin expression in endothelial cells and increased the number ofadhered monocytes in cell adhesion assay.Our data suggest that LRG accelerates the onset of colonic inflammation at least in part byenhancing leukocyte trafficking through the upregulation of TGF-β-induced endoglin expressionin vascular endothelial cells.

a90072 [1. Poster only]New markers for celiac disease: anti-neo-epitope human and microbial transglutaminases

1AESKU.KIPP Institute, Mirkoforum Ring 2, 55234 Wendelsheim, Germany,2B. Rappaport School ofMedicine, Technion-Israel Institute of Technology, Haifa, IsraelTorsten Matthias1,Aaron Lerner2,Patricia Jeremias1,Sandra Neidhöfer1

Objectives: Microbial transglutaminase (mTg) and human tissue Tg (tTg) complexed to gliadinpeptides present neo-epitopes. Antibodies against these complexes are called tTg neo-epitopeand mTg neo-epitope. Reliability of antibodies against the non-complexed and complexed formsof both transglutaminases to reflect intestinal damage and to diagnose pediatric Celiac Disease(PCD) was compared.

Methods: 95 PCD patients, 99 normal children (NC) and 79 normal adults (NA) were tested usingthe following ELISAs detecting IgA, IgG or both IgA+IgG combined: AESKULISA® tTg (tTg, RUO),AESKULISA® tTg New Generation (tTg neo-epitope (tTg-neo)), AESKULISA® mTg (RUO) andAESKULISA® mTg neo-epitope (mTg-neo, RUO). Marsh criteria were used for the degree ofintestinal injury.

Results: All anti-mTg-neo and anti-tTg-neo levels were higher (p<0.001) compared to the singleantigens. tTg-neo IgA and IgG+IgA were higher than mTg-neo IgA and IgA+IgG (p<0.0001). Theantibody activities reflecting best the increased intestinal damage were: mTg-neo IgA> mTg-neoIgA+IgG > tTg-neo IgG ≥ mTg-neo IgG > tTg-neo IgA> tTg-neo IgA+IgG. Taken together, mTg-neo IgG and tTg-neo IgA & IgA+IgG correlated best with intestinal pathology (r=0.5633,r=0.6165 & r= 0.6492; p<0.0001, p<0.0001 & p<0.0001 respectively).

Conclusion: The complexed forms of both transglutaminases exhibited a higher OD activity andbetter reflected intestinal damage in PCD, compared to the non-complexed forms. mTg isimmunogenic in children with CD and by complexing to gliadin its immunogenicity and pathologyreflection is enhanced.

a90073 [1. Poster only]COMPARISON OF THE RELIABILITY OF CELIAC DISEASE SEROLOGY TO REFLECT INTESTINALDAMAGE.1AESKU.KIPP Institute, Wendelsheim, Germany,2B. Rappaport School of Medicine, Technion-IsraelInstitute of Technology, Haifa, IsraelTorsten Matthias1,Aaron Lerner2,Sandra Neidhöfer1,Patricia Jeremias1

Objectives and study: In view of the increasing importance of the serological biomarkers for thescreening and diagnosis of celiac disease (CD), the reliability of those isolated or combinedantibodies to reflect the intestinal damage in children with CD was evaluated.

Methods: 95 pediatric CD patients (mean age 8.3), 45 nonspecific abdominal pain children (AP)(mean age 7.3), 99 normal children (NC) (mean age 8.5) and 79 normal adults (NA) (mean age 28)were tested by the following ELISAs, detecting IgA, IgG or both, IgA and IgG: AESKULISA®Gliadin (AGA), AESKULISA® tTg (tTG; RUO), AESKULISA® DGP (DGP) and AESKULISA® tTg NewGeneration (Neo-epitope tTg complexed to gliadin=tTg-neo). The results were compared to thedegree of intestinal injury, using revised Marsh criteria. Scatter diagrams and regressionanalysis comparing the 12 antibodies’ optical density (OD) activities to the degree of theintestinal damage were correlated.

Results: Most of the assays were able to differentiate patients with low and high degree ofintestinal damage. Comparing the different correlations between CD associated IgA and IgGantibodies’ isotypes, the tTg-neo IgA (r2=0.968, p<0.0025) and tTg-neo/DGP IgGs (r2=0.989,p<0.0001/ r20.985, p<0.0001, respectively) stood out, significantly, as the best indicators ofthe intestinal damage in CD.The highest OD values (medium 2.94±1.2, p<0.0001) were achievedby using the tTg-neo IgA ELISA in patients with Marsh 3c.

Conclusion: tTg-neo IgA/IgG antibodies should be preferably used to diagnose and reflectintestinal damage in childhood CD.

a90074 [1. Poster only]TISSUE/MICROBIAL TRANSGLUTAMINASE NEO-EPITOPES SHOW SIMILAR IMMUNO-EPITOPES INCELIACS1AESKU.Diagnostics GmbH & Co. KG,2AESKU.KIPP Institute, Wendelsheim, Germany,3B.RAPPAPORT SCHOOL OF MEDICINE, TECHNION-ISRAEL INSTITUTE OF TECHNOLOGY, HAIFA,ISRAELTorsten Matthias1, 2,Aaron Lerner3,Sandra Neidhöfer1,Patricia Jeremias1

Introduction: Microbial transglutaminase (mTg) is able to modulate a high variety of proteinsand is used in food industry to enhance quality of food products. mTg de/transamidates peptidesin the same way as the endogenous tissue transglutaminase (tTg), the autoantigen in celiacdisease (CD).

Aims: To compare the complexes of mTg and tTg docked gliadin peptides (resulting in mTg or tTgneo-epitopes) whether they form structural or linear immunoreactive epitopes in CD patients.

Methods: mTg, tTg, gliadin peptides, mTg neo-epitope and tTg neo-epitope complexes werestudied after preincubation with different additives (defolding chemicals and digestingenzymes) as digested or linear structures of all immunoreactive epitopes. Immunoreactivity ofthe preincubated antigens was compared to untreated complexes. Sera of CD patients were usedto compare serological activity against structural and linear epitopes.

Results: The overall immunoreactivity of tTg/ mTg neo-epitopes treated with digesting enzymeswas reduced to 37±20 and 59±11 % for IgA and 25±12 and 53 ±18 % for IgG, respectively,compared to no additive treatment (p<0.001). After chemical treatment no decrease inserological activity against mTg neo-epitopes 109±11 % and tTg neo-epitope slightly decreaseof 93±9 % (p=0.003, p=0.0045, IgA and IgG) was found.

Discussion: Complexes formed by crosslinking and modulating food proteins are strongstructural epitopes. These molecules formed during industrial food processing might leading toan enrichment of immunopotent structures, thus, affecting CD patients.

a90075 [1. Poster only]3-D STRUCTURES OF HUMAN AND MICROBIAL TRANSGLUTAMINASES COMPLEXED TO GLIADINARE SIMILAR1AESKU.Diagnostics GmbH & Co. KG,2AESKU.KIPP Institute

Torsten Matthias2,Sandra Neidhöfer1,Patricia Jeremias2

Objective: In the food industry, microbial transglutaminase (mTg) is used to modulate textureand improve the properties of food products. Due to their common enzymatic functions, thequestion has arisen whether complexes of mTg formed by transamidation reactions could berelevant for celiac patients. Linear and three dimensional structures of the complexes formed bytTg or mTg and gliadin (tTg neo-epitopes or mTg neo-epitopes, respectively) were compared.

Methods: mTg/ tTg neo-epitopes were formed, separated by size and confirmed by SDS-PAGE.For structural alignment and docking experiment the program YASARA was used. Structuralalignment was performed with the primary structures of mTg and tTg by using the MUSTANG-algorithm.

Results: No alignment on the α-C atoms of the protein backbones and the 3D structure betweenmTg and tTg were observed. Glutamine or other positive residues are directed to the activecentre due to a mainly negatively charged surface. After docking of the gliadin peptide to mTgand tTg, both enzymes show adjacent partial-positive charges. Moreover, both neo-epitopecomplexes show a superimposed epitope similarity, even though homology at the entrance to theactive centre is still low.

Conclusion: Only after docking of gliadin peptides to mTg or tTg, similarities in charges andstructure of epitopes appear. If molecular mimicry leads from mTg neo-epitopes to tTg-neo-epitopes there must exist a similarity. If an antibody-paratope exists, an anti-mTg neo-epitopeantibody might show cross-reactivity to a tTg neo-epitope.

a90076 [1. Poster only]ANTI ENTEROCYTE AUTOANTIBODIES IN CELIAC DISEASE.

1AESKU.KIPP Institute, Germany,2Massachusetts General Hospital, Harvard Medical School, MA.USATorsten Matthias1,Aaron Lerner1,Patricia Jeremias1,Kushak Rafail2,Harland Winter2

Introduction: Endoscopic and histological changes of autoimmune enteropathy (AIE) are similarto celiac disease before treatment, but these are not altered by any form of dietary restriction,including a gluten-free diet. Even antibodies to tissue transglutaminase have been described inover 30% AIE patients, but no anti enterocyte antibodies (AEA) were studied in pediatric CD.

Methods: CD (No=33) was diagnosed based on positive celiac serology (anti-neo-epitope tissuetransglutaminase (Aesku*…) and/or anti endomysial antibodies) and small intestinal biopsy. Ageand sex matched controls (No=48) were defined by abdominal pain, negative celiac serology,normal upper endoscopy and normal small intestinal biopsies. AEA test was performed usingWestern blot. Homogenates from normal human intestinal mucosa were electrophoreses on 7.5%SDS-PAGE and transferred on nitrocellulose membranes. Blots were treated with serum fromceliac patients and controls and immunodeveloped using ELISA kit.

Results: 3/33 (9%) and 6/48 (12.5%) were positive for AEA in the pediatric celiac compared tothe control group, respectively. The frequency of AEA positivity in abdominal pain control groupwas higher than in the celiac group (P<0.0001).

Conclusions: Despite the clinical, endoscopic and histological overlap between AIE and CD, AEAare more frequent in non CD, non-specific abdominal pain children with normal intestinalhistology. Most probably the AEA in CD represent an epiphenomenon. The presence of AEA in thenon-CD abdominal pain children remains to be investigated.

a90077 [1. Poster only]GLUTEN IMMUNOLOGICAL SIDE EFFECTS ARE DETRIMENTAL TO HUMAN HEALTH.

1AESKU.KIPP Institute, Germany,2B. Rappaport School of Medicine, Technion-Israel institute ofTechnology, Haifa, IsraelTorsten Matthias1,Aaron Lerner2

Objectives and study: Evolution was accompanied by enrichment of gluten content in the wheatand today 80% of the proteins are gluten. In parallel, some unwanted effects induced by glutenconsumption in non-celiac affected populations were recently described Aims: to summarize theliterature for gluten consumption and withdrawal effects on autoimmune diseases.

Methods: A systematic review was performed, using Medline, Google, and Cochrane Librarydatabases.

Results: The following conditions respond to gluten free diet (GFD): Transaminasemia, type 1diabetes, rheumatoid arthritis, dermatitis herpetiformis, thyroiditis, gluten ataxia and multiplesclerosis. Several pathophysiological avenues were described for the detrimental effects ofgluten: breach of intestinal tight-junction integrity, decreased viability and apoptosis inductionin human cell-lines, induction of neutrophil migration, decrease in NKG2D and ligand expression,increase Th17 population, affects regulatory T-cell subsets, change innate immunity anddendritic cell functions and change diversity of the microbiome.

Conclusions: multiple non-celiac autoimmune diseases and conditions respond, to a variabledegree, to GFD. The protective mechanisms of GFD are constantly unraveled and involve multipleimmunoregulatory pathways. Since transglutaminase 2 is pivotal in post translationalmodification of gluten and autoimmunogenesis, GFD might slow its progression. Severalpathophysiological pathways can explain the detrimental health effects of gluten consumptionin humans.

a90079 [1. Poster only]THE INTESTINE AS PRIMARY SITE FOR POST-TRANS MODIFICATION IN SYSTEMIC AUTOIMMUNEDISEASE1AESKU.KIPP Institute, Germany,2B. Rappaport School of Medicine, Technion-Israel institute ofTechnology, Haifa, IsraelTorsten Matthias1,Aaron Lerner2

Autoimmune Diseases share multiple autoimmune pathogenic features. End/ exogenous enzymeshave the capacity of intestinal enzymatic neo-antigen generation by post-translationalmodifications (PTM) of proteins.

To describe the endogenous and exogenous intestinal enzymatic production of immunoactiveneo-epitope peptides.

Peptides crosslinking, de/amination/deamidation, de/phosphorylation, a/deacetylation,de/tyrosination, glycosylation, arginylation, methylation, citrullination and carbamylation takesplace in the intestine. Intestinal human tissue and microbial transglutaminases (tTg, mTg) inducemultiple neo-epitopes on the Tg-peptide docked complex or citrullination by peptidylargininedeiminase resulting in autoantibodies formation in celiac disease and rheumatoid arthritis,respectively. Taken together, the highest lymphocyte concentration, the enzymatic capabilities,the huge environmental luminal load of food nutrients and additives, infectious agents andtoxins increaseing intestinal permeability, the dysbiosis and intestinal enzymes that modifypeptides and available substrates, make the gut an ideal prime candidate for autoimmunogenesisby PTM. Individual patients may undergo initiation events at gut, but still converge on differentend organs as the time and disease process evolves.

The gut is an ideal place for neo-epitoped peptide formation that drives local and systemicautoimmunity. Suitable substrates, the corresponding endo-/ exo- enzymes, the antigenic loadand the shared genes set the stage for PTMs, resulting in intestinal and systemicautoimmunogenesis.

a90080 [1. Poster only]Peripheral organs’ autoimmunity is highly connected to the intestinal ecosystem’s events

1AESKU.KIPP Institute, Germany,2B. Rappaport School of Medicine, Technion-Israel Institute ofTechnology, Haifa, IsraelTorsten Matthias1,Aaron Lerner2

Objectives and study: The human gut possess all the components necessary to start theautoimmune cascade. The aim was to characterize the multiple gut-remote organ autoimmuneaxes.

Methods: A systematic review was performed to identify Studies referred to gut multiple organsaxes, using Medline, Google, and Cochrane Library databases.

Results: The specific dysbiota and tight junction dysfunction are primary defects in autoimmunediseases. The end result of the passage of none-self- proteins, from the luminal compartment tothe sub epithelial one, initiates the autoimmune cascade. The richness of the mucosal milieu inimmune components, cells and systems, blood and lymphatic vessels, entero-neuronal andendocrine network and mural endo-mesoderm cohabitation, constitute an ideal place to initiate,maintain and propagate the autoimmune process. The mucosal committed immune cells, modifiedproteins, proinflammatory cytokines and lymphokines circulate via the local vessels, distributingautoimmune messages to remote organs, thus creating a gut-extraintestinal axes. Brain, joint,bone, endocrine, kidney, lung, liver, heart and skin, is directionally relayed to the intestinalevents taking place in the genetically susceptible individuals.

Conclusions: The intestine is a major site of changing tolerance to autoimmunity. The diseasespecific dysbiota, its post translational capacity to modify proteins, the plethora of substrates,the leaky gut, the local immune, neuroendocrine, vascular and lymphatic systems make theintestine a prime candidate to drive systemic autoimmunity.

a90138 [1. Poster only]Evaluation of liver tissue as substrate for detection of endomysium antibodies

1Institute of Laboratory Medicine, Medical Faculty of the University and University Hospital,Leipzig, Germany,2EUROIMMUN AG Labormedizinische Diagnostika AG Dassow,Germany,3Children`s Hospital of the Clinical Centre "Sankt Georg" Leipzig,Germany,4Translational Gastroenterology Unit, Experimental Medicine, University of Oxford,John Radcliffe Hospital, Oxford, England,5University Children`s Hospital Dresden,Germany,6University Children`s Hospital Graz, Austria,7University Children`s Hospital Gießen,Germany,8University Children`s Hospital Tübingen, Germany,9University Children`s HospitalFechner Kai2,Wolf Johannes1,Jahnke Annika2,Richter Thomas3,Uhlig Holm H.4,Laass MartinW.5,Hauer Almuthe6,Laffolie Jan de7,Stern Martin8,Flemming Gunter9,Mothes Thomas1

Current guidelines prescribe detection of antibodies (IgA) against endomysium (EmA) by indirectimmunofluorescence (IIF) as one confirmatory test to diagnose paediatric coeliac disease (CD)without duodenal biopsy. Primate oesophagus presents the gold standard as respective IIFsubstrate; however, other tissues as well are rich in the major EmA antigen tissuetransglutaminase. We tested the potential of primate liver as alternative substrate for EmAdetection.

Sera of 298 biopsy-proven CD patients and 574 disease controls (including also type 1 diabetesmellitus, inflammatory bowel disease, selective IgA deficiency) were tested for IgA EmA by IIF onprimate oesophagus and liver cryosections (Euroimmun AG, Germany).

Comparison of test results showed that oesophagus and liver perform comparably sensitive(95.3% vs 95.6%, p=0.69) and specific (98.1% vs 97.2%, p=0.32) with similar positive andnegative predictive values (PPV: 96.3% vs 94.7%, p=0.95; NPV: 97.6% vs 97.9%, p=0.72) fordetection of IgA EmA. Moreover, staining pattern of EmA along liver sinusoids was more distinctthan staining of oesophageal connective tissue layers considering patterns caused by potentiallyco-existing autoantibodies, e.g. anti-smooth muscle antibodies.

In conclusion, detection of IgA EmA is likewise sensitive and specific on liver as it is onoesophagus. Since the EmA staining pattern on liver is highly characteristic and thus lesssusceptible to interferences by other autoantibodies, fluorescence read out can be facilitated.Thus, liver may constitute a reasonable alternative substrate in EmA IIF.

a90099 [1. Poster only]Autoimmune CD8 T cell-mediated interface dermatitis is regulated by Langerhans cells

1Department of Dermatology, Faculty of Medicine, University of Tsukuba,2Institute forMolecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz,55131 Mainz, GermanyNoriko Kubota1,Naoko Okiyama1,Akimasa Saito1,Yosuke Ishitsuka1,Rei Watanabe1,Björn E.Clausen2,Manabu Fujimoto1

Interface dermatitis (IFD) is associated with different autoimmune inflammatory skin diseasesand its histologic features exhibit dermal infiltration of lymphocytes and epidermal vacuolardegeneration. Previous reports showed that mucocutaneous IFD during acute graft-versus-hostdisease (aGVHD) and lupus erythematosus is mediated by cytotoxic CD8 T cells. As a murinemodel of IFD, we generated transgenic (Tg) mice that express membrane-bound chickenovalbumin (OVA) under control of a keratin 14 promoter (K14-mOVA Tg mice), which developaGVHD-like disease after OVA-specific CD8 T cell (OT-I) transfer. To determine whetherLangerhans cells (LCs) regulate autoimmune mucocutaneous disease with IFD, we transferred1×106 OT-I cells into Langerin-diphtheria toxin (DT) receptor knock-in/K14-mOVA double Tg(DTg) mice. LC-depleted DTg mice developed markedly exacerbated aGVHD-like disease (meanskin score, MSS: 4.18 ± 2.08) as compared to LC-sufficient animals (MSS: 2.10 ± 1.14, p<0.05;u-test). In addition, Proliferation of OT-I cells stimulated with OVA peptide (SIINFEKL) wasinhibited upon co-culture with LCs isolated from the epidermis of K14-mOVA Tg mice (opticaldensity, OD: 1.04 ± 0.02) as compared to splenic dendritic cells (OD: 1.79 ± 0.26, p<0.05; t-test). In contrast, LCs did not curb the production of granzyme B and Fas ligand by the OT-Icells. The inhibition of OT-I cell proliferation did not require cell-cell contact, indicating thatLCs regulate mucocutaneous IFD via soluble factor(s) attenuating the proliferation ofkeratinocyte-specific autoimmune CD8 T cells.

a90104 [1. Poster only]Perforin is essential for induction of keratinocytes death in a murine model of autoimmunemucocutaneous disease with interface dermatitis1Department of Dermatology, University of Tsukuba

Akimasa Saito1,Naoko Okiyama1,Noriko Kubota1,Yosuke Ishitsuka1,Rei Watanabe1,ManabuFujimoto1

Interface dermatitis (IFD) is a set of histological finding including dermal infiltration oflymphocytes and death of epidermal basal cell layer keratinocytes, in which autoimmunemechanism involves. In various skin diseases with IFD, perforin and granzyme released fromautoaggressive cytotoxic CD8 T cells are considered to induce keratinocytes death. Thetransgenic mice that express membrane-bound OVA under the control of a keratin 14 promoter(K14-mOVA Tg mice) develop mucocutaneous disease with IFD by transfer of chicken ovalbumin(OVA)-specific CD8 T cell (OT-I-cell). In the murine model of IFD, even K14-mOVA Tg micetreated with depleting antibodies to CD4 (GK1.5) developed mucocutaneous injury, while β2microglobulin-knockout (KO) K14-mOVA Tg mice without the expression of MHC class I did not.To investigate the role of perforin in the pathogenesis of IFD, we transferred perforin (PRF1)-KO OT-I cells to K14-mOVA Tg mice. The mice did not develop mucocutaneous disease with IFD.Flow cytometry analysis showed that PRF1-KO OT-I cells obtained from skin-draining lymphnodes of K14-mOVA Tg mice 5 days after the transfer proliferated, activated and producedinterferon-γ as well as wild-type OT-I cells. Immunohistochemical staining with anti-greenfluorescent protein (GFP) antibodies revealed that GFP+ PRF1-KO OT-I cells infiltrated only inthe dermis, while wild-type GFP+ OT-I cells attacked to interface of the epidermis. In aggregate,perforin plays a critical role in IFD.

a90157 [1. Poster only]A multivariant profile ELISA for one-step diagnostics of autoimmune bullous dermatoses

1Institute for Experimental Immunology, affiliated to EUROIMMUN AG, Luebeck,Germany,2Department of Dermatology, University of Luebeck, Germany,3Lübeck Institute ofExperimental Drematology (LIED), University of Luebeck, GermanyMende Miriam1,Daehnrich Cornelia1,van Beek Nina2,Johannsen Nora1,Goletz S.2, 3,DworschakJ.2,Schlumberger Wolfgang1,Zillikens Detlef2,Schmidt Enno2, 3

Autoimmune bullous dermatoses (AIBD) are characterized by autoimmunity to structural proteinsof desmosomes (pemphigus) and dermal-epidermal junction (pemphigoid disease) resulting inextensive blistering of skin and/or mucous membranes. Major antigens are BP180, BP230,collagen type VII, desmoglein 1 and 3, and envoplakin. Here we present an ELISA for paralleldetermination of relevant serum autoantibodies.

The ELISA is based on recombinant fragments of the immunodominant region of BP180, BP230,collagen type VII, desmoglein 1 and 3, and envoplakin as antigenic substrates, each applied in aseparate reagent well. Its performance was compared to conventional stepwise diagnosticdecision strategy including immunofluorescence with frozen tissue sections, antigen dots, andimmunoblots.

Analyzing 158 sera from AIBD patients and 145 control individuals, sensitivities from 60% (anti-BP230, bullous pemphigoid) to 100% (anti-desmoglein 3, pemphigus vulgaris) and specificitiesfrom 97.3% (anti-BP180 NC16A) to 100% (anti-collagen type VII) were reached. In a prospectivestudy with 289 consecutive sera from patients with suspected AIBD, the ELISA and theconventional diagnostic strategy yielded identical results in 89% of patients.

The ELISA represents a sensitive and specific multiparametric test system for one-stepdiagnostics of pemphigus and pemphigoid diseases, in high accordance to conventional stepwisediagnostics. Discrepancies could be attributed to the absence of certain target structures (e.g.p200, laminin 332, BP180 ectodomain), and the lack of IgA autoantibody detection.

a90119 [1. Poster only]IgG4-positive cells in the affected skin of alopecia areata

1Department of Dermatology, Wakayama Medical University

Takaharu Ikeda1,Takashi Yoshimasu1,Fukumi Furukawa1

【Background】Recently we experienced a case of IgG4-related disease (IgG4-RD) mimicking with alopecia areata(AA). Ordinary AA is a T cell-mediated organ-specific autoimmune disease targeting unknownautoantigens in the hair follicles. AA is considered to be a Th1/Tc1 disease, and the pathogenesisis naturally different from that of IgG4-RD. B cell linkages including plasma cell in the affectedskin of AA are not focused to discuss so much. We investigated about the presence of IgG4-positive cells in AA.【Methods】The case of IgG4-RD mimicking with AA and the cases of AA who had visited the alopecia clinic ofour department were included. Tissue samples were taken from each skin lesion by punchbiopsies. The sections were provided for hematoxylin and eosin and toluidine blue staining andimmunohistochemical analysis.【Results】In IgG4-RD there were IgG4-positive cells densely perifollicular, periappendage, and perivascularinfiltration. The ratio of IgG4-positive cells to IgG-positive cells (IgG4+/IgG+ ratio) was 60 to70%. The number of IgG4-positive cells per HPF was over 200. In AA there were IgG4-positivecells sparsely perifollicular infiltration. But the IgG4+/IgG+ ratio in each follicular unit was 40to 70%. Mast cells infiltrated in the parts where other cells accumulated in IgG4-RD and mainlyin or near the follicular units in AA.【Conclusion】We will discuss the similarities or the differences between IgG4-positive cells of ordinary AAand those of IgG4-RD.

a90152 [2. Poster or Oral]Diversity of humoral responses to the centromere proteins in with chronic liver diseases

1Department of Medical Technology, Kagawa Prefectural University of HealthSciences,2Department of Dermatology, Nagoya University Graduate School ofMedicine,3Department of Gastroenterology and Neurology, Kagawa University School of MedicineTakashi Himoto1,Yoshinao Muro2,Koji Fujita3,Takako Nomura3,Joji Tani3,AsahiroMorishita3,Hirohito Yoneyama3,Tsutomu Masaki3

Background: Anticentromere antibodies (ACAs) have been observed in autoimmune liver diseases.However, little is known about the differences in immune responses to the centromere proteinsamong the liver diseases,Methods: By synthesizing recombinant proteins consisting of the N- and C-termini of majorcentromere proteins, we investigated the humoral responses in patients with HCV-relatedchronic liver disease (CLD-C), primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH).Results: Eight of the 754 (1%) patients with CLD-C, 14 of the 57 (25%) patients with PBC and 6of the 38 (16%) patients with AIH had ACAs. There were no significant differences in ACA titersdetermined by an indirect immunofluorescent method among the autoimmune liver diseases.However, the titers of IgG-subclass autoantibodies against the C-terminus of centromereprotein (CENP)-B were significantly higher in the CLD-C patients than in the AIH patients.Likewise, the titers of IgM-subclass autoantibodies against the N-terminus of CENP-A weresignificantly higher in the PBC group than in the CLD-C group. The ACA-positive patients whodeveloped liver cirrhosis had significantly higher titers of the IgA-subclass autoantibodiesagainst the C-terminus of CENP-C than those who did not.Conclusion: These findings suggest that immunoreactivities against the fragments ofcentromere proteins show distinct patterns among CLD-C, PBC and AIH and that thedetermination of immunoreactivities against the centromere proteins may be useful for theprediction of disease progression.

a90180 [2. Poser or Oral]

Multiplex autoantibody detection for autoimmune liver diseases and autoimmune gastritis

J. Vanderlocht1, L. Bakker-Jonges2,3, J. Damoiseaux4

1 Tissue Typing Laboratory, Department of Transplantation Immunology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Center+, Maastricht, The Netherlands

2 Medical Laboratories, Reinier de Graaf Gasthuis, Delft, The Netherlands

3 Stichting Kwaliteitsbewaking Medische Laboratoria, Nijmegen, The Netherlands

4 Central Diagnostic Laboratory, Maastricht University Medical Center, Maastricht, The Netherlands.

Autoantibody detection for autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and

autoimmune gastritis (AIG) is traditionally performed by IIF on a combination of tissues. Multiplex

line/dot blots (LIA/DIA) offer multiple advantages, i.e. automation, objective reading, no

interfering reactivities, no coincidental findings.

In the current study we evaluated automated DIA (D-Tek) for detecting autoantibodies related to

autoimmune diseases of the gastrointestinal tract. We tested samples of the Dutch EQC program

and compared the results with the consensus of the participating labs. For the autoimmune liver

diseases and AIG, respectively, 46 and 33 samples were tested.

For anti-mitochondrial and anti-smooth muscle antibodies a concordance rate of 96% and 93%

was observed, respectively. The concordance rate for anti-parietal cell antibodies was 88% when

samples without EQC consensus (n=9) were excluded. For antibodies against intrinsic factor a

concordance of 94% was observed. For all these antibodies discrepancies were identified that

relate to the different test characteristics and the preponderance of IIF utilizing labs in the EQC

program.

In conclusion, we observed good agreement of the tested DIA blots with the consensus results of

the Dutch EQC program. Taken together with the logistic advantages these blots are a good

alternative for autoantibody detection in the respective diseases. A large prospective multicenter

study is warranted to position these novel tests further in the whole spectrum of assays for the

detection of these antibodies.

Jan Damoiseaux will be presenting author; preference: oral presentation

a90088 [2. Poster or Oral]Jade-2 is a novel component of autoantigenic 'rod and ring' intracellular structures

1Department of Oral Biology, University of Florida

S. John Calise1,Thuy Nguyen1,Edward K.L. Chan1

Anti-rods/rings autoantibodies (anti-RR) are most often detected in hepatitis C (HCV) patientsafter treatment with interferon-α/ribavirin, although anti-RR seropositivity has beendemonstrated in non-HCV patients and healthy individuals. Autoantigenic 'rod and ring'structures (RRs) assemble when purine biosynthesis is disturbed, and are composed of inosine 5′-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GMP biosynthesis.However, not all sera react with IMPDH, suggesting there are other components of RRs yet to bedetermined. We have identified a protein known as Jade-2 as a novel component of RRs. Jade-2 isa factor in the HBO1 histone acetyltransferase complex, and its paralogue Jade-1 regulates thecell cycle and binds tumor suppressor von Hippel-Lindau protein (pVHL) and protein kinase B(AKT1), both previously shown to interact with IMPDH. A variety of mammalian cell lines(including HeLa, Huh7, Hep3B, and others) were treated with small-molecule inhibitors known toinduce RR formation. RRs were positively detected using anti-Jade-2 in multiple cell linesindependent of induction method. Jade-2 also localized to RRs as early as 15 min aftertreatment with 1 mM ribavirin. Our data demonstrate that Jade-2 colocalizes with IMPDH earlyin the RR assembly process, and its presence in RRs is conserved across different cell types andconditions of induction. The functions of Jade family proteins in histone acetylation and the cellcycle suggest a potential function of RR beyond their established role as markers of guaninenucleotide deficiency.

a90146 [1. Poster only]Myositis-specific and myositis-associated autoantibodies in patients with myositis.

1Division of Hematology, Rheumatology and Respiratory Medicine, Department of Internalmedicine, Faculty of Medicine, Kagawa UniversityShusaku Nakashima1,Atsushi Kondo1,Risa Wakiya1,Hiroki Ozaki1,Hiromi Shimada1,MiharuIzumikawa1,Tomohiro Kameda1,Hiroaki Dobashi1

Idiopathic inflammatory myopathies (IIMs) including dermatomyositis (DM) and polymyositis (PM)are inflammatory disease of the skeletal muscle. Autoimmune mechanisms play important roles inIIMs, especially in DM and PM. Myositis-specific autoantibodies (MSA) and myositis-associatedautoantibodies (MAA) are autoantibodies detected in PM/DM. These autoantibodies havevalidated for better clinical categorization of IIMs at diagnosis. However, the measurement ofthese autoantibodies respectively is not approved in Japan. Therefore, there is no analysis abouttheir clinical profile with Japanese patients.Objective: We investigate the prevalence of MSA and MAA, and analyze their characteristics inour patientsMethod: We enrolled 69 patients that suspected myositis with/without interstitial lung disease(ILD). The autoantibodies were analyzed using Euroline Myositis Profile3 (Euroimmun, Lübeck,Germany).Results: The positive rate of autoantibodies in all 69 patients was 68.1%. Ro52 (49.2%) wasfound frequently, followed by PL-7 (14.5%), PM-Scl75 (10.1%), Jo1 (8.7%), SRP (5.8%), Ku(5.8%), EJ (4.3%), Mi-2 (2.9%), PL-12 (1.4%) and PM-Scl100 (1.4%). All patients with anti-ARS(Jo-1, PL-7, PL-12 and EJ) had ILD, and two patients of those with Jo-1 and Ku, PL-7 andPL12had rapid progressive interstitial pneumonia. On the other hand, Mi-2 positive patientsdidn’t have ILD. In addition, anti-ARS were associated with arthritis and mechanic’s hands.Conclusion: The result of autoantibodies by Euroline Myositis Profile3 was associated withclinical feature.

a90121 [1. Poster only]Comparison of Prognosis in Interstitial Lung Disease Between Polymositis/ DermatomyosisPatients with Anti-Jo-1 Antibody and Anti-EJ Antibody1Division of Rheumatology, Department of Internal Medicine, Tokai University School ofMedicine,2Division of Rheumatology, Department of Internal Medicine, Tokai University HachiojiHospitalShinji Sato1,Yasushi Koyama1,Sho Sasaki1,Takayoshi Kurabayashi1,Noriko Sasaki2,NaofumiChinen1,Takayuki Wakabayashi2,Chiho Yamada1,Yasuo Suzuki1

Background/Objective: Anti-aminoacyl-tRNA synthetase (anti-ARS) autoantibodies arepolymyositis/dermatomyositis (PM/DM) specific autoantibodies and are known to be associatedwith similar clinical features called as anti-synthetase syndrome. However, recent studiesdemonstrated that distinct differences in the clinical characteristics were present among theseantibodies. In this study, we examined differences in prognosis of interstitial lung disease (ILD)between anti-Jo-1 and anti-EJ positive PM/DM.Methods: Twenty-nine patients diagnosed as having PM/DM and ILD with anti-Jo-1 or anti-EJantibody at Tokai University Hospital between 2011 and 2015 were selected. Autoantibodieswere identified by immunoprecipitation assays and all clinical and laboratory data were examinedfrom medical records. Cumulative survival rates were calculated using the Kaplan-Meier method.Results: Of the 29 patients, 19 (66%) were positive for anti Jo-1 and 10 (34%) were positivefor anti-EJ. Induction rates of home oxygen therapy during follow up period was higher in anti-EJ antibody group than in anti-Jo-1 antibody group (10.5% vs. 30.0%: P=0.31). Also, mortalityrates and recurrence rates were higher in anti-EJ positive group (15.8% vs. 30.0%: P=0.63 and26.3% vs. 50.0%: P=0.24, respectively). Cumulative survival rate was worse in PM/DM patientswith anti-EJ antibody than in anti-Jo-1 antibody.Conclusion: These results suggest that PM/DM patients with anti-EJ antibody had worseprognosis of ILD compared to those with anti-Jo-1 antibody.

a90149 [2. Poster or Oral]The analysis of inflammatory myopathies complicated with interstitial lung disease with anti-Jo-1 antibody1Department of Stem Cell and Immune Regulation, Yokohama City University Graduate School ofMedicine, Yokohama, Japan,2Y-CURD Study Group, Japan,3Center for Rheumatic Diseases,Yokohama City University Medical Center, Yokohama, JapanYumiko Sugiyama1, 2,Ryusuke Yoshimi1, 2,Maasa Tamura1, 2,Naoki Hamada1, 2,Hideto Nagai1, 2,YukoTatekabe1, 2,Naomi Tsuchida1, 2,Yutaro Soejima1, 2,Yosuke Kunishita1, 2,Daiga Kishimoto1, 2,HirotoNakano1, 2,Reikou Kamiyama1, 2,Kaoru Minegishi2, 3,Yukiko Asami1, 2,Yohei Kirino1, 2,Shigeru Ohno2,

3,Hideaki Nakajima1

Background: Interstitial lung disease (ILD) is one of the predominant causes of death inpolymyositis/dermatomyositis (PM/DM). We have already reported that interstitial lesions inlarger region of lungs are an independent prognostic factor for PM/DM with ILD (PM/DM-ILD),and most common causes of death in early stage are respiratory failure and infection. We alsoshowed that high initial dose of prednisolone (PSL) was a risk factor for infections. Here weinvestigated the relationship between positive anti-Jo-1 antibody and patients’ characteristicsto optimize the treatment for PM/DM-ILD. Methods: We retrospectively analyzed clinicalfeatures, laboratory and high-resolution computed tomography findings, initial therapeuticregimens, and episode of serious infection of PM/DM-ILD who had received initial treatment atthe two hospitals belonging to Yokohama City University from 1993 to 2015. The lungs werehorizontally divided into 4 zones and severity was evaluated in each zone. Results: One hundredsix patients (PM 19, DM 49, clinically amyopathic DM (CADM) 38), whose mean age was 55 ± 14years, were recruited. Sixteen had anti-Jo-1 antibody. Positive anti-Jo-1 antibody wassignificantly related to the consolidation in smaller region of lungs by multivariate analyses (p =0.049, hazard ratio 9.64), although it was not significantly associated with mortality, infectionrates, and treatment regimen. Conclusion: A lower initial dose of PSL can be sufficient andbetter for treatment for PM/DM-ILD patients with positive anti-Jo-1 antibody for reducing riskof infection.

a90161 [1. Poster only]Anti-aminoacyl-tRNA synthetases (ARS) antibody seems to be associated with the severity ofmuscular manifestation and alveolar-endothelial damages.1Department of Rheumatology, Infectious Diseases and Laboratory Medicine, University ofMiyazakiAyako Kawano1,Kunihiko Umekita1,Motohiro Matsuda1,Shunichi Miyauchi1,Kazuyoshi Kubo1,MaoKomura1,Kosho Iwao1,Ichiro Takajo1,Yasuhiro Nagatomo1,Akihiko Okayama1

Objectives: The purpose of this study was to clarify the association between the diseaseactivity of interstitial lung diseases (ILD) and blood coagulation disorders in patients withpolymyositis and dermatomyositis (PM/DM).

Methods: This study is retrospective observation study. The medical records of 22 patients whowere diagnosed as having PM/DM admitted to our hospital from April 2012 to March 2015 werereviewed in present study. Diagnosis of ILD was evaluated by chest high-resolution CT. Wereviewed the laboratory findings and autoantibody profile associated with PM/DM.

Results: Eighteen of 22 (81.8%) patients with PM/DM were diagnosed as having ILD.Autoantibodies associated with PM/DM were evaluated in 14 patients among 18 patients withPM/DM-ILD. Anti-aminoacyl-tRNA synthetases (ARS) and anti-MDA5 antibody was positive in8/14 patients (57%) and 4/14 patients (29%), respectively. Anti-Jo-1 antibody was detected in3 patients (38%), anti-PL7 in 3 patients (38%), anti-OJ in one patient (12%), and anti-EJ in onepatient (12%) in 8 patients with anti-ARS antibody positive PM/DM-ILD. The levels of creatininekinase (CK), KL-6 and D-dimer in anti-ARS antibody positive patients tended to be higher thanthose in anti-ARS antibody negative patients. Additionally, there is significantly positivecorrelation in the levels of between serum KL-6 and plasma D-dimer (R= 0.58, p= 0.0008).

Conclusion: These results suggest that anti-ARS antibody, especially anti-Jo-1 and anti-PL7,seems to be associated with the severity of muscular manifestation and alveolar-endothelialdamages.

a90089 [2. Poster or Oral]Anti-MDA5 Antibody Positive Juvenile Dermatomyositis with Hemophagocytic Syndrome (a casereport and literature review)1Department of Rheumatology and Allergy, Hyogo Prefectural Kobe Children's Hospital

Yoshihiro Hishitani1,Kazuko Kasai1,Yasuo Nakagishi1

(Case Report) An 11 years old girl was admitted to our institute. She had disseminated eruptionsfor 3 months, and persistent fever and general fatigue for 1 month. Hemophagocytosis,myositis, and heart failure were detected by bone marrow examination, elevation of muscleenzymes in the serum and MRI of legs, and echocardiogram, respectively. So she was diagnosed asjuvenile dermatomyositis and hemophagocytic syndrome (HPS). Her HPS was so persistent thatshe needed intensive immunosuppressive therapy, including high dose oral prednisolone,methylprednisolone pulse therapy, weekly oral methotrexate, continuous intravenouscyclosporine A, intravenous lipo-dexamethasone, IVIG, plasma exchange, IVCY, and oraltacrolimus to get remission. During the course, anti-MDA5 antibody turned out to be positive.After remission, immunosuppression was tapered, causing a relapse with mild interstitial lungdisease 6 months later. But this relapse was able to be controlled with glucocorticoid andtacrolimus.(Discussion) Here we present a case of anti-MDA5 antibody positive juvenile dermatomyositiswith severe hemophagocytic syndrome and mild interstitial lung disease. Anti-MDA5 antibody isknown to have relationship with clinically amyopathic dermatomyositis and rapidly progressiveinterstitial lung disease, in adult patients. But in patients of juvenile dermatomyositis, therelationship seems unclear. This difference might be a key for understanding the disease.

a90153 [2. Poster or Oral]Nailfold vascular findings of anti-melanoma differentiation-associated gene 5 antibody-positivepatients with dermatomyositis

1Tsuruga Hospital

Kazuhiro Komura1

Rapidly progressive interstitial lung disease is complicated in majority of patients withdermatomyositis who are positive for anti-melanoma differentiation-associated gene 5 antibody(MDA5). Clinically amyopathic dermatomyositis with Heliotrope rash and Gottron’s sign canoffer an implication for MDA5-positivity and the critical treatment before uncovering the resultof blood testing, since these patients can survive only if they received immediate and intensivetherapy. We observed nailfold capillary formation of two acute patients with anti-MDA5antibody using dermatoscopy in the current study in order to test the capability to predict theMDA5 positivity. Marked haemorrhages and enlarged capillaries were observed in almost allnailfolds of both hands, while only upto three nailfolds in patients with antibodies against Jo-1,TIF1, or centromere. However, loss of capillaries was not detected under the dermatoscopy atall, while these were detected in capillary scope in the literature in anti-MDA5 antibody positivepatients. In conclusion, the results of the current study suggest that nailfold findings usingdermatoscopy have a potential to diagnose the patients anti-MDA5 antibodies at their firstvisit, although there is a limitation in number of patient samples in this study. Likewise, thenailfold findings on dermatoscopy may provide the visible information for the pathogensis ofinterstitial pneumonitis in these patients as well.

a90020 [1. Poster only]Predictive factors of poor prognosis and utility of plasmaexchange in anti-MDA5(+)dermatomyositis with interstitial lung disease under immunosuppressive therapy

1Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School ofMedicine, Kyoto,2Center for Rheumatic Diseases, Kyoto University Hospital, Kyoto,3Departmentof Rheumatology and Clinical Immunology, Wakayama Medical University Hospital, Wakayama,JapanMirei Shirakashi1,Ran Nakashima1,Yuji Hosono1,Kosaku Murakami1,Motomu Hashimoto2,NaoichiroYukawa3,Yoshitaka Imura1,Hajime Yoshifuji1,Masao Tanaka2,Koichiro Ohmura1,Tsuneyo Mimori1, 2

Background: It has been reported that anti-MDA5(+) dermatomyositis(DM) often accompaniesrapidly progressive interstitial lung disease(ILD) with poor prognosis. We developed an intensiveregimen of combined immunosuppressive therapy (high dose glucocorticoids, oral calcineurininhibitors and intravenous cyclophosphamide) and confirmed its efficacy in anti-MDA5(+) DM-ILD patients. But some of them are resistant to the intensive therapy. We examined the utility ofplasmaexchange(PE) in such intractable cases and intended to investigate the prognostic factorsof them.Methods: We evaluated the serum cytokines, ferritin and KL-6 levels by ELISA or multiplex assaybefore treatment in 32 patients who received the intensive immunosuppressive regimen. Thepatients were divided into three groups; the survivors without PE (n=22, group A), the survivorswith PE (n=4, group B) and the deceased (n=6, group C).Results: 10 of anti-MDA5(+) patients were resistant to the intensive regimen and PE wasunderwent in 5 patients. Among 5, 4 were survived and 1 was deceased. Serum levels of IL-6, IL-12p70, IL-18, IFNα, ferritin and KL-6 before treatment were significantly higher in group B thanin group A. Serum levels of IL-6, IL-18 and soluble CD163 before treatment were significantlyhigher in group C than in group A.Conclusion: PE seems to be one of the effective treatment in anti-MDA5(+) DM-ILD. We may beable to predict the patients who will show resistance to immunosuppressive therapy using acombination with serum levels of IL-6, IL-12p70, IL-18, IFNα, ferritin and KL-6 beforetreatment.

a90109 [1. Poster only]In situ expression of transcriptional intermediary factor-1γ (TIF-1γ) in patients with cancer-associated myositis (CAM)1Department of Allergy and Rheumatology, Nippon Medical School

Hiroko Kadota1,Yuichiro Shirai1,Kiyotaka Nagahama1,Yuka Okazaki1,Mitsuhiro Takeno1,AkiraShimizu1,Masataka Kuwana1

Objective. To clarify protein expression of TIF-1γ in cancer and non-cancer tissue samples formCAM patients in the presence or absence of anti- TIF-1γ antibody. Methods. This study enrolled4 patients with CAM diagnosed between 2007 and 2015, whose tumor tissue was available forhistological evaluation. RNA immunoprecipitation (IP) and specific IP-immunoblot assays wereused to identify myositis-specific autoantibodies. The expression of TIF-1γ was assessed in bothcancer and non-cancer parts from the same tissue samples by immunohistochemistry. Results.Two patients (carrying cholangiocarcinoma or breast cancer) were positive for anti-TIF-1γ andthe remaining two (carrying gastric cancer or ovarian cancer) were negative for any myositis-specific antibodies, including anti-TIF-1γ. Immunohistochemical study showed that TIF-1γexpression was detected in residential cells as well as in cancer cells identified by nuclei withcomparably large size and apparently dysplasia morphology. There was no difference inintracellular distribution or staining intensity of TIF-1γ between cancer and non-cancer cellswithin the same samples. In addition, similar findings were observed in CAM patients,irrespective of the presence of absence of anti-TIF-1γ antibody. Conclusion. It is highly likelythat autoantibody response to TIF-1γ observed in CAM patients is not explained simply byoverexpression of TIF-1γ in cancer tissue.

a90102 [2. Poster or Oral]Clinical features and prognosis in anti-SRP and anti-HMGCR necrotizing myopathy

1Department of Neurology, Keio University School of Medicine,2Department of NeuromuscularResearch, National Institute of Neuroscience, and Department of Genome Medicine Development,Medical Genome Center, National Center of Neurology and PsychiatryShigeaki Suzuki1,Akinori Uruha2,Yurika Watanabe1,Jin Nakahara1,Kohei Hamanaka2,KazukoTakayama2,Norihiro Suzuki1,Ichizo Nishino2

Objective: To elucidate the common and distinct clinical features of immune-mediatednecrotizing myopathy (IMNM) associated with autoantibodies against signal recognition particle(SRP) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR).Methods: We examined a cohort of 460 patients with idiopathic inflammatory myopathies (IIM)by a muscle biopsy-oriented registration study in Japan. Study entry was strictly determined bythe comprehensive histological assessment to exclude other neuromuscular disorders.Results: Of 460 IIM patients, we diagnosed 73 (16%) as having inclusion body myositis (IBM). Thefrequencies of anti-SRP and anti-HMGCR antibodies in 387 patients with IIM other than IBMwere 18% and 12%, respectively. One patient had both autoantibodies. Severe limb muscleweakness, neck weakness, dysphagia, respiratory insufficiency, and muscle atrophy were morefrequently observed in patients with anti-SRP antibodies than with anti-HMGCR antibodies.Histology was characterized by necrosis and regeneration of muscle fibers and was consistentwith IMNM except in one HMGCR-positive IBM patient. Most patients were initially treated withcorticosteroids: however, additional immunosuppressive drugs were required, especially in thepatients with anti-SRP antibodies.Conclusion: Anti-SRP antibodies are associated with severe neurological symptoms, more thananti-HMGCR antibodies. Although these autoantibodies are independent serological markersassociated with IMNM, patients bearing either share common characteristics.

a90038 [2. Poster or Oral]Interstitial Lung Disease in Patients with Anti-Signal Recognition Particle Antibodies

1Department of Rheumatology and Clinical Immunology, Kyoto University Hospital

Keiichiro Kadoba1,Kosaku Murakami1,Ran Sasai1,Yoshitaka Imura1,Hajime Yoshifuji1,KoichiroOmura1,Tsuneyo Mimori1

Background: Anti-signal recognition particle antibodies (aSRP) are myositis-specificautoantibodies associated with severe necrotizing myopathy. Patients with aSRP sometimeshave interstitial lung disease (ILD), but the characteristics of ILD in patients with aSRP areunclear.Objectives: To determine the clinical and serologic features of ILD in patients with aSRP.Methods: We reviewed clinical data of 26 patients with aSRP. aSRP was detected by 7SL-RNA inRNA immunoprecipitation (RIP) using HeLa cells.Results: Of the 26 patients, 20 had PM/DM, 3 had RA, 3 had Sjögren’s syndrome, 1 had SLE, and1 had Takayasu arteritis. Among 21 patients for whom radiographic data were available, 10patients had ILD. All of them showed non-UIP (usual interstitial pneumonia) pattern on chest CTscans. Serologically, 7 of 21 patients were anti-Ro/SS-A antibody-positive (detected by RIP),and the occurrence of ILD was more frequent in patients with anti-Ro/SS-A antibodies than innegative patients (86% vs. 29%, respectively; P<0.05). In all ILD patients, ILD was stable orslowly progressive and glucocorticoid therapy was only partly effective. None of the ILDpatients died during follow-up (mean 11 years).Conclusion: ILD is not a rare clinical manifestation in patients with aSRP, and non-UIP pattern isa typical radiologic finding. Positive anti-Ro/SS-A antibodies correlated with more frequentoccurrence of ILD. Clinically, ILD is associated with stable or slow progression, poor response toglucocorticoid but good life prognosis.

a90148 [2. Poster or Oral]Presence of anti-cN-1A (Mup44, NT5c1A) IgG is specific for Sporadic Inclusion Body Myositis

1RDL Reference Laboratory Inc., Los Angeles, CA, USA,2EUROIMMUN AG, Luebeck, Germany

Mende Miriam2,Cho EunByul 1,Shen Guo1,Karayev Dmitry1,Metzger Allan L. 1,Morris Robert I.1,Kramp Sabine L. 2,Dähnrich Cornelia2,Schlumberger Wolfgang2

Sporadic Inclusion Body Myositis (sIBM) is an autoimmune disease manifesting with muscledegeneration, inflammatory infiltrates and inclusion vacuoles. Diagnosis of sIBM is hampered byits imprecise characteristics, at times indistinguishable from other Idiopathic InflammatoryMyopathies, but may now be assisted by detection of sIBM-specific autoantibodies targetingmuscle antigen Mup44, identified as cytosolic 5’-nucleotidase 1A (cN-1A; Mup44; NT5c1A). Thisstudy evaluated sensitivity and specificity of an anti-cN-1A IgG serological assay in sera frompatients with and without sIBM.

Serum from patients with clinically and pathologically diagnosed sIBM (n=68), suspected sIBM(n=15), myositis controls [including dermatomyositis (n=4), polymyositis (n=7); unspecifiedmyositis without sIBM (n=94), muscle atrophy (n=1), myonecrosis (n=4)], from patients withSLE (n=33), scleroderma (n=20), Sjogren’s (n=20), rheumatoid arthritis (n=20) and fromhealthy controls (n=254) were tested for anti-cN-1A IgG using an anti-cN-1A ELISA (full-lengthantigen, Euroimmun AG).

Anti-cN-1A was most frequent among definite sIBM (41.2%). The overall specificity was 96.3%with individual specificities from 90% (scleroderma) to 100% (PM or DM).

The presence of anti-cN-1A in serum appears to be disease-specific for sIBM. These antibodiesare found at a moderate prevalence, but are only rarely detected in other autoimmune conditions.Thus, anti-cN-1A ELISA may support the diagnostics of sIBM and accelerates the suspecteddiagnosis in cases of positivity, where muscle biopsy is delayed or unfeasible.

a90162 [2. Poster or Oral]Clinical Significance of Cyclic Citrullinated Peptide-specific Antibody and Rheumatoid Factor inPatients with Polymyositis/Dermatomyositis

1Division of Rheumatology, Department of Internal Medicine, Tokai University School ofMedicine,2Division of Rheumatology, Department of Internal Medicine, Tokai University HachiojiHospitalShinji Sato1,Sho Sasaki1,Takayoshi Kurabayashi1,Yasushi Koyama1,Noriko Sasaki2,NaofumiChinen1,Takayuki Wakabayashi2,Chiho Yamada1,Yasuo Suzuki1

Background: The presence of autoantibodies against aminoacyl tRNA synthetases (ARS) isassociated with polymyositis/dermatomyositis (PM/DM) with a high frequency of arthritis.However, anti-cyclic citrullinated peptide (CCP) antibody and rheumatoid factor (RF) can also befound in patients with PM/DM. The clinical significance of anti-CCP antibody and RF in patientswith PM/DM has not been clearly established.Methods: Serum samples from 60 patients diagnosed with PM/DM were screened for anti-CCPusing ELISA, for RF using latex coagulating nephelometry assay and for anti-ARS using animmunoprecipitation assay. Associations between anti-CCP, RF and anti-ARS and clinicalfeatures were analyzed.Results: Sera from 25 (8 with PM, 17 with DM) of the 60 patients (42%) with PM/DM were foundto contain anti-ARS (14 with anti-Jo-1, 4 with anti-PL-12, 4 with anti-EJ and 3 with anti-PL-7).Ten of 60 (17%) had anti-CCP (4 with PM, 6 with DM) and 12 of 60 (20%) had RF (3 with PM, 9with DM). Five patients with anti-CCP (50%) had arthritis, of which 4 were also diagnosed as RA.In anti-ARS-positive patients, the presence of anti-CCP or RF were significantly increasedcompared with anti-ARS-negative (32% vs. 6% P=0.012, 38% vs. 9% P=0.019, respectively).However, associations between arthritis and the presence of anti-CCP or RF were notstatistically significant in patients with PM/DM (P=1.00, P=0.32, respectively).Conclusions: These results suggest that anti-CCP antibody and RF are closely associated withanti-ARS antibody in patients with PM/DM.

a90066 [2. Poster or Oral]Autoantibodies to Su/Argonaute 2 in Japanese patients with inflammatory myopathy

1Department of Dermatology, Nagoya University Graduate School of Medicine,2Department ofDermatology, Japan Community Health Care Organization Chukyo Hospital, Nagoya, Japan.,3Department of Clinical Nursing, University of Occupational and Environmental Health,Kitakyushu, JapanMariko Ogawa-Momohara1,Yosinao Muro1,Masashi Akiyama1,Masanari Kodera2,Minoru Satoh3

Background: Anti-Su antibodies, originally defined in the ’80s, are found in 10-20 % of varioussystemic autoimmune rheumatic diseases (SARD), including SLE, scleroderma, polymyositis(PM)/dermatomyositis (DM) and Sjögren's syndrome. The 100kD Su antigen was identified asargonaute2 (Ago2) that plays a major role in RNA interference. However, immunoprecipitation(IP) has been the only method to detect anti-Su and its clinical significance is uncertain. Aim: Toestablish anti-Ago2 ELISA and analyze its clinical significance in PM/DM. Methods: Forty sampleseach of anti-Su positive and negative sera, defined by IP using K562 cell extract, were used toestablish in-house ELISA using recombinant Ago2 protein. Sera from PM/DM (n = 222; 89classical DM, 63 clinically amyopathic DM, 24 cancer-associated DM, 17 juvenile DM, 22 PM, and 7myositis overlap) were screened for anti-Ago2. Serum samples above or around the ELISA cut-offvalue were tested by IP. Results: Eighteen of 222 (8.1%) sera from PM/DM showed ELISA unitvalue > mean+5SD of the negative controls. By IP, 14/18 (77%) of them were positive, indicating~5.5% of PM/DM patients have anti-Su/Ago2. Among the 14 patients with anti-Su/Ago2, 9 casesalso had myositis-specific autoantibodies, 5 anti-ARS, 3 anti-MDA5, 1 anti-TIF1-g. Conclusion: Anew anti-Ago2 ELISA is useful for screening of anti-Su/Ago2 antibodies based on a goodcorrelation of the results by ELISA and IP . Further study on patients with broad spectrum ofSARD is necessary to clarify clinical significance of the antibody.

a90026 [1. Poster only]Antibody to Ribonuclease-H2 is a novel autoantibody specifically recognized in SLE; Importanceof intramolecular epitope spreading for autoantibody production1Department of Rheumatology, Juntendo University

Kazuhisa Nozawa1,Kentaro Doe1,Kaori Uomori1,Ken Yamaji1,Naoto Tamura1,Yoshinari Takasaki1

Objective:We have previously published an autoantibody to chromatine assembly factor-1(CAF-1), whichbelongs to the PCNA-binding proteins, is specifically recognized in SLE. Ribonuclease-H2 (RNase-H2) also belongs to the PCNA-binding proteins and functions as a regulating factor for celldivision. Therefore, we assumed that RNase-H2 is a candidate of autoantigens specificallyrecognized in SLE.Materials & Methods:Immunoreactivities against RNase-H2 and CAF-1 recombinant antigens were evaluated by ELISAand were further confirmed by immunoblotting in patient’s sera consisting of SLE, otherconnective tissue diseases, sera reacting with standard autoantigens, and normal healthycontrols (NHCs).Results:Average of serum anti-RNase-H2 antibody titer was significantly elevated in patients with SLEand patients sera reacting with double strands ds DNA. In addition to the titer, the prevalence ofanti- RNase-H2 antibody was 33.9% and it was significantly higher especially in SLE patients andthe sera reacting with ds DNA. Moreover, the titer of anti-RNase-H2 antibody was stronglycorrelated with titer of anti-CAF-1 antibody in regression analysis.Conclusion:We identified anti-RNase-H2 antibody as a novel autoantibody specifically recognized in serawith SLE. Moreover, the strong correlation in these antibodies suggested that intermolecularepitope spreading plays important role for the autoantibody production and diversification inSLE.

a90164 [2. Poster or Oral]Association of anti-TPI antibodies with aseptic meningitis in patients with NPSLE

1Department of Gastroenterology and Rheumatology, Fukushima Medical University School ofMedicineShuzo Sato1,Makiko Yashiro1,Tomoyuki Asano1,Hiroko Kobayashi1,Hiroshi Watanabe1,HiromasaOhira1

Background: We have reported that autoantibodies to triosephosphate isomerase (TPI), which isan important glycolytic enzyme in red blood cells or neuronal cells, are associated withneuropsychiatric systemic lupus erythematosus (NPSLE) pathogenesis. However, the clinicalfeatures regarding anti-TPI antibody (anti-TPI) positive NPSLE were yet known. The aim of thisstudy is to investigate clinical features of anti-TPI positive NPSLE patients using anti-TPI indexvalues determined by enzyme-linked immunosorbent assay (ELISA). Methods: Thirty one NPSLEpatients treated in our department were included in this study. Serum samples were collectedand serum anti-TPI tites were measured by ELISA. The anti-TPI index values were defined as(OD405 of samples - negative control) / (OD405 of positive control - negative control) ×100. Theanti-TPI index values greater than 2 standard deviation above the mean of healthy controls (N =18) were regarded to be positive. Clinical features were compared between anti-TPI positive andanti-TPI negative NPSLE. Results: Ten of 31 NPSLE patients (32.3%) were positive for anti-TPI.The clinical features of anti-TPI positive NPSLE were comparable with those of anti-TPInegative NPSLE, except for higher frequency of aseptic meningitis (40% vs 4.8%). Laboratorydata in patients with anti-TPI positive NPSLE showed significantly higher serum IgG levels andanti-TPI index values positively correlated with serum IgG levels. Conclusion: These resultsindicate that anti-TPI may be associated with the pathogenesis of aseptic meningitis in NPSLE.

a90065 [1. Poster only]Antibodies to Microtubule Associated Protein-2 in the Cerebrospinal Fluid are a UsefulDiagnostic Biomarker for Neuropsychiatric Systemic Lupus Erythematosus1Department of Rheumatology, Juntendo University,2Department of Rheumatology,Rheumatology and Internal Medicine, Sasaki Institute, Kyoundo Hospital, Tokyo, JapanKazuhisa Nozawa1,Yusuke Yamada1,Soichiro Nakano1,Yukiko Mitsuo1,Ken Yamaji1,NaotoTamura1,Kenjiro Yamanaka2,Yoshinari Takasaki1

Objective: Previous reports indicate that serum anti-microtubule associated protein 2 (MAP-2)antibodies are common in sera from patients with neuropsychiatric systemic lupuserythematosus (NPSLE). Differential diagnosis of NPSLE is occasionally difficult because ofdifferential diagnosis which can mimic NPSLE. Therefore, specific biomarkers for NPSLE areneeded. We conducted this study to clarify whether cerebrospinal fluid (CSF) anti-MAP-2antibodies are a useful diagnostic biomarker for NPSLE.Methods: ELISA was conducted to measure CSF concentrations of anti-MAP-2 and anti-ribosomalP antibodies and of IL-6 in NPSLE patients and non-NPSLE controls.The non-NPSLE controlsconsisted of SLE patients with neuropsychiatric symptoms caused by non-NPSLE conditions andpatients with other connective tissue diseases.Results: Significantly higher anti-MAP-2 antibody titers were found in the CSF of patients withNPSLE versus non-NPSLE controls. The prevalence of anti-MAP-2 antibodies was 33.3% in NPSLEpatients, when a positive cut-off value was 3 standard deviations above the mean OD of non-NPSLE controls. None of the controls had anti-MAP-2 antibodies in their CSF. Both anti-ribosomal P antibody titers and concentration of IL-6 in the CSF were significantly higher inpatients with NPSLE having anti-MAP-2 antibodies than in patients with non-NPSLE controls.Conclusions: Anti-MAP-2 antibodies could be detected in the patients with NPSLE, and itspresence was highly specific for NPSLE. We propose that CSF anti-MAP-2 antibodies are a noveland useful diagnostic biomarker for NPSLE.

a90108 [1. Poster only]Novel autoantigens associated with lupus nephritis

1Department of Hematology, Clinical Immunology and Infectious Diseases, Ehime UniversityGraduate School of Medicine,2Department of Pathology, Gifu University Hospital,3AdvancedResearch Support Center, Ehime University,4Department of Cardiology, Pulmonology,Hypertension and Nephrology, Ehime University Graduate School of Medicine,5Proteo-ScienceCenter, Ehime University,6Department of Rheumatology and Clinical Immunology, KyotoUniversity Graduate School of MedicineSachiko Araki1,Endy Adnan1,Jun Ishizaki1,Tatsuhiko Miyazaki2,Yuki Tanaka3,MasachikaShudou3,Takuya Matsumoto1,Koichiro Suemori1,Takafumi Okura4,Hiroyuki Takeda5,TatsuyaSawasaki5,Nozomi Ishigooka6,Takao Fujii6,Tsuneyo Mimori6,Masaki Yasukawa1,Hitoshi Hasegawa1

[Purpose] We investigated the characterization of new lupus nephritis (LN)-associatedautoantigens. [Method] LN-associated autoantigens were screened from an N-terminalbiotinylated protein library created from a wheat cell-free protein-production system by usingserum from patients with LN and identified by immunoprecipitation and immunohistochemicalstaining of the renal tissue samples. The specificity of the identified autoantigens was analyzedby ELISA with serum from various autoimmune diseases patients. Furthermore, the renal tissuesof C57BL/6 mice were examined after an intravenous injection of the purified proteins. Theseautoantigens and the previously reported 6 autoantigens (ribosomal P, C1q, Sm, dsDNA,nucleosome, actinin) were compared using the serum samples from patients with LN diagnosed byrenal biopsy. [Result] Two LN-associated autoantigens, ribosomal RNA-processing protein 8(RRP8), and spermatid nuclear transition protein 1 (TNP1), were identified byimmunoprecipitation and immunofluorescence of the renal tissue samples of patients. Immunecomplex-deposited glomerulonephritis was also recognized in C57BL/6 mice injected with RRP8or TNP1. Patients with inactive LN were negative for anti-RRP8 antibodies, whereas some ofthose with active LN showed high titers of these antibodies. [Conclusion] We have identifiedRRP8 and TNP1 as the new LN autoantigens. Further investigations will be necessary forevaluating the performance of panels of LN-associated autoantibodies for diagnosis, monitoring,and prognostic stratification of patients with LN.

a90014 [1. Poster only]Cleaved Form of Osteopontin in Urine as a Clinical Marker of Lupus Nephritis

1Department of Rheumatology and Clinical Immunology, Graduate School of Medicine, KyotoUniversity, Kyoto, Japan,2Center for Innovation in Immunoregulative Technology andTherapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan,3Research Portfolio &Science, Astellas Pharma Inc., Tokyo, Japan,4Department of Nephrology, Graduate School ofMedicine, Kyoto University, Kyoto, JapanKoji Kitagori1, 2,Hajime Yoshifuji1,Takuma Oku2, 3,Chiyomi Sasaki2,Hitomi Miyata4,ToshikiNakajima1,Yoshitaka Hirayama2, 3,Tsuneyo Mimori1

Several studies reported that osteopontin (OPN) is increased in plasma and urine of patientswith systemic lupus erythematosus (SLE),and correlates with disease activity, implyingassociation of OPN with pathophysiology of SLE. We assessed utility of two forms of OPN (OPNfulland its cleaved form, OPN N-half) in plasma and urine as markers of disease activity of lupusnephritis (LN). Samples were collected from patients with SLE (LN: N = 29, non-LN: N = 27),minimal change nephrotic syndrome (MCNS) (N = 4), diabetic nephropathy (DN) (N = 5) andhealthy volunteers (HC) (N = 17). While there was no significant difference in urine OPN fullconcentration among patient groups, urine OPN N-half concentration was significantly higher inpatients with LN than HC (p < 0.01). Moreover, urine OPN N-half was higher in LN patients withovert proteinuria (urine protein/creatinine ratio: P/C > 0.5) than LN patients with minimalproteinuria (P/C < 0.5, p < 0.0001), and also higher than in MCNS/DN patients with overtproteinuria (P/C > 0.5, p < 0.05). Urine thrombin activity correlated with urine OPN N-halfconcentration (p < 0.0001), but not with urine OPN full concentration. These results suggestthat urine OPN N-half level reflects renal inflammation, and that the pathophysiology of LN isdifferent from that of MCNS/DN. Thus, urine OPN N-half may be a disease activity marker forLN.

a90047 [1. Poster only]Role of Mucosal-associated Invariant T (MAIT) Cells In Lupus Dermatitis.

1Department of Internal Medicine and Rheumatology, Juntendo University School ofMedicine,2Department of Immunology, Juntendo University School of MedicineGoh Ebata Murayama1, 2,Asako Chiba2,Hirofumi Amano1,Ken Yamaji1,Naoto Tamura1,SachikoMiyake2

Background/Purpose:Mucosal-associated invariant T (MAIT) cells are innate T cells that are restricted by MHC-relatedmolecule-1 (MR1) and express a semi-invariant TCRa chain: Va7.2-Ja33 in humans and Va19-Ja33in mice.Systemic lupus erythematosus (SLE) and psoriasis are chronic inflammatory diseases with severecomplications such as scarring skin lesions. In this study, we conducted this study to clarifyfunctions of MAIT cells in a lupus model by using FcgRIIB-/- Yaa mice and a imiquimod(IMQ)-induced psoriasis model.Methods:Lupus model mice, which are FcgRIIB-/- Yaa mice, were crossed to MR1 deficient mice lackingMAIT cells, and disease progression was compared between MR1-/- FcgRIIB-/- Yaa and FcgRIIB-/-

Yaa mice. MR1-/- and MR1+/+C57BL/6J mice were topically treated with 5% IMQ.Results:There was a significant worsening of dermatitis score in MR1-/- FcgRIIB-/- Yaa mice compared toFcgRIIB-/- Yaa mice. The histopathological analysis revealed there was a significant worsening ofdermatitis and the higher dermatitis score in MR1-/-C57BL/6J mice compared to control MR1+/+

mice.Conclusion: The present study demonstrates that dermatitis was exacerbated in MR1-/- mice inboth lupus model and psoriasis model mice, indicating that MAIT cells may have a suppressiveeffect on skin inflammation. Because MAIT cells have recently been found in human skin, MAITcells may be useful therapeutic targets for the treatment of lupus dermatitis and psoriasis.

a90171 [1. Poster only]Angioimmunoblastic T-cell lymphoma with autoimmune hemolytic anemia and thrombocytopeniamimicking SLE1Division of Rheumatology, National Hospital Organization Kyoto Medical Center,2Division ofGeneral Internal Medicine, National Hospital Organization Kyoto Medical Center,3Division ofHematology, National Hospital Organization Kyoto Medical CenterMikiko Hashimoto Iguchi1,Shuko Kaito2,Masashi Goto2,Takao Odagaki2,Yoshiaki Okuno3,HiroshiKoyama2

Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of T-cell lymphoma, 1% to 2 % ofnon-Hodgkin’s lymphoma, clinically characterized by generalized lymphadenopathy, extranodaldisease, hypergammaglobulinemia and autoimmune hemolytic anemia.We present a 73-year-old man who had AITL with autoimmune hemolytic anemia andthrombocytopenia. He was positive for anti-nucleolar antibody and had a high-titer anti-double-strand DNA antibody. He had generalized lymphadenopathy and splenomegaly and biopsy ofcervical lymph node revealed AITL.Because of severe anemia and thrombocytopenia, pulse methylprednisolone therapy wasadministered initially, followed by CHOP therapy. Despite this cytopenias progressed andintravenous immune globulin therapy was given. Since then his immune dysregulation improveddramatically.AITL mostly affects the elder populations and its prognosis is generally poor. A number ofautoimmune phenomena have been reported in association with AITL, including autoimmunehemolytic anemia, vasculitis, polyarthritis, and autoimmune thyroid disease.Elderly onset autoimmune disease with lymphadenopathy should be considered possibility ofAITL.

a90086 [1. Poster only]A patient-derived autoimmune IgG monoclonal anticardiolipin antibody that binds to beta 2glycoprotein domain I but not to total beta 2 glycoprotein I molecule1Division of Rheumatology, Endocrinology and Nephrology Hokkaido University Graduate Schoolof MedicineKenji Oku1,Yusaku Kanetsuka1,Olga Amengul1,Ryo Hisada1,Kazumasa Ohmura1,MasaruKato1,Toshiyuki Bohgaki1,Tetsuya Horita1,Shinsuke Yasuda1,Tatsuya Atsumi1

Background Anti-cardiolipin/β2 glycoprotein I (aCL/β2GPI) antibodies are the group of antibodiesthat target CL/β2GPI complex. However, aCL is often regarded as with lower specificity.Recently, antibodies to β2GPI domain I are reported as more specific to antiphospholipidsyndrome(APS) (aDI). We examined the diversity of aCL/β2GPI in new patient-derived monoclonalaCL. Methods B cells from APS patients were immortalized with EBV infection to clone aCL-producing cells. A stable cell line containing an IgG monoclonal antibody(EV35102) wasestablished. The presence of a homologous antigen of EV35102 was examined by ELISA. Thepathogenicity of EV35102 was evaluated in antibody treated monocytes: tissue factor (TF)mRNA and intracellular signal protein activation were analyzed with real-time PCR and array kit,respectively. APL profiles of 182 APS patients and 141 connective tissue diseases patients wereanalyzed. Results EV35102 bound to CL only in presence of β2GPI, but did not recognize totalβ2GPI. Surprisingly, EV35102 bound to β2GPI domain I. EV35102, significantly induced monocyteTF mRNA or p38 MAP kinase phosphorylation. Positive aCL with negative aβ2GPI was found in20/182 APS and 5/141 non-APS. In these groups, 4/20 and 0/5 patients were aD1 positive.Conclusion: EV35102 is a new subset of aCL/β2GPI with its epitope on β2GPI domain I, butundetectable by aβ2GPI ELISA. EV35102 had procoagulant property thus this subset is possibleto be pathogenic. The diversity in aCL/β2GPI should be recognized and aCL assay is still usefulfor screening this subset.

a90124 [1. Poster only]Clinical Analysis of 12 Pediatric Patients of Antiphospholipid Syndrome with PulmonaryEmbolism1Department of Pediatrics,Peking Union Medical College Hospital

Hongmei Song1,Jingran Ma1

Objective: To identify the characteristics of pediatric APS patients with pulmonary embolism.Methods: Among 47 pediatric APS patients from Peking Union Medical College Hospital (PUMCH)during the year of 2000 to 2014, 12 patients were diagnosed of pulmonary embolism who wereinvestigated. Results: 25.5% of the pediatric APS patients had pulmonary embolism, which wasthe first presenting manifestation in half of them. Among patients with pulmonary embolism,83% suffered from deep vein thrombosis at the same time. Thrombotic recurrence accounted for16.7%, which happened only in primary APS patients. Proportions of positive anticardiolipin(ACL) and lupus anticoagulant (LA) were 83% and 75% respectively. Among pediatric APSpatients with pulmonary embolism, primary APS accounted for 50% and the other patients wereall associated with systemic lupus erythematosus (SLE), and 67% of primary APS patients hadpositive anti-nuclear antibodies (ANA). Conclusion: Pulmonary embolism is not common inpediatric APS patients, but in patients with pulmonary embolism, it can be the first symptom,which should be paid more attention for early diagnosis. Rethrombosis is prominent due toirregular treatment. Multiple tests for antiphospholipid antibody (aPL) and other autoantibodiesare proposed.

a90049 [2. Poster or Oral]Genetic mutation of phospholipase D4 disturbs the homeostasis and immunological tolerance ofB cells in mice.

1Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School ofMedicine,2Rheumatic Disease Cencter, Kyoto University,3Center for Genomic Medicine, KyotoUniversityShuji Akizuki1,Chikashi Terao3,Koichiro Ohmura1,Namiko Okabe1,Takashi Matsuo1,KosakuMurakami1,Ran Sasai1,Motomu Hashimoto2,Yoshitaka Immura1,Hajime Yoshifuji1,MasaoTanaka2,Tsuneyo Mimori1, 2

Genetic studies identified that phospholipase D4 (PLD4) has a causative role for thepathogenesis of human autoimmune diseases. PLD4 is a member of PLD superfamily which, in thecase of classical PLD1, and PLD2, enzymatically hydrolyze phosphatidylcholine to produce cholineand phosphatidic acid, and is involved in diverse cellular function, including signal transduction,endocytosis, and membrane vesicle trafficking. The transcription of PLD4 is preferentiallyobserved within lymphoid systems, especially in B cells, macrophages, and dendritic cells.However, in contrast to classical PLDs, the functional investigation of PLD4 is limited. In thisstudy, to elucidate the role of PLD4 for immune system, we examined the mutant mouse thatpossesses premature stop codon at exon3 of PLD4. The mutant mice exhibited peripherallymphoid organ hyperplasia with spontaneous formation of germinal center from their young age.In addition, hypergammaglobulinemia, the production of anti-nuclear antibody, and deposition ofimmunoglobulin within renal glomeruli were observed. Enlarged lymphoid organs were dominatedby increased number of matured B cells, accompanied by higher serum concentration ofBAFF/BlyS, collectively displaying phenotypical similalities with human and mouse model ofsystemic lupus erythematosus. Although the mechanism underlying immunological disturbance ofthe mutant mouse has been still under investigation, our results, in line with human geneticstudies, proposed the importance of PLD4 for maintaining immune tolerance and thedevelopment of autoimmunity.

a90179 [2. Poster or Oral]Clinical and serological features of anti-centromere antibody-positive systemic sclerosis inChinese patients1School of Life Sciences, Fudan University

Jiucun Wang1,Xiangxiang Chen1,Yue Ding1,Haiyan Chu1,Yanyun Ma1,Yuan Li1,Li Jin1,Hejian Zou1

Objectives. To identify the association of clinical and serologic features in anti-centromereantibody (ACA)-positive Chinese patients with systemic sclerosis (SSc).

Methods. Five hundred and five Han Chinese SSc patients from a multicenter study were included.Specific clinical characteristics including autoantibodies and some biochemical indicators weredetected. The demographic, clinical and serologic features were compared between ACA+ andACA- patients.

Results. There were 93 (18.4%) ACA+ patients in the recruited 505 SSc patients, whichconsisted of 73 males and 432 females, 256 lcSSc and 249 dcSSc. Multivariate analysis revealedthat, compared with ACA- patients, ACA+ patients had older age at onset (42.57±12.56 vs38.13±13.73, p<0.001), lower modified Rodnan’s skin score (9.73±8.90 vs 15.34±10.26, p =0.002), and higher frequency of fingertip ulcer (19.1% vs 9.6%, p = 0.029). Besides, ACA+ SSccorrelated positively with hand tumefaction (75.4% vs 58.7%, p = 0.034), skin thickening(70.6% vs 36.3%, p<0.001), and sclerodactyly (80.6% vs 52.3%, p<0.001), while negatively withpigmentation or loss (67.6% vs 82.5%, p = 0.017) and cardiopulmonary involvement (47.8% vs69.1%, p<0.001). Additional findings that decreased IgG level (6.9% vs 0.4) in ACA+ andincreased C3 level (9.5% vs 22.2%) in ACA- group were observed which were rarely reportedpreviously.

Conclusions. ACA may be a marker of poor prognosis in Chinese SSc for fingertip ulcer, handtumefaction, and sclerodactyly. ACA were also a marker of good prognosis for cardiopulmonaryinvolvement.

a90147 [1. Poster only]Anti-activin type IIB receptor intracellular domain (ACVRIIB-in) Ab may relate the cause ofSystemic sclerosis (SSc) with anti-topoisomerase-I antibody (ATA).1Dept. of Respiratory Medicine, Allergy and Rheumatic Disease, Graduate School of Medicine,Osaka UniversityMichihito Katayama1,Toru Hirano1,Yoshimasa Hamano1,Atsushi Kumanogoh1

Anti-centromere Ab (ACA), ATA and anti-RNA polymerase III Ab (ARA) are well knownautoantibodies in SSc. In spite of the longtime efforts of researchers, the relationships betweenthese antibodies and etiology of SSc are still unknown. To identify new autoantibodies which aremore close to the cause of SSc, firstly, we conducted protein array assays and we identifiedanti-ACVRIIB-in Ab as a new candidate autoantibody for SSc. ACVRIIB is a receptor of activinswhich are the members of TGF-β superfamily. TGF-β is known as a key factor in the pathogenesisof SSc. Next, we measured the anti-ACVRIIB-in Ab and serum activin A in SSc patients (n= 20)and healthy controls (HC, n= 20) with ELISA. We found the elevations of anti-ACVRIIB-in Abtiter (AU/ml, SSc 3.7 [2.4, 6.4], HC 2.4 [1.4, 3.4], p= 0.012) and activin A (ng/ml, SSc 0.42[0.38, 0.67], HC 0.23 [0.17, 0.31], p< 0.001) in SSc patients. Then, we measured the anti-MHCclass II DR/ACVRIIB-in complex (DR/ACVRIIB-in) Ab titer with flow cytometry in patients withSSc (ACA+: 7, ATA+: 18, ARA+: 13) and compared the Ab titer and clinical manifestations. Wefound that DR/ACVRIIB-in Ab titers (AU/ml) were high in ATA+ patients (10 [5.2, 12]) and low inARA+ patients (-1.4 [-2.5, -0.33]) (p < 0.001). Those of ACA+ were moderate (6.1 [3.1, 19]) (vs.ATA+ p= 0.012, vs. ARA+ p= 0.003). Any relationship between topoisomerase-I and ACVRIIBisn’t reported until now and there isn’t amino acid sequence homologies between them.Investigation of relationship between SSc and DR/ACVRIIB-in Ab may give a new insight of SSc.

a90129 [2. Poster or Oral]Serum Calprotectin levels in Japanese patients with diffuse and limited systemic sclerosis

1Dept Research and Development, Inova Diagnostics,2Dept Dermatology, Institute MedicalPharmaceutical Health Sciences, Kanazawa University,3Dept Dermatology, University of TsukubaGary L Norman1,Zakera Shums1,Jay Milo1,Susan Encabo1,Yashuhito Hamaguchi2,KazuhiroTakehara2,Michael Mahler1,Manabu Fujimoto3

Introduction:Systemic sclerosis is a systemic autoimmune rheumatic disease resulting inprogressive accumulation of extracellular matrix elements in the skin and internal organs.Diffuse cutaneous SSc(dcSSC) generally progresses more rapidly and with more severeconsequences than the limited cutaneous(lcSSc) form. Biomarkers to predict progression andresponse to therapies are needed. Increased levels of serum calprotectin(sCP), a proteinproduced by cells of myeloid lineage and whose presence is a biomarker of inflammation, havebeen reported in SSc patients. We have examined the levels of sCP in a well-characterized cohortof Japanese SSc patients.Methods: Serum from 225 consecutive SSc patients(108 dcSSc and 117 lcSSc) meeting AmericanCollege Rheumatology criteria for SSc or having ≥3 CREST features and anti-centromerepositivity visiting Kanazawa Univ. Hosp. were tested with 84 healthy controls(HC). sCP wasmeasured using a research use only calprotectin ELISA (Inova Diagnostics, USA).Results: Overall, 34% (76/225) of SSc patients had values ≥30 ng/ml. Patients with dcSSc weremore than twice as likely as those with lcSSc(49.1% vs.19.7%) to have levels ≥30ng/ml. Medianvalues of sCP were 29.6, 16.9 and 10.6 ng/ml for dcSSc, lcSSc and HC(p<0.0001).Conclusions: sCP levels are increased in patients with SSc and the frequency of increased levelsis significantly higher in dcSSc compared to lcSSc (p<0.001). sCP is a potential new non-invasivebiomarker for assessing patients with SSc, although additional studies will be necessary toassess its clinical utility.

a90097 [1. Poster only]Biomaker of YKL-40 for the Presence of Interstitial Pneumonia and Pulmonary ArterialHypertension in Systemic Sclerosis1Division of Rheumatology, Department of Internal Medicine, Hyogo college of Medicine

Tetsuya Furukawa1,Kiyoshi Matui1,Masayasu Kitano1,Yuichi Yokoyama1,MasahiroSekiguchi1,Naoto Azuma1,Hajime Sano1

Background: Systemic Sclerosis (SSc) is an intractable connective tissue disease that causesfibrosis of the skin and organs, and its prognosis is affected by interstitial pneumonia (IP) andpulmonary arterial hypertension (PAH). Chitinase-like protein YKL-40 has been implicated ininflammation and tissue remodeling. A previous study in our department demonstrated elevatedblood levels of YKL-40 in SSc patients. Methods: Subjects comprised 33 SSc patients examined inour department between August 2014 and March 2016 who had no complications (Group1), 21patients with IP (Group2), 4 patients with PAH (Group3), and 13 patients with both IP and PAH(Group4), as well as a control group of 16 healthy individuals. Immunohistochemical staining(IHC) was performed on normal skin and stored samples of SSc skin. Serum levels of YKL-40 weremeasured by ELISA, and age percentile strata of YKL-40 were calculated. Results: Normal skinremained unstained by IHC, whereas the subcutaneous vascular endothelium was prominentlystained and the epithelium was lightly stained in SSc. Serum YKL-40 levels were 73.6±45.4 ng/ml(YKL-40 age percentile 53.7 ± 29.3 ) in Group 1, 122.1 ± 89.0 ng/ml ( 68.8 ± 27.9 ) in Group 2,183.5 ± 120.4 ng/ml ( 90.7 ± 11.1 ) in Group 3, and 225.9 ± 161.0 ng/ml ( 89.5 ± 16.3 ) in Group4, all significantly elevated above the level of 38.1 ± 18.1 ng/ml ( 24.9 ± 18.5 ) in healthyindividuals, and highest in Groups 2, 3 and 4. Conclusion: YKL-40 may be useful as a marker offibrosis in uncomplicated SSc.

a90100 [2. Poster or Oral]Efficacy of autologous stem cell transplantation in the treatment of systemic sclerosis

1Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate Schoolof MedicineToshio Odani1,Shinsuke Yasuda1,Masaru Kato1,Kenji Oku1,Toshiyuki Bohgaki1,OlgaAmengual1,Tetsuya Horita1,Tatsuya Atsumi1

Objectives: We aimed to elucidate the efficacy and safety of autologous stem celltransplantation (HSCT) for severe systemic sclerosis (SSc).Patients and Methods: In our phase 2 trial, a total of 14 early diffuse SSc patients wereenrolled, Median age was 44.5 years (range 19-57), disease duration was 21 months (8-36) andmodified Rodnan skin score (mRSS) was24.5 (13-38). Peripheral blood stem cells (PBSCs) werecollected by cyclophosphamide (CY: 2 g/m2) for 2 days, followed by administration ofgranulocyte colony stimulating factor. After collecting PBSCs, patients were treated with CY(50 mg/kg) for 4 days followed by HSCT. The primary outcome was improvement at 1 year’sfollow-up, defined as a decrease >25% from baseline in mRSS. The secondary outcome wasimprovement of 2 years mRSS, pulmonary function and high resolution computed tomography(HRCT) findings. We also assessed the long term efficacy and safety after HSCT.Results: Among 14 SSc patients, one died due to treatment related mortality. Thirteen patientswere followed-up at least 2 years after HSCT. A significant improvement in mRSS was achievedin 77% of patients at 1 year’s follow-up, in 77% at 2 years and in 67% at 4 years. Althoughpulmonary function parameters did not change significantly, HRCT findings were improved in63%of patients at 2 years and in 60% at 4 years. After a median follow-up of 4.5 (2-9.3) years,no one died and 50% of patients had event-free survival without mortality, relapse orprogression at 4 years.Conclusions: This study confirmed the efficacy of HSCT in patients with severe SSc.

a90178 [2. Poster or Oral]Regenerating Gene Protein as a Novel Autoantigen in the Pathogenesis of Sjögren’s Syndrome

1The Center for Rheumatic Diseases, Nara Medical University,2Department of General Medicine,Nara Medical University,3Department of Diagnostic Pathology, Nara Medical University,4

Department of Biochemistry, Nara Medical UniversityTakashi Fujimoto1,Takanori Fujimura1,Kiyomi Yoshimoto2,Maiko Takeda3,Akiyo Yamauchi4,AsakoItaya-Hironaka4,Yasuhito Tanaka1,Shin Takasawa4

Background: Sjögren’s syndrome (SS), an autoimmune disease characterized by exocrine glanddysfunction. The regenerating gene, Reg, was originally isolated from a rat regenerating isletcDNA library, and its human homologue was named REG Iα. Recently, we discovered that REG IαmRNA, as well as its product, was overexpressed in ductal epithelial cells in the salivary glandsof SS patients. Objective: To measure autoantibodies against REG Iα in the sera of SS patients,and explore the differences between patients who were positive and negative for the anti-REGIα antibodies (anti-REG). Furthermore, to elucidate the role of cytokines and the subsequentintracellular mechanism for induction of REG Iα in the salivary glands. Results: Anti-REG in thesera were found in 11% (13 of 117) of SS and 2.3% (6 of 271) of control subjects (P = 0.0001).The anti-REG positive showed significantly lower saliva secretion (0.7 g/2 min) than thenegative (2.0 g/2 min) in Saxon test (P = 0.007). Human and rat salivary ductal cells weretreated with several cytokines (IL-6, IL-8, etc.), and the transcriptional activity was measuredby luciferase assay. IL-6 stimulation significantly enhanced the REG Iα promoter activity in bothcells. Deletion analysis revealed that the region of the REG Iα gene containing a consensussequence for STAT binding was responsible for the promoter activation by IL-6. Conclusion: ThisIL-6/STAT dependent REG Iα induction and the following autoimmunity to REG Iα may play animportant role in the pathogenesis of SS.

a90115 [2. Poster or Oral]Serum ganglionic acetylcholine receptor antibodies in patients with Sjögren’s syndrome

1Department of Neurology, Kumamoto University Hospital, Kumamoto, Japan,2Department ofClinical Research, Nagasaki Kawatana Medical Center, Nagasaki, Japan,3Department of Neurology,Nagasaki Kawatana Medical Center, Nagasaki, JapanAkihiro Mukaino1,Shunya Nakane1,Osamu Higuchi2,Yasuhiro Maeda2, 3,Waka Sakai2, 3,HidenoriMatsuo3,Yukio Ando1

Background: It remains unclear whether autonomic neuropathy in Sjögren’s syndrome (SS) isrelated to anti-ganglionic acetylcholine receptor (gAChR) antibodies.Objective: To investigate the relationship between autonomic dysfunction in SS and anti-gAChRantibodies.Methods: We measured anti-α3 and β4 gAChR antibodies by luciferase immunoprecipitationsystems (LIPS) test. (1) First, we tested for the presence of gAChR antibodies in serum samplesfrom 39 patients with SS (absent information regarding autonomic symptoms) and healthyvolunteers. (2) Second, we measured gAChR antibodies in serum samples from 10 SS patients whoshowed autonomic symptoms. (3) Finally, we combined the data of seropositive SS patients withautonomic symptom from the first study with all of the patients from the second study, andanalyzed the clinical features.Results: (1) Anti-α3 and β4 gAChR antibodies were detected in 23.1% (9/ 39). Five of nine SSpatients had autonomic symptoms. (2) Anti-α3 and β4 gAChR antibodies were also detected in80.0% (8/ 10). These seropositive patients had predominant and severe autonomic symptoms andhad autonomic neuropathy. (3) Thirteen of fifteen SS patients with autonomic symptoms(86.7%) were seropositive for anti-gAChR antibodies, and we confirmed sicca complex,orthostatic hypotension, upper and lower gastrointestinal symptoms, and bladder dysfunction athigh rates.Conclusion: The possibility of anti-gAChR antibodies aiding the diagnostics of SS with autonomicdysfunction.

a90040 [1. Poster only]Alteration of clinical course by Immunosuppressants in children with positive anti-Ro antibodiesand with chronic nonspecific complaints1Department of Pediatrics, Nippon Medical School

Yasuhiko Itoh1,Tomoko Shigemori1,Shingo Yamanishi1,Hidehiko Nagasaki1,Yusuke Ozaki1,YujiroTenable1,Tohru Igarashi1

Anti-Ro antibodies are occasionally found in children with chronic nonspecific complaints (CNC)such as fatigue. Although a few of them showed positive lip biopsy findings equivalent to thatof Sjögren's Syndrome (SS), the majority of such children exhibited no evidence of SS. To clarifythe pathogenic role of anti-Ro in the future development of dryness, those children have been 　followed for more than 10 years, both clinically and immunologically.The patients included in this study were : 1) 27 children with CNC, with anti-Ro, and with 　negative lip biopsies; 2) 10 children with CNC, with anti-Ro, and with positive biopsies; 3) 9children with SLE and with positive anti-Ro; 4) three children with MCTD and with positive anti-Ro. Anti-Ro antibodies were measured by using either the Ouchterlony, ELISA, Western blot or, insome cases, RNA-immunoprecipitation.Twenty-three of 27 patients in group 1 showed no evidence of SS even after more than 10 years.Most of the patients in group 2 who had not been treated by immunosuppressants have graduallydeveloped dryness. Patients who had been treated have developed no or little dryness. Patientsin the groups 3 and 4 have developed no dryness. All patients in groups 3 and 4 had been treatedby immunosuppressants against their basic diseases and developed no dryness. Most patientswith only CNC treated with immunosuppressants did not develop dryness.These results suggest that there may be a chance to prevent SS with immunosuppressants inchildren with positive anti-Ro if the treatment is initiated before they develop dryness.

a90123 [2. Poster or Oral]Detection of ANCA: a multicenter EUVAS evaluation of the value of indirect immunofluorescenceversus antigen-specific immuno-assays1Central Diagnostic Laboratory, Maastricht University Medical Center, TheNetherlands,2Department of Rheumatology and Immunology, Klinikum Bad Bramstedt,Germany,3Department of Autoimmune Serology, Statens Seruminstitute, Copenhagen,Denmark,4Rheumazentrum Schleswig-Holstein Mitte, Neumünster, Germany,5Department ofInternal Medicine, Section Nephrology and Immunology, Maastricht University Medical Center,Maastricht, The Netherlands,6Department of Rheumatology, Rigshospitalet, Copenhagen,Denmark,7Clinical Department of Laboratory Medicine, University Hospitals Leuven, 3000Leuven, Belgium; Department of Cardiovascular Sciences, KU Leuven, Leuven ,Belgium,8ClinicalDepartment of General Internal Medicine, Research Department of Microbiology and Immunology,Laboratory of Clinical Infectious and Inflammatory Disorders, University Hospitals Leuven,Leuven, Belgium,9Maastricht University, The Netherlands,10Department of Microbiology andImmunology, KU Leuven, and Department of Laboratory Medicine, University Hospitals Leuven,Jan Damoiseaux1,Elena Csernok2,Niels Rasmussen3,Frank Moosig4,Pieter van Paassen5,BoBaslund6,Pieter Vermeersch7,Daniel Blockmans8,Jan Willem Cohen Tervaert9,Xavier Bossuyt10

Objective: This multicenter study was performed to evaluate the diagnostic accuracy of a widespectrum of novel technologies nowadays available for detection of myeloperoxidase (MPO)- andproteinase 3 (PR3)-antineutrophil cytoplasmic antibodies (ANCA).Methods: Sera (obtained at the time of diagnosis) from 251 patients with ANCA associatedvasculitis (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis(MPA), and from 924 disease controls were tested for the presence of C-/ P- and /A-ANCA byindirect immunofluorescence (IIF) (at two sites) and for the presence of PR3- and MPO-ANCA by8 different immuno-assays.Results: The area under the curve (AUC) of the receiver operating characteristic curve todiscriminate AAV from controls was 0.923 (95% CI 0.902-0.944) and 0.843 (95% CI 0.814-0.871) for the two IIF methods. For the antigen-specific immuno-assays, the AUC varied between0.936 (95% CI 0.912-0.960) and 0.959 (95% CI: 0.941-0.976), except for one immuno-assay forwhich the AUC was 0.919 (95% CI: 0.892-0.945).Conclusion: Our comparison of various ANCA detection methods showed (i) large variabilitybetween the two IIF methods tested and (ii) a high diagnostic performance of PR3- and MPO-ANCA by immuno-assay to discriminate AAV from disease controls. Consequently, dualIIF/antigen-specific immuno-assay testing of each sample is not necessary for maximaldiagnostic accuracy. These results indicate that the current international consensus on ANCAtesting for AAV needs revision.

a90114 [1. Poster only]Effects of urea treatment on the antineutrophil anticytoplasmic antibodies (ANCA)

1Immunology Department, Hospital Dr. Oscar Alende, Mar del Plata, Argentina,2LaboratoryDepartment, Hospital Don Victorio Tetamanti,Mar del Plata, ArgentinaEstela Motta1,Laura Dominguez1,Paola Rolandi2,Marcelo Zamora1

Objective: To evaluate the avidity of anti-PR3 and anti-MPO and its association with clinicalstatus. Materials and Methods: The avidity was studied in 9 patients who had positive results byELISA, 7 of them with anti-PR3 and 2 with anti-MPO. Low avidity antibodies were stripped bywashing the ELISA wells with urea and the IIF was performed with urea in ethanol and formalin-fixed neutrophils in 2 patients. Results: 5/7 samples anti-PR3 positive presented avidity lessthan 30% (1 ulcerative colitis, 1 granulomatosis with polyangiitis (GPA), 1 C-ANCA vasculitis, 1lung disease and 1 unknown). The other 2 samples showed avidity higher than 80% (1 C-ANCAvasculitis and 1 GPA). The 2 samples with anti-MPO had similar avidities with 50% average (1rapidly progressive glomerulonephritis and 1 unknown). One of the patients with anti-PR3 lowavidity (11.9%) had P-ANCA in ethanol-fixed slide and C-ANCA in formalin slide. After ureatreatment no pattern was observed in ethanol slides meanwhile in formalin the titer decreased.In contrast, in a patient with anti-PR3 high avidity (86.3%) no change was observed in C-ANCApattern with urea. Conclusions: The avidity of anti-PR3 is not related to the clinic status. Thepresence of P-ANCA with PR3 specificity could be associated with low avidity antibodies. In thepatient studied, a different performance was observed in the avidity of antibodies in formalinslide compared with ethanol slide and ELISA.

a90025 [2. Poster or Oral]Myeloperoxidase/HLA class II complexes as targets for autoantibodies in microscopic polyangiitis

1Department of Rheumatology and Clinical Immunology,Graduate School of Medicine, KyotoUniversity,2Laboratory of Immunochemistry, World Premier International (WPI) ImmunologyFrontier Research Center,3Department of Immunochemistry, Research Institute for MicrobialDiseases, Graduate School of Medicine, Osaka University,4Department of Geriatric Medicine andNephrology, Osaka University Graduate School of Medicine,5Department of Medicine II, HokkaidoUniversity Graduate School of Medicine, Sapporo, Japan,6Center for Genomic Medicine, GraduateSchool of Medicine, Kyoto UniversityRyosuke Hiwa1, 2, 3,Koichiro Ohmura1,Noriko Arase2, 3,Hui Jin2, 3,Kouyuki Hirayasu2, 3,MasakoKohyama2, 3,Tadahiro Suenaga2, 3,Fumiji Saito2, 3,Hirotsugu Iwatani4,Tatsuya Atsumi5,ChikashiTerao6,Tsuneyo Mimori1,Hisashi Arase2, 3

Autoantibodies against myeloperoxidase (MPO) expressed in neutrophils play an important rolein the pathogenesis of microscopic polyangiitis (MPA). However, MPO is not expressed on thesurface of normal neutrophils and thus molecular mechanisms how autoantibodies cause MPAhave remained unclear. We have recently found that cellular misfolded proteins are transportedto the cell surface by HLA class II molecules and the misfolded proteins associated with HLAclass II molecules are targeted by autoantibodies in rheumatoid arthritis and antiphospholipidsyndrome, suggesting that HLA class II molecules play an important role in autoantibodyrecognition. Here, we demonstrate that MPO is expressed on the cell surface by associating withHLA class II molecules. MPO expressed on the cell surface complexed with HLA class II moleculeswere recognized by MPO-anti-neutrophil cytoplasmic antibody (ANCA). Furthermore,autoantibody binding to MPO/HLA-DR complexes was significantly correlated with odds ratio forMPA by each HLA-DR allele. Indeed, MPO/HLA class II complexes were detected in neutrophilsfrom MPA patients as well as cytokine-stimulated neutrophils from healthy donors with MPA-susceptible HLA-DR allele. Furthermore, cells expressing MPO/HLA-DR complex was activated byMPO-ANCA. Our findings suggest that MPO complexed with HLA class II molecules are involved inthe pathogenesis of MPA as targets for MPO-ANCA.

a90059 [1. Poster only]PR3-ANCA in children and adolescents with inflammatory bowel disease

1Institute of Immunology, Medical Faculty Carl Gustav Carus, Technical University Dresden,Dresden, Germany,2University Children's Hospital, University Hospitals Carl Gustav Carus,Technical University Dresden, Dresden, GermanyNadja Roeber1,Josefine Jahn2,Karsten Conrad1,Martin W. Laass2

The aim of this study was to determine the prevalence and relevance of PR3-ANCA in childrenand adolescents with inflammatory bowel disease (IBD). Sera from 326 patients with IBD,including 187 Crohn’s disease (CD), 121 ulcerative colitis (UC), and 18 indeterminate colitis (IC)patients, and 164 controls were tested for ANCA by indirect immunofluorescence, for PR3-ANCAby chemiluminescence immunoassay (CIA), and for ASCA-IgA by ELISA. Their association withclinical phenotypes according to Paris classification, liver manifestations, especially primarysclerosing cholangitis (PSC) and overlap of PSC with autoimmune hepatitis (AIH), wereinvestigated. The prevalence for UC, CD and IC patients was 41%, 11% and 11%, respectively,whereas none of the controls was positive. In contrast to ANCA associated vasculitides, therewas no correlation between ANCA pattern and PR3-ANCA results obtained by CIA. PR3-ANCA wasmore prevalent and the titers were higher among UC patients compared to CD patients. Anextensive inflammation in the form of backwash ileitis and pancolitis was associated with higherprevalence of PR3-ANCA in children and adolescents with UC. Furthermore, the prevalence ofPR3-ANCA was higher in UC patients with PSC, PSC/AIH overlap and elevated liver enzymes thanin those without additional liver manifestation. In summary, the CIA test for PR3-ANCA can aidin the diagnosis of IBD and, in combination with other IBD associated antibodies like ASCA, helpto differentiate early between UC and CD. Additionally, PR3-ANCA may indicate livermanifestations in IBD patients.

a90141 [1. Poster only]DNA-BOUND LACTOFERRIN IS THE MAJOR TARGET FOR P-ANCA IN ULCERATIVE COLITIS

1EUROIMMUN AG, Institute for Experimental Immunology, Lübeck, Germany

Fechner Kai1,Komorowski Lars1,Teegen Bianca1,Niemann Stephanie1,Probst Christian 1,StöckerWinfried1

Different antigens have been proposed to be a target for anti-neutrophil perinuclearcytoplasmic antibodies (p-ANCA), which are present in up to 67% of ulcerative colitis cases (UC).We found a method to clearly prove that UC-associated p-ANCA are predominantly directedagainst DNA-bound lactoferrin.

Slides with ethanol-fixed human granulocytes were incubated in 1 M MgSO4 to strip off their p-ANCA targets, including lactoferrin. Subsequently, the cells were repleted by incubation withlactoferrin purified from human colostrum. Success of lactoferrin immobilization was controlledby incubation with an antibody against recombinant lactoferrin. The cells were used in IIFT, inparallel to ethanol-fixed granulocytes, to detect ANCA by a standard procedure. Sera of 39patients with UC, 96 with Crohn’s disease (CD), and from 50 healthy blood donors (HBD) wereanalysed.

Using standard ethanol-fixed granulocytes, p-ANCA were detected with a prevalence of 72% inUC (CD 11%, HBD 0%). Reactions with the lactoferrin-repleted substrate were as follows: UC72%, CD3%, HBD 0%. Of the UC sera tested p-ANCA negative on standard ethanol-fixed granulocytes,55% reacted positive with the lactoferrin-repleted substrate. Therefore, with a combination ofboth substrates, the sensitivity of IIFT was increased to 87% for UC.

By recruitment of the new lactoferrin-repleted substrate additionally to ethanol-fixedgranulocytes, the sensitivity of p-ANCA can be raised by 15% in UC without a significantreduction of specificity.

a90031 [1. Poster only]IL-12p40 in Pathophysiology of Takayasu Arteritis

1Department of clinical immunology and rheumatology, Kyoto University, Kyoto-city,Japan,2Center for Genomic Medicine. Kyoto-University, Kyoto, JapanToshiki Nakajima1,Hajime Yoshifuji1,Chikashi Terao2,Kosaku Murakamai1,Nobuo Kuramoto1,RanNakashima1,Yoshitaka Imura1,Masao Tanaka1,Koichiro Ohmura1,Tsuneyo Mimori1

【Backgrounds】Takayasu arteritis (TAK) is a type of large vessel arteritis, which affects aortaand its main branches. We previously found a SNP rs6871626 in IL12B region as a susceptibilitygene to TAK by a genome-wide association study. IL12B encodes IL-12p40, but there has been noinvestigation of IL-12p40 in TAK.【Objectives】1) To investigate the expression of IL-12p40, 2) To examine the influence of SNPrs6871626 on the cytokine production.【Methods】1) Plasma was collected from 40 TAK patients and 19 healthy controls (HCs), andthe plasma concentration of IL-12p40 was measured. 2) Monocytes were obtained from 18 TAKpatients and 14 HCs and incubated with IL-6, IL-10 and M-CSF. Then the concentration of IL-12p40 was measured after stimulating INF-γ and LPS.【Results】Plasma concentration of IL-12p40 in TAK patients was significantly higher than inHCs (p=0.031). Plasma IL-12p40 of TAK patients was correlated weakly with ESR (ρ=0.40,p=0.018) and dose of prednisolon (ρ= -0.34, p=0.032). IL-12p40 in supernatant tended to behigher of TAK patients than of HCs . After categorization of TAK patients according to the riskallele at rs6871626, IL-12p40 in supernatant was significantly higher of TAK patients with riskallele than of patients without risk allele (p=0.044).【Conclusion】IL-12p40 may be play an important role in the pathophysiology of TAK. The riskallele of IL12B can influence on the IL-12p40 production of TAK patients.

a90043 [1. Poster only]Three cases of varied size vessel vasculitis; Large vessel vasculitis with small or medium vesselinvolvement.1Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate Schoolof Medicine, Sapporo, JapanMichihiro Kono1,Shinsuke Yasuda1,Hiroyuki Nakamura1,Sanae Shimamura1,Yuka Shimizu1,MasaruKato1,Kenji Oku1,Toshiyuki Bohgaki1,Tetsuya Horita1,Tatsuya Atsumi1

Background: Classification criteria of vasculitis are based on the predominantly involved type ofvessels. However, it is very important to realize that vasculitis of each category can affect anysize artery. We showed clinical course of three cases complicated with varied size vesselvasculitis.Case 1: A 31-year-old woman presented with saddle nose, scleritis and multiple skin ulcers inboth lower legs, was diagnosed as small vessel vasculitis. Despite immunosuppressive therapy,aneurysms in left posterior tibial and right peroneal artery and a threatened rupture of thoracicaortic aneurysm were revealed. Aortic root replacement was performed and she was diagnosed ashaving Takayasu arteritis based on pathological findings of her artery.Case 2: A 68-year-old woman was diagnosed as anti-neutrophil cytoplasmic antibody (ANCA)associated vasculitis (AAV) with recurrent optic neuritis and pleuritis with positivemyeloperoxidase ANCA. She was treated with prednisolone (PSL) plus intravenouscyclophosphamide. During the tapering of PSL, computed tomography newly demonstrated aorticwall thickening, which resulted in a diagnosis of aortitis complicated with AAVCase 3: A 74-year old woman was presented with otitis media, engorgement of superficialtemporal arteries and pachymeningitis. The left temporal artery biopsy was performed, resultingin a diagnosis of giant cell arteritis.Conclusion: Our cases remind physicians of the importance in evaluating systemic lesionregardless of the mainly affected vessel size, when treating patients with systemic vasculitis.

a90131 [2. Poster or Oral]Regulation of autoimmune responses through TRIM-regulated precision autophagy and secretion.

1Department of Nephrology, Osaka University,2Department of Molecular Genetics &Microbiology, University of New MexicoTomonori Kimura1,Vojo Deretic2

Autophagy has multiple, and sometimes conflicting, immunological manifestations, includingsuppression or activation of inflammasome and type I interferon response through mechanismsthat are not fully understood. Here we report that a subset of TRIMs dictate immune responsethrough autophagy. Among TRIMs, TRIM20 and TRIM21, two of strongly disease-related membersof the large family of TRIM (tripartite motif) proteins, act as receptor-regulators to bridgespecific targets for autophagic degradation directly to autophagic machinery; by beingrecognized by TRIMs, the targets of autophagy (the inflammasome components NLRP3, pro-caspase 1 and NLRP1 for TRIM20, and IRF3 for TRIM21, respectively) were directly recruited toautophagic machinery for the degradation, and this process is mitigated by the FamilialMediterranean Fever-causing mutations of TRIM20. On the other hand, IL-1beta, a leaderlessprotein, is recognized by TRIM16 for autophagic secretion. These findings reveal a previouslyunknown mode of action of TRIMs as receptor-regulators for highly-selective type of autophagy(termed ‘precision autophagy’) and as receptors for unconventional secretion of cytokines.Regulations of TRIMs would provide great opportunity for the regulation of autoimmunediseases.

a90118 [2. Poster or Oral]Histone H1: a novel alarmin-like mediator and molecular target for immune regulation

1Graduate Institute of Clinical Medical Sciences, Chang Gung University College ofMedicine,2Liver Transplantation Center and Department of Surgery, Kaohsiung Chang GungMemorial Hospital,3Basic Medical Science of Nursing, Department of Nursing, Josai InternationalUniversity,4Hiroshima Research Center for Healthy Aging (HiHA), Department of MolecularBiotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima UniversityToshiaki Nakano1, 2,Shigeru Goto2, 3,Takeshi Goto3,Seiji Kawamoto4,Chao-Long Chen2

The release of nuclear antigens from damaged cells and/or activated immune cells, such asdendritic cells (DCs) and macrophages, into the blood stream has been associated with theprogression of several diseases, including infectious diseases, proinflammatory disorders,malignancies, and traumas. In previous studies, we have demonstrated elevated levels ofcirculating histone H1 and high-mobility group box 1 (HMGB1) in the course of liver transplantrejection. These nuclear antigens may act as “nuclear weapon” in the induction ofproinflammatory immune responses. Thus prevention of those nuclear antigen-driven innateimmune signals would be a therapeutic target for proinflammatory disorders. We havedemonstrated the transient induction of autoantibody (auto-Abs) against histone H1 andHMGB1, which may be related to long-term liver allograft acceptance. Our in vitro studies havealso elucidated the unique role of nuclear proteins and their Abs in both individual cells cell-to-cell interactions among DCs, T cells, mast cells and mesenchymal stem cells. In this presentation,we would like to discuss the impact of nuclear histone H1 as a novel alarmin-like endogenousmediator and its potential as a molecular target for amelioration of transplant rejection,fibrosis and allergic responses.

a90145 [1. Poster only]Extracellular linker histone H1 deteriorates IgE-mediated allergic hypersensitivity

1Hiroshima Research Center for Healthy Aging (HiHA), Department of Molecular Biotechnology,Graduate School of Advanced Sciences of Matter, Hiroshima University, Japan,2GraduateInstitute of Clinical Medical Sciences, Chang Gung University College of Medicine, Taiwan,3LiverTransplantation Center and Department of Surgery, Kaohsiung Chang Gung Memorial Hospital,Taiwan,4Department of Nursing, Josai International University, JapanTakashi Fujimura1,Toshiaki Nakano2, 3,Shigeru Goto3, 4,Takeshi Goto4,Kazuhisa Ono1,Chao-LongChen3,Seiji Kawamoto1

Nuclear materials act as autoantigens to trigger proinflammatory immune responses.Extracellular histones are known to be an alarmin to induce proinflammatory responses. Here, weprovide evidence that extracellular linker histone H1 directly acts on mast cells to enhance IgE-mediated immediate hypersensitivity. We found that systemic immunization by antigen withalum, but not by alum only, increased circulating histone H1 level. Interestingly, histone H1-specific monoclonal antibody not only suppressed IgE-mediated mast cell degranulation, but alsoameliorated passive cutaneous anaphylaxis (PCA) and allergic rhinitis. Conversely, co-sensitization with histone H1 exacerbated mast cell degranulation, PCA, and allergic rhinitis.These results suggest that extracellular linker histone H1 represents an alarmin to amplify mastcell-mediated hypersensitivity. Furthermore, blocking of the histone H1 offers alternativetherapeutic strategy for intervention against type I allergic inflammation.

a90151 [1. Poster only]Anti-nuclear antibody positive rate in familial Mediterranean fever patients in Kagawauniversity hospital1Department of Internal Medicine, Hematology, Rheumatology and Respiratory Medicine, Facultyof Medicine, Kagawa University,2Departments of Pediatrics, Kyoto University Graduate School ofMedicine, Kyoto UniversityMiharu Izumikawa1,Tomohiro Kameda1,Shusaku Nakashima1,Hiromi Shimada1,Hiroki Ozaki1,RisaWakiya1,Atsushi Kondo1,Norimitsu Kadowaki1,Ryuta Nishikomori2,Dobashi Hiroaki1

Purpose: Autoinflammatory diseases are a relatively new category of diseases that are differentfrom autoimmune diseases and it has been defined that anti-nuclear antibody (ANA) is not foundin autoinflammatory diseases patient. However, autoimmune and autoinflammatory diseasesshare common characteristics. Familial Mediterranean fever (FMF) is a disease of the frequencyhighest in autoinflammatory diseases and a single gene disorder characterized by recurrent,self-limiting episodes of fever accompanied by peritonitis, pleuritis, synovitis, and erysipelas-like skin lesions. FMF is an important discrimination disease in collagen disease area though FMFis a rare disease. We think that each case's detailed analysis and examination are useful for thediagnosis and treatment in the future.Materials and methods: Patients were diagnosed accordingto the Tel-Hashomer criteria by March, 2016 in this hospital.We collected data that relates toMEFV gene mutations, CRP level, serum amyloid value, and ANA and efficacy of colchicine withthe clinical records of them, and did the statistical examination.Results: 6 patients were MEFV-mutation positive. The average of onset age was 25.14±13.78 years old. ANA were present in28.6 %(at a dilution of 1:80 or 1:40) and 14.2%(at a dilution of 1:160 or more).Conclusion: Thepossibility that the FMF patient has ANA might be the same as it of healthy person.

a90096 [2. Poster or Oral]A case of drug hypersensitivity mimicking adult-onset still's disease

1Immuno-Rheumatology Center, St. Luke's International Hospital

Tokutaro Tsuda1,Masato Okada1

Case is 65 year-old male. He has past histories of hyperlipidemia, hypertension and transientcerebral ischemic attack . Since Mar. 2010, he was prescribed low dose aspirin, rosuvastatin andunisia combination tablet (candesartan and amlodipine). In the beginning of June 2015, he visitedto the previous hospital because of general malaise, polyarthralgia and persistent fever for 2weeks. Lab. data showed elevated inflammatory markers(WBC 20000/μＬ, neutrophils 88%, CRP19.74mg/dl) and liver dysfunction (AST 131 IU/L, ALT 196 IU/L, LDH 273 IU/L). He was admittedto the department of gastroenterology, but various viral titers were all negative, and liverbiopsy revealed only mild inflammatory change. Although the gastroenterologists stopped allmedication, the inflammation and liver enzyme elevation remained. Furthermore they foundhyperferritinemia (2127 ng/ml), so he was diagnosed with adult-onset still’s disease.He was referred to us in the end of July 2015. Until then his clinical symptoms and lab dataabnormalities were spontaneously improved without immunosuppressive therapy. In Sep. 2015serum ferritin was once normalized, but CRP and ferritin were getting worse after telmisartanhad been introduced by another cardiologist in Oct. 2015. We recommended stopping allmedication again in Jan 2016, then his CRP and ferritin levels were completely normalized for amonth. We performed drug-induced lymphocyte stimulation tests for telmisartan and Unisia,which revealed all negative, but stimulation indices were slightly high (166% and 133%,respectively).

a90071 [1. Poster only]Leucine rich α-2 glycoprotein promotes lung fibrosis by modulating TGF-β signaling

1Laboratory of Immune Signal, National Institutes of Biomedical Innovation, Health andNutrition,2Department of Animal Bioscience, Nagahama Institute of Bio-Science andTechnology,3Division of Translational Research, Integrated Center for Advanced MedicalTechnologies (ICAM-Tech), Kochi Medical School, Kochi UniversityHiromi Honda1,Minoru Fujimoto1,Shintaro Nomura2,Ayako Masumoto2,Chisato Nakayama2,HayatoUrushima1,Tomoharu Ohkawara1,Satoshi Serada1,Tetsuji Naka1, 3

ObjectiveLung diseases relating to collagen disorder often accompanies fibrosis and TGF-b is one of theessential mediators in tissue fibrosis. In this study, we aimed to investigate the involvement ofLRG, recently reported as a modulator of TGF-b, in a murine model of lung fibrosis.MethodsMice were intratracheally treated with bleomycin and fibrosis induction was evaluatedhistochemically and biochemically. TGF-β signaling was investigated using L929 mouse fibroblastcell line.ResultsIntratracheal administration of bleomycin in wild type (WT) mice increased LRG protein inbronchial alveolar lavage fluids (BALF) and lung tissues. When compared to WT mice, fibrosis wassignificantly inhibited in LRG knockout (KO) mice. Although there was no significant differencein BALF TGF-b levels between WT and LRG KO mice, phosphorylation and nuclear translocation ofSmad2 protein were attenuated in the lung of LRG KO mice. In vitro experiments using L929revealed that stably-expressed LRG or recombinant LRG could enhance TGF-b-induced Smad2phosphorylation and the expression of downstream genes. Knockdown of endoglin, one of TGF-b-related receptor that interacts with LRG failed to reverse enhanced Smad2 phosphorylation byLRG.ConclusionThese data suggest that LRG enhances TGF-b signaling in fibroblasts by an endoglin-independentmechanism, leading to promote fibrosis in a murine model of lung fibrosis.

a90112 [1. Poster only]Anti-GM-CSF/MHC class II complex antibody in autoimmune pulmonary alveolar proteinosis

1Dept. of Respiratory Medicine, Allergy and Rheumatic Disease, Graduate School of Medicine,Osaka University,2National Hospital Organization Kinki-Chuo Chest Medical Center,3Laboratoryof Immunochemistry, World Premier International Research Center (WPI), Immunology FrontierResearch Center, and Department of Immunochemistry, Research Institute for Microbial Disease,Osaka University,4Biostatics & Data Management, National Cerebral and Cardiovascular CenterYoshimasa Hamano1,Hiroshi Kida1,Masaki Hirose2,Akiko Matsumuro2,Tadahiro Suenaga3,HanaSarashina1,Toshimitsu Hamasaki4,Yoshikazu Inoue2,Hisashi Arase3,Atsushi Kumanogoh1

Inhibition of alveolar macrophage maturation by anti-GM-CSF antibody is known as themechanism of autoimmune pulmonary alveolar proteinosis (aPAP). However, the titer of anti-GM-CSF autoantibody does not reflect the disease severity score (DSS). Recently, we have reportedthat MHC class II presents misfolded self-proteins on the cell surface without processing topeptides, and protein/MHC class II complex could be the target of autoantibodies in autoimmunediseases. We examined whether this mechanism also works for GM-CSF. Full-sized GM-CSFbinding to HLA-DR was detectable on the cell surface, when GM-CSF and HLA-DR plasmids weretransfected into 293T cells. The efficiency of GM-CSF presentation was different depending onapplied HLA-DR alleles. Sequencing analysis disclosed two critical amino acid positions of HLA-DR, β47 and β71. Immunohistochemistry showed that HLA-DR was highly expressed in alveolarmacrophages and respiratory epithelial cells in aPAP lung. Anti-GM-CSF/HLA-DR complexautoantibody by flow-cytometry was found to distinguish patients with aPAP from healthycontrols (sensitivity 100%; specificity 90.5%). Although anti-GM-CSF autoantibody levelsmeasured by ELISA were not co-related, anti-GM-CSF/HLA-DR complex autoantibody levels weresignificantly co-related with DSS in 21 patients with aPAP. From these data, we conclude thatthe autoantibody against GM-CSF presented by MHC class II is the pathogenetic autoantibody inaPAP. We also suggested the possibility of involvement of certain specific MHC class II alleles inthe onset of aPAP.

a90045 [1. Poster only]The clinical significance of autoantibody-producing CD180-lacking plasmablasts in systemicautoimmune and immune-based inflammatory disorders

1Department of Rheumatology, Saga University

Syuichi Koarada1,Akihide Ohta1,Yoshifumi Tada1

CD180 (RP105) molecule expressed on mature B cells is a Toll-like receptor (TLR) associatedmolecule. RP105 is deeply associated with B cell function, survival and death. RP105-lacking Bcells produce autoantibodies, including anti-ds-DNA antibodies in Systemic Lupus Erythematosus(SLE). Large population of plasmablasts lacking RP105 are found in patients with activesystemic autoimmune diseases and take part in pathophysiology. We have studied clinicalsignificance of RP105-negative B cells including plasmablasts in various immune-based diseases.We also analyzed the 5 sub-populations of plasmablasts in the diseases. Increased RP105-negative B cells, high levels; SLE, ANA-negative SLE, Sjögren’s syndrome, dermatomyositis, IgG4-related disease (IgG4-RD), ANCA-associated vasculitis. low levels; rheumatoid arthritis (RA),systemic sclerosis (SSc), Behçet's disease, mixed connective tissue disease (MCTD), polymyositis(PM). RP105-negative B cells, especially plasmablasts, play crucial roles in systemic autoimmuneand immune-based inflammatory disorders. Also, distribution of sub-population and differenceof expressed antigens in RP105-lacking plasmablasts may be associated with pathophysiology inindividual disorders.

a90064 [1. Poster only]The role of novel lipid mediators in connective tissue diseases

1Department of Rheumatology and Clinical immunology,Kyoto University Graduate School ofMedicine, Kyoto, Japan,2Department of the Control for Rheumatic Diseases,Kyoto UniversityGraduate School of Medicine, Kyoto, JapanIsao Murakami1,Kosaku Murakami1,Akiko Yoshida1,Takashi Usui1,Nobuo Kuramoto1,RanSasai1,Motomu Hashimoto2,Yoshitaka Imura1,Hajime Yoshifuji1,Masao Tanaka2,KoichiroOhmura1,Tsuneyo Mimori1

Background： Omega3 polyunsaturated fatty acids (PUFAs) are reported to be effective inrheumatoid arthritis. Specialized Proresolving Mediators (SPMs) are generated from omega3PUFAs. SPMs are related to the resolution of acute inflammation and tissue repair. However, therole of SPMs in chronic inflammation including connective tissue diseases remains to bedetermined.Purpose：To clarify the role of SPMs in connective tissue diseases.Method：Peripheral blood was collected from patients with PM/DM (n=12) and RA (n=13） inKyoto University Hospital and healthy donors (n=5）. The surface expression of SPM receptors(FPR2, BLT1, and CMKLR1) on white blood cells was determined by flow cytometrical analysis.The expression profiles of SPM receptors were compared between patients and healthy donors.Result：The expression profile of BLT1 and CMKLR1 in CTD patients were same as in healthydonors. The expression of FPR2 on T cells was higher in CTD patients than in healthy people. Theexpression of FPR2 on granulocytes, monocytes and B cells was comparable between CTDpatients and healthy donors.Conclusion: FPR2 was upregulated on T cells in CTD patients.

a90174 [1. Poster only]The Effect of Quality Indicator Monitoring for Glucocorticoid–Induced Osteoporosis

1Immuno-Rheumatology Center, St. Luke's International University Hospital

MASATO OKADA1,Masei Suda1,Yasuhiro Suyama1

Objective: The rate of appropriate prescription for glucocorticoid-induced osteoporosis (GIOP)is low worldwide. This study aimed to evaluate the effect of quality indicator (QI) monitoring.Methods: We studied all patients prescribed prednisolone (as low as 7.5 mg daily) or itsequivalent for equal to or more than 3 months at our institution from 2010 to 2016. Thepatients were divided into 3 groups: Group A, males; Group B, females <50 years old; and GroupC, females ≧50 years old. The QI for GIOP was defined as the prescription rate of theappropriate anti-osteoporotic drug .Results: The numbers of participants were 401, 420, 520, and 513 in 2010, 2011, 2012 and2013, respectively., with pooled rates of the QI of 45.8%, 51.3%, 54.7%, and 55.1%,respectively. The change between 2010-2011 and 2010-2013 was statistically significant(p<0.05). Sub-analyses showed statistically significant improvements in the QI in Groups A and Cand that the QI widely varied between departments.Conclusion: Despite institution- wide announcements of QI goals, a substantial information andfidelity gap may exist between subspecialties involved in GIOP care. QI monitoring for GIOPsignificantly improved appropriate anti-osteoporotic drug prescriptions.

a90056 [1. Poster only]Is hormone replacement therapy an effective treatment for arthralgia in post-/ peri-menopausalwomen? Two case reports from an ongoing trial.

1Rheumatology and Women's Health, Keigu Clinic,2Health Sciences Research Institute,3MonashHealth RheumatologyKiyomitsu Miyachi1, 2,Belinda Sasse3,Shinichi Mashiba2

(Aim) Recently it has been shown that estrogen replacement inhibits the production of TNFthrough suppression of NFkB activity in human macrophages. In this trial, the efficacy ofhormone replacement therapy (HRT) for the treatment of arthralgia in menopausal andperimenopausal women was examined. Case 1. A 55 year old female presented in 2014complaining of bilateral wrist and small finger joint pain, and was found to be RF positive (52).She had undergone menopause 6 years previously. NSAIDs were ineffective for her arthralgia. Hersimplified menopausal index SMI was high (94) and HRT was commenced in April, 2014. Twomonths later, the patient’s visual analogue scale for joint pain reduced to 20 from 100. She hasnot experienced arthralgia since, nor developed rheumatoid arthritis (RA). Case 2. A 42 year-oldfemale presented in 2013 with onset of irregular menstruation. HRT was commenced in June,2013. Three months later, most symptoms had improved. Review a further 3 months later at theage of 44 found her to have normal E2 levels and so HRT was ceased in Dec. 2014. It was howevernoted that she had a positive ANA and anti-SS-A. Three months later, she visited the clinic withnew onset of dry mouth and eyes, and recurrence of her menopausal symptoms. Although theyresolved with reinstitution of cyclical HRT for 3 months, her sicca symptoms remained and shewas later diagnosed with Sjogren’s syndrome. (Discussion) Further research is being conductedto ascertain if such treatment may reduce the proportion of these women who progress to thedevelopment of RA.