Hiromasa Nakano

Proteomic surveillance of autoantigens in patients with Behcet's disease by a proteomic approach

To promote an understanding of autoimmunity in BD, we surveyed autoAgs in patients with BD and investigated the prevalence and clinical significance of the identified autoAbs. Specifically, proteins, extracted from peripheral blood mononuclear cells and separated by 2DE, were subjected to WB, using five serum samples from patients with BD. The detected candidate autoAgs were identified by mass spectrometry. As a result, 17 autoantigenic spots were detected by the 2DE‐WB, out of which eight spots were identified. They are enolase‐1, cofilin‐1, vimentin, Rho‐GDI β protein, tubulin‐like protein, and actin‐like proteins. The autoAbs to one of the identified proteins, cofilin‐1, were investigated by WB using a recombinant protein in 30 patients with BD, 35 patients with RA, 32 patients with SLE, and 16 patients with PM/DM. The autoAbs to cofilin‐1 were detected by WB in four (13.3%) of the 30 patients with BD, five (14.3%) of the 35 patients with RA, two (6.3%) of the 32 patients with SLE, and eight (24.2%) of the 33 patients with PM/DM. Our data indicate that the generation of autoAbs to cofilin‐1 may reflect common immunological disorders in BD, RA, and PM/DM. Our data would help understanding of the immunopathology of BD. In addition, the proteomic approach would be a useful way to investigate autoAgs.

Clinical subsets associated with different anti-aminoacyl transfer RNA synthetase antibodies and their association with coexisting anti-Ro52

Abstract Objective: To characterize clinical features of polymyositis/dermatomyositis (PM/DM) patients with different anti-aminoacyl transfer RNA synthetase (ARS) antibodies and their association with anti-Ro52. Methods: Autoantibodies in sera from 97 Japanese patients (36 PM, 56 DM, and 5 clinically amyopathic DM), who satisfied Bohan and Peter or modified Sontheimers criteria, were characterized by immunoprecipitation and enzyme-linked immunosorbent assay. Clinical information was from medical records. Features associated with different anti-ARS and anti-Ro52 antibodies were analyzed. Results: The prevalence of anti-ARS was similar to other studies (Jo-1, 22%; EJ, 4%; OJ, 1%; PL-12, 1%), except for a high prevalence of anti-PL-7 (12%), which allowed us to characterize patients carrying this specificity. Serum creatine kinase >3000 IU/l was less common in anti-PL-7-positive patients (57%) than anti-Jo-1-positive patients (18%) (p = 0.0328) and was not found in anti-EJ-positive individuals. Interstitial lung disease was common in anti-ARS-positive patients (97%) (p < 0.0001 vs. 48% in anti-ARS-negative). Anti-Ro52 antibodies were frequently detected with anti-ARS (59%) (57% in anti-Jo-1, 67% in anti-PL-7) (vs. 21% in anti-ARS-negative, p < 0.0002). Anti-Ro52 was associated with overlap syndrome (26%) (vs. 7% in anti-Ro52-negative, p = 0.0119). Conclusions: Patients with different anti-ARS in combination with anti-Ro52 appear to be associated with distinctive clinical subsets.

Comparative proteomic analysis of neutrophils from patients with microscopic polyangiitis and granulomatosis with polyangiitis

UNLABELLED Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils play a key role in their pathology. In this study, in order to discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of 11 MPA patients and 9 GPA patients and 10 healthy controls (HC) were analyzed by 2D-DIGE. In all the 864 spots detected, intensity of 55 spots was significantly different (p<0.05) among the three groups by ANOVA. 31 out of the 55 spots were identified by mass spectrometry. Orthogonal partial-least-squares-discriminate analysis revealed that the abundance profile of the protein spots discriminated the AAV group from the HC group, and the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various pI values, while a considerable part of spots whose intensity was higher in GPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a-kinase anchor protein 7 isoforms beta had a high diagnostic potential. BIOLOGICAL SIGNIFICANCE In this study, protein profiles of polymorphonuclear cells (PMNs) of microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) patients and healthy controls (HC) were investigated by 2D-DIGE, and MS analysis. As a result, we found that the protein profiles of PMNs were useful for distinguishing between patients (MPA and GPA) and HC, and between patients with MPA and patients with GPA. Especially, we found that the 13 protein spots that consisted of 10 proteins considerably contributed to the discrimination between MPA and GPA. This is the first to demonstrate that protein profiles of PMNs are different among MPA, GPA and healthy control. The 10 proteins we identified in this study would be new biomarkers for the diagnosis of the diseases, and may be reflect the pathology difference between MPA and GPA.

Cutaneous polyarteritis nodosa associated with HLA-B39-positive undifferentiated spondyloarthritis in a Japanese patient

We present the case of a 43-year-old man diagnosed with HLA-B39-positive spondyloarthritis who developed cutaneous lesions consistent with cutaneous polyarteritis nodosa (CPN). Previous studies indicated an elevated incidence of HLA-B39 in HLA-B27-negative Japanese patients with spondyloarthritis. This case suggested that CPN may also occur in association with forms of HLA-B39-positive spondyloarthritis. The rarity of this association is emphasized. Therapy with corticosteroid and methotrexate improved both the cutaneous lesions and the clinical symptoms of spondyloarthritis.

Behçet’s Disease with Vascular Involvement: The Contribution of Anticardiolipin Antibodies and Thrombomodulin

Patients were included by clinical records between 1998 and 2002 at our division. All patients met with international criteria of BD. Lupus anticoagulant (LA) assay was performed by Russell’s viper venom time (dRVVT) and dilute APTT (dAPTT). Anticardiolipin antibodies (aCl-beta2GP I) were assayed with specific EIA for beta2-glycoprotein I (Yamasa kit, Tokyo, Japan). “Vascular involvement” included deep venous thrombosis, subcutaneous thrombophlebitis, arteritis, and various types of venous occlusion (Table 1).

FRI0313 Detection of anti-granulocyte macrophage colony-stimulating factor autoantibody in systemic lupus erythematodes

Background Systemic lupus erythematodes(SLE) causes various symptoms in the respiratory system. Ground glass opacity (GGO) with SLE is conventionally considered interstitial pneumonia caused by the deposition of immune complexes. However, in most cases, ground glass opacity is not related to disease activity or inflammation. It is thus hypothesized that GGO is due to pulmonary alveolar proteinosis (PAP). Objectives Anti-Granulocyte Macrophage colony-stimulating Factor(GM-CSF) autoantibody is a cause of PAP. We therefore measured anti-GM-CSF autoantibody in sera of patients with SLE to test the hypothesis that PAP occurs in lupus pulmonary disease. Methods The samples were obtained from a total of 64 japanese patients in our hospital. They were diagnosed according to the 1997 criteria for SLE. The enzyme-linked immunosorbent assay was performed according to a modified version of the method described previously (1) with sera being diluted 100-fold with phosphate-buffered saline. 50μl diluted sera were transferred to a plate coated with 1μg/ml of GM-CSF (Escherichia coli derived) and the plate was kept at 4 degrees temperature for 8 h. After washing, autoantibodies captured by GM-CSF were detected by peroxidase-labeled anti-human IgG antibodies. Color was detected using o-Phenylenediamine and the absorbance was measured at 450 nm. Results Anti-GM-CSF autoantibodies were detected in 53 percent of SLE patients, with the cuttoff level being 2SD (standard deviation) of the value of the healthy serum. The titers varied, and were significantly higher in SLE patients than in patients with systemic scleroderma. Furthermore, GGO in high-resolution CT was significantly related to the anti-GM-CSF autoantibody titers (p=0.01). The patient with the highest titers had decreased respiratory function with massive ground glass opacity in both lungs. Bronchoalveolar lavage was performed, and PAP was confirmed. The presence of anti-GM-CSF autoantibodies may be a predictor of the onset of PAP because increased levels of autoantibodies were detected two years prior to onset of pulmonary disease in this patient. Conclusions This is the first report to systematically measure anti-GM-CSF autoantibodies in patients with SLE. PAP was present in pulmonary disease that was conventionally considered interstitial pneumonia in patients with SLE. It is important to measure anti-GM-CSF autoantibodies because the treatment differs for PAP and interstitial pneumonia in patients with SLE. References Kitamura, T et al. 1999. Idiopathic pulmonary alveolar proteinosis as an autoimmune disease with neutralizing antibody against granulocyte. macrophage colony stimulating factor. J. Exp. Med. 190:875.880. Disclosure of Interest: None Declared

AB0153 Comparative proteomic analysis of neutrophils from patients with microscopic polyangiitis and granulomatosis with polyangiitis

Background Both microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA) belong to ANCA-associated vasculitis (AAV), in which neutrophils are thought to be involved in their pathology. Clinically, it is often difficult to distinguish MPA from GPA. To discriminate between MPA and GPA, protein profiles of peripheral blood polymorphonuclear cells (PMNs) of MPA and GPA patients and healthy controls (HC) were analyzed. Objectives In this study, proteomic profilesof PMN of AAV patients and healthy controls (HC) were analyzed using two-dimensional difference gel electrophoresis (2D-DIGE), in order to confirm whether the profiles are useful to discriminate between AAV and HC, or MPA and GPA. Methods Proteins extracted from peripheral blood PMNs of 11 MPA patients, 9 GPA patients, and 10 HC were separated by two-dimensional difference gel electrophoresis (2D-DIGE). Differentially expressed protein spots were identified by mass spectrometry analysis. Then, to find biomarker candidates which discriminate between MPA and GPA, the obtained protein profiles were subjected to the multivariate data analysis using SIMCA-P+ containing principal component analysis (PCA) and orthogonal partial-least-squares-discriminate analysis (OPLS-DA), and subjected to the receiver operating characteristic (ROC) analysis. Results In all the 864 protein spots detected, intensity of 55 spots was found to be significantly different (p < 0.05) among the three groups by an analysis of variance (ANOVA). 31 out of the 55 spots were identified by mass spectrometry. The OPLS-DA analysis revealed that the expression profile of the protein spots discriminated the AAV group from the HC group completely and also discriminated the MPA group from the GPA group completely. 13 protein spots were considered as biomarker candidates to distinguish between MPA and GPA. In those, spots whose intensity was higher in MPA than in GPA included actin with various pI values, while a considerable part of spots whose intensity was higher in GPA than MPA were proteins related with the activity of neutrophils. Among the candidate proteins, ROC analysis showed that a combination of neutrophil gelatinase-associated lipocalin and a kinase anchor protein 7 isoforms beta had a high diagnostic potential. Conclusions In the study, we determined that the protein profile of the neutrophil was clearly different between AAV and HC, and between MPA and GPA. In Particular, GPA was characterized by high expression level of the proteins associated with the activity of the neutrophil. Disclosure of Interest None Declared

THU0410 Characterisation of interstitial lung disease associated with adult still’s disease

Background Adult Still’s disease (ASD) is an inflammatory disorder characterized by persistent fevers, arthritis, and an evanescent rash. Interstitial lung disease (ILD) is rare, reported in 6%1) of ASD. Some patients progressed to acute respiratory distress syndrome. However, clinical characteristics and treatment strategies of ILD associated with ASD are not established. Objectives To characterize clinical features and outcome of ASD-associated ILD (ASD-ILD). Methods We retrospectively reviewed clinical charts of all the patients diagnosed with ASD according to Yamaguchi criteria for ASD at our institutes from 2005 to 2011. ILD was diagnosed based on chest high-resolution CT (HRCT). We extracted the patients with ASD-ILD and compared their clinical characteristics with the non-complication group. Also, we characterized the HRCT findings of ASD-ILD. Results Total 84 cases of ASD was identified, male to female ratio was 22:62. Most cases fall into two age groups (20-30 and 50-60 y.o.), the average age was 42.4 (16-86 y.o.). ILD was identified in 7 out of 84 cases (8.3%), male to female ratio was 1:6. The average age was 63.6 (36-76 y.o.). Patient number (Pt) 1-3 developed ASD and ILD at the same time. The rest of the cases (Pt4-7) developed ASD-ILD about 1 year after suffering ASD. HRCT showed marked thickening of the interlobular septi, the bronchovascular bundles, and the pleura in all cases. These finding involved whole of the lungs evenly in all cases but one which had lower lobe predominance. There was no honeycombing formation. Three (43%) out of 7 patients (Pt1-3) suffered from dyspnea with hypoxemia, which paralleled with the activity of ASD. As ILD complication rapidly progressed, intravenous cyclosphophamide (IVCY) or tocilizumab (TCZ) were introduced with an increasing doze of glucocorticoid (GC), resulting in an improvement of dyspnea and ILD. Even though Pt4-6 did not show respiratory symptom, HRCT finding of ILD was worsen with the recurrence of ASD. To deal with the recurrence of ASD, increasing dosage of GC and ciclosporin was used. Pt7 did not show any respiratory symptom and his ILD was stable during the course. As for other clinical characteristics of ILD complication, it was of note that hemophagocytic syndrome was complicated in 5 cases with ILD (71%). Six patients in ILD (85.7%) showed a recurrence during the course. All of them were remitted after the intensified treatment. There was no death. Conclusions ASD-ILD tended to occur in older female. ILD complicated at the initial diagnosis of ASD was symptomatic and progressive, which required an aggressive immunosuppressive therapy such as IVCY and TCZ. HRCT finding included marked thickening of the interlobular septi, the bronchovascular bundles, and the pleura, which could be pathognomonic imaging characteristics of ASD-ILD. HRCT findings suggest massive infiltration of macrophages and lymphocytes2) with lymphangiectasis in the inter septal space. Histologic investigation is definitely needed. References Ohta A et al. Adult Still’s disease: a multicenter survey of Japanese patient. J Rheumatol. 1990;17:1058-63. Sato H et al. A case of Adult onset Still’s disease complicated with cryptogenic organizing pneumonia. Inten Med. 2011;50:247-51. Disclosure of Interest None Declared

350 The Vasculitis Induced by the Cerebrovascular Coil.

Background The treatment of a cerebrovascular coil is the popular treatment for the cerebral aneurysm. It is treatment to insert a metal coil in aneurysm. It is not reported the allergy of cerebrovascular coil. In Dec, 2019, a 57-year-old Japanese woman came to the neurosurgery hospital. The cerebral aneurysm was treated by coil embolization. Two days later, she was returned to hospital by left facial paralysis. Vasculitis was detected on the peripheral artery of the coil by MRI (magnetic resonance imaging). It was treated by prednisolone 60 mg/d, and paralysis was improved, although slightly remained sequelae. Object To understand of necessity of long term steroid therapy, we investigated metal allergy for coil. Methods The steroid was tapered and discontinued. The MRI was checked every 3 months. When vasculitis recurs, tapering is canceled. Three kinds of coils were used by embolization. Their coil consists of platinum and tungsten mainly, but it is unknown about the other component metal. After the steroid was discontinued, a patch test for 3 coil and LTT (lymphocyte transformation test) for the contrast medium was indicated. It was interpreted using ICDRG (INTERNATIONAL CONTACT DERMATITIS RESEARCH GROUP) criteria. It was given her informed consent to this study. Results After treatment of steroid, only Potassium bichromate was positive and other 17 metals, including the platinum was negative by the patch test. Two coils of the same type, was positive. The contrast medium was negative by LTT. One month later, new lesion of vasculitis was detected on the peripheral artery of the coil by MRI. Conclusions It is a metal coil at risk of causing vasculitis. When it was treated by coil embolization, the check of the allergy to metal is recommended, because it was possible to experience permanent sequelae.