Seido Ooka

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.

Autoantibodies to low-density-lipoprotein-receptor-related protein 2 (LRP2) in systemic autoimmune diseases

We previously reported that autoantibodies (autoAbs) to the main epitope on CD69 reacted to its homologous amino acid sequence in low-density-lipoprotein-receptor-related protein 2 (LPR2), a multiligand receptor for protein reabsorption. In this study, we have investigated the prevalence, autoepitope distribution, and clinical significance of the autoAbs to LRP2 in patients with systemic autoimmune diseases. Using six recombinant proteins (F2–F7) for LRP2 and one for CD69, we detected autoAbs to LRP2 in sera of patients with rheumatoid arthritis (RA), systemic lupus erythematosus, Behçets disease, systemic sclerosis, and osteoarthritis and then mapped autoepitopes by Western blotting. The autoAbs to LRP2 were detected in 87% of the patients with rheumatoid arthritis, 40% of those with systemic lupus erythematosus, 35% of those with systemic sclerosis, 15% of those with osteoarthritis, and 3% of those with Behçets disease. Multiple epitopes on LRP2 were recognized by most of the anti-LRP2+ serum samples. All of the tested anti-CD69 autoAb+ samples reacted to LRP2-F3 containing the homologous sequence to the main epitope of CD69; however, only 38% of the anti-LRP2-F3+ samples reacted to CD69. Clinically, the existence of the autoAbs to LRP2-F4, -F5, and -F6 correlated with the presence of proteinuria in RA. This study revealed that LRP2 is a major autoantigen in RA. The autoAbs to LRP2 are probably produced by the antigen-driven mechanism and the autoimmunity to LRP2 may spread to include CD69. The anti-LRP2 autoAbs may play pathological roles by inhibiting the reabsorbing function of LRP2.

Identification of β‐Tubulin Isoform V as an Autoantigen in Allergic Rhinitis by a Proteomic Approach

Autoantibodies to IgE and p2‐adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2‐dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, β‐tubulin isoform V (β‐tubV), using a recombinant protein and then measured prevalence of the anti‐β‐tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti‐β‐tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and β‐tubV is one of the major autoantigens in allergic rhinitis.

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.

Altered posttranslational modification on U1 small nuclear ribonucleoprotein 68k in systemic autoimmune diseases detected by 2D Western blot

Anti‐ribonucleoprotein (anti‐RNP) antibodies are one of the representative autoantibodies detectable in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Generally, posttranslational modifications (PTMs) on autoantigens are proposed to be involved in the production of autoantibodies. In this study, we tried to detect the alteration in PTMs on a U1 small nuclear RNP 68k subunit (U1‐68k), a major antigen of anti‐RNP antibodies. Peripheral blood mononuclear cells (PBMCs) were obtained from patients with MCTD, SLE, and rheumatoid arthritis (RA), and from healthy donors. U1‐68ks in the PBMCs were detected by 2D Western blot (WB), where extracted nuclear proteins were separated by 2DE, followed by the detection of U1‐68k using WB. More than 20 PTM isoforms were detected with different molecular weights of 65.0 , 66.5, and 68.0kDa, and different pIs between 6.0 and 8.5. Importantly, the relative intensity of the spot with 66.5 kDa and pI 7.5 was significantly increased in the MCTD and SLE groups compared to the RA and healthy groups. Further, this U1‐68k isoform, in particular, in its RS domain, was found to have significantly decreased phosphorylation compared to the other isoforms. The PTM alternation may be one of the steps to generate the anti‐RNP antibodies.

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.

Autoantibodies to CD69 in Patients with Chronic Hepatitis Type C: A Candidate Marker for Predicting the Response to Interferon Therapy

Objective: To understand the autoimmunity associated with chronic hepatitis C (CHC), we investigated autoantibodies (autoAbs) to CD69. Methods: With this aim, we tested the reactivity of serum samples from patients with CHC and asymptomatic carriers of hepatitis C virus (HCV), as well as from patients with chronic hepatitis B (CHB) and autoimmune hepatitis (AIH), to recombinant CD69 molecules. Results: Frequencies of anti-CD69 autoAbs were 38.7% in CHC, 15.8% in AIH and 12.3% in CHB. None of the tested asymptomatic HCV carriers had autoAbs to CD69. It is important clinically that the presence of anti-CD69 autoAbs was found to be associated with a poor response to interferon-α (IFN-α) therapy. In the epitope analysis, multiple epitopes were mapped on CD69, indicating antigen-driven production of the autoAbs. Conclusion: We evidenced existence of anti-CD69 autoAbs in patients with CHC, and found that the anti-CD69 autoAb may have potential for predicting responses to IFN-α therapy.

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).

FRI0493 Improved Detection of Early Pulmonary Hypertension in Patients with Connective Tissue Diseases by Simple Exercise Doppler Echocardiography

Background Several excellent screening methods for pulmonary hypertension (PH) or pulmonary arterial hypertension (PAH) in connective tissue diseases (CTD) such as systemic sclerosis (SSc) have been proposed (1)(2). We previously introduced a formula for estimating mean pulmonary arterial pressure (mPAP) using simple exercise Doppler echocardiography for CTD patients (3)(4). Objectives To validate the applicable formula for selecting CTD patients who need RHC based on the results of their exercise Doppler echocardiography. We also investigated whether exercise Doppler echocardiography contributed to the more accurate detection of the patients who need RHC. Methods A total of 231 CTD patients having either dyspnea or lower carbon monoxide diffusing capacity (DLCO) were performed Doppler echocardiography before and after exercise with the Masters double two-step test. Of 231 patients, 31 underwent RHC. Using the data from the 31, we derived a formula using tricuspid regurgitation pressure gradient at 3 minutes after exercise Doppler echocardiography for estimating mPAP (=0.270+0.387 x TRPG at 3 min. after exercise)(r2 =0.5367, P<0.0001), which was previously described (3). Validation with the additional 25 patients was performed. We compared its diagnostic value with Schreibers formula (1) and DETECT algorithm (2). Results Applying the formula to the validation cohort of 25 patients, which included 7 patients with borderline mPAP and 3 with manifest PH, gave a good correlation between the estimated and actual mPAP by RHC (Spearman r =0.7516, p<0.0001). In receiver operating characteristic, the area under the curve was 0.952. Using an estimated threshold of 17.5 mmHg for diagnosis of PH, the sensitivity and specificity were 100% and 79%, respectively. Among our 31 SSc patients, the sensitivity and specificity of Schreibers formula for diagnosing PH were 86% and 31%, respectively. Applying DETECT algorithm to our 23 SSc patients with DLCO <60%, the sensitivity and specificity for diagnosing PAH were 83% and 41%, respectively. Conclusions The provided formula for estimating mPAP gave a good correlation with actual mPAP by RHC. By adding this method as a part of screening for PH, selection of CTD patients potentially having PH would be more efficient. References Schreiber B et al. Improving the detection of pulmonary hypertension in systemic sclerosis using pulmonary function tests. Arthritis Rheum, 2011; 63: 3531. Coghlan JG et al. Evidence-based detection of pulmonary arterial hypertension in systemic sclerosis: the DETECT study. Ann Rheum Dis, 2014; 73: 1340. Yamasaki Y et al. Good detection of early pulmonary hypertension using exercise Doppler echocardiography in patients with connective tissue diseases and its comparison with other screening tool. EULAR 2014. Suzuki K et al. Simple exercise echocardiography using a Masters two-step test for early detection of pulmonary hypertension. J Cardiol, 2013; 62: 176. Disclosure of Interest None declared