Yoshihiro Arimura

A Panel Set for Epitope Analysis of Myeloperoxidase (MPO)-Specific Antineutrophil Cytoplasmic Antibody MPO-ANCA Using Recombinant Hexamer Histidine-Tagged MPO Deletion Mutants

A major target protein of antineutrophil cytoplasmic antibody with a perinuclear staining pattern (P-ANCA) has been identified as myeloperoxidase (MPO). Recombinant deletion mutants of MPO, eight fragments of the heavy-chain subunit, and two fragments of the light chain subunit were expressed in E. coli using a pQE expression vector. The recombinant hexamer histidine-tagged fragments were partially purified as the denatured proteins on a Ni2+-charged nitrirotriacetic acid column. The recombinant fragments were reacted with a rabbit polyclonal antibody to human MPO in Western blotting. In addition, the reactivities of the proteins with MPO-ANCA-positive sera of four patients with renal diseases were examined by Western blotting. The profile of the reactivity showed that different sera recognized different sets of fragments of the heavy chain, whereas no serum reacted with the fragments of the light chain. These results indicate that the sera of patients with MPO-ANCA-positive diseases showed varied reactivities with the different fragments. Furthermore, an ELISA system using a set of the fragments completely purified by Sephacryl S-200HR column chromatography was established. The panel set is useful for subclassification of MPO-ANCA-related diseases.

Analysis of risk epitopes of anti-neutrophil antibody MPO-ANCA in vasculitis in Japanese population.

Autoantibodies to myeloperoxidase (MPO) are a subset of anti‐neutrophil cytoplasmic antibody (ANCA, MPO‐ANCA) detected in the sera of some patients with primary systemic vasculitis. The titer of MPO‐ANCA does not always reflect disease activity and this inconsistency may be attributable to differences in epitopic specificity by MPO‐ANCA among various patients with vasculitis. Epitope analysis may also explain the occurrence of MPO‐ANCA in different vasculitic syndromes. We screened the sera of 148 MPO‐ANCA positive patients from six vasculitic syndromes: rapidly progressive gromerulonephritis (RPGN), microscopic polyangiitis (MPA), idiopathic crescentic glomerulonephritis (I‐CrGN), classic polyangiitis nodosa (cPAN), Churg‐Strauss syndrome (CSS), Kawasaki disease (KD); and from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The sera were collected by the Intractable Vasculitis Research Project Group in Japan. No serum showed epitopes La and Lb of light chain of MPO, and sera with 68.6% of patients showed a positive reaction to one or more epitopes in heavy chain of MPO. Analysis of binding level showed that RPGN, I‐CrGN and MPA sera mainly reacted to the Ha epitope at the N‐termimus of the MPO heavy chain, CSS sera reacted to Ha and the Hf epitope close to the C‐terminus of the MPO heavy chain, KD reacted mainly to Hf, while SLE and RA sera reacted to all epitopes. These results suggest that MPO‐ANCA recognizing specific regions of the N‐terminus of the MPO H‐chain confer an increased risk of vasculitis RPGN, I‐CrGN, MPA and CSS. Furthermore, the epitopic specificity of MPO‐ANCA differentiates vasculitic from non‐vasculitic syndromes associated with MPO‐ANCA positivity and differentiates in the cirtain type of vasculitis from various vasculitic syndromes. In particular, vasculitic syndromes associated with kidney involvement had similar epitopic reactivity which suggests that this pattern confers an increased risk of vasculitis.

Position paper: Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis

Anti-neutrophil cytoplasmic antibodies (ANCAs) are valuable laboratory markers used for the diagnosis of well-defined types of small-vessel vasculitis, including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). According to the 1999 international consensus on ANCA testing, indirect immunofluorescence (IIF) should be used to screen for ANCAs, and samples containing ANCAs should then be tested by immunoassays for proteinase 3 (PR3)-ANCAs and myeloperoxidase (MPO)-ANCAs. The distinction between PR3-ANCAs and MPO-ANCAs has important clinical and pathogenic implications. As dependable immunoassays for PR3-ANCAs and MPO-ANCAs have become broadly available, there is increasing international agreement that high-quality immunoassays are the preferred screening method for the diagnosis of ANCA-associated vasculitis. The present Consensus Statement proposes that high-quality immunoassays can be used as the primary screening method for patients suspected of having the ANCA-associated vaculitides GPA and MPA without the categorical need for IIF, and presents and discusses evidence to support this recommendation.

Tubulointerstitial nephritis without glomerular lesions in three patients with myeloperoxidase-ANCA-associated vasculitis

BackgroundMyeloperoxidase–antineutrophil cytoplasmic antibody (MPO–ANCA)-associated vasculitis frequently induces crescentic glomerulonephritis. However, a few cases have so far been reported to have only tubulointerstitial (TI) nephritis without any apparent glomerular lesions. We recently treated three similar cases. Therefore, their pathological features as well as clinical manifestations were studied in detail.MethodsThe pathological study was performed with immunohistochemical staining using various antibodies to the vascular endothelial cell surface markers, von Willebrand factor, type IV collagen, cytokeratin, E-cadherin, and MPO in addition to the routine histochemical examination.ResultsThe study disclosed the loss of CD34 endothelial cell surface markers with and without the destruction of type IV collagen (capillary basement membrane) in the peritubular capillaries, even though the glomeruli showed good staining of these factors. Electron microscopy showed breaks in the capillary basement membrane. The loss of CD34 staining was associated with the infiltration of a few mononuclear cells and neutrophils in the lumen of peritubular capillaries and the surrounding interstitial tissues. The cytokeratin staining in the tubular epithelial cells was also diminished around these areas. Tubulitis was demonstrated with or without the destruction of the tubular basement membrane. The clinical manifestations of these three cases were only a few red blood cells and granular casts in the urinary sediment as well as slightly increased β2-microglobulin in the urine, but no proteinuria.ConclusionBased on these findings, the loss of CD34 vascular endothelial markers occurs in the early phase of the disease because of the MPO, which is presumed to have burst out from the infiltrated, activated neutrophils. This MPO, which releases proteolytic enzymes and radical oxygen species, acts on tissue destruction, namely the lysis of endothelial cell membranes as well as vascular basement membranes in the peritubular capillary. This mechanism eventually proceeds to the destruction of the peritubular capillary walls (vasculitis). This pathogenesis is thought to play an important role in the pathogenesis of TI nephritis, which is associated with MPO–ANCA vasculitis.

A nationwide survey on the epidemiology and clinical features of eosinophilic granulomatosis with polyangiitis (Churg-Strauss) in Japan

Abstract Objective. We conducted a cross-sectional nationwide survey to determine eosinophilic granulomatosis with polyangiitis (Churg-Strauss) (EGPA) prevalence and clinical features in Japan. Methods. Data for EGPA patients in 2008 were collected from 1,564 hospitals. In total, 965 patients were reported from 365 departments. In a second survey, clinical data for 473 patients were obtained. Results. We estimated that 1,866 (95% CI: 1,640–2,092) patients have EGPA in Japan (prevalence, 17.8/1,000,000). Of the 473 patients in the second survey, 315 fulfilled American College of Rheumatology (ACR) criteria or Lanhams criteria for EGPA. The mean age (± SD) of the 315 at onset was 55 ± 14 years, male to female ratio 1:2. 93% of patients had neurological manifestations, which were the organ system most frequently involved. Among 277 patients tested for myeloperoxidase (MPO)-/p anti-neutrophil cytoplasmic antibody (ANCA), 139 (50%) were positive, while only 6 of 238 were positive for proteinase3 (PR3)-/cANCA. MPO-ANCA-positive patients had renal involvement, mucous membrane or ophthalmological symptoms, and ENT symptoms more frequently, whereas cutaneous lesions and cardiovascular involvement were less common. Conclusion. The prevalence of EGPA and the frequency of MPO-/p-ANCA-positivity in Japanese EGPA patients were mostly similar to those of Western countries. However, female predominance and a high frequency of neurological manifestations characterized Japanese patients.

Risk Factors Associated with Relapse in Japanese Patients with Microscopic Polyangiitis

Objective. We retrospectively studied the risk factors associated with relapse during remission maintenance therapy for myeloperoxidase-antineutrophil cytoplasmic autoantibody (MPO-ANCA)-positive microscopic polyangiitis (MPA). Methods. Sixty-two patients diagnosed with MPA according to the European Medicines Agency classification algorithm during a 2-year period from January 1, 2005, to December 31, 2006, and who achieved remission after the first remission-induction therapy, were examined (registration no. UMIN000001785). Results. The patient group comprised 25 men and 37 women aged 70.0 ± 8.9 years. The mean observation period was 30.2 ± 15.9 months. The rate of relapse was 24.2% (15/62), and mean interval between remission and relapse was 16.9 ± 13.5 months. During maintenance therapy following remission, the risk of relapse increased when the reduction rate of prednisolone increased above 0.8 mg/month (OR 12.6, 95% CI 2.2–97.9). Proteinuria at the start of maintenance therapy (regression coefficient 1.991 ± 0.758, p < 0.05) and the change in red blood cell counts in urine during the period from the start of maintenance therapy to the final observation (regression coefficient 0.126 ± 0.040, p < 0.01) were identified as risk factors influencing the vasculitis damage index. Conclusion. In Japan, relapse of MPO-ANCA-positive MPA may be associated with the reduction rate of oral prednisolone administration during maintenance therapy.

Renal pathology of ANCA-related vasculitis: proposal for standardization of pathological diagnosis in Japan

BackgroundIn Japan, systematic evaluation of the histologic parameters of anti-neutrophil cytoplasmic autoantibodies (ANCA)-related vasculitis has been performed according to the Japanese classification by Shigematsu et al. However, this classification is quite different from that of the European Vasculitis Study Group (EUVAS) classification. Therefore, a histological common basis is needed to compare Japanese histological data with the international database.MethodHistological parameters concerning glomerular, tubulointerstitial, and vascular lesions of ANCA-related vasculitis, which are indispensable for clinical management, were elucidated and defined by reviewing, utilizing the merits of, and amending the two scoring systems.Results and conclusionA new comprehensive and standardized scoring system, by which histological quantitative assessment can provide evidence for therapy planning, has been developed for renal biopsy of Japanese ANCA-related vasculitis.

Increased release of myeloperoxidase in vitro from neutrophils of patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody (MPO-ANCA) related glomerulonephritis

Summary: In order to elucidate the role of neutrophils in the pathogenesis of MPO‐specific anti‐neutrophil cytoplasmic antibody (MPO‐ANCA) related glomerulonephritis (GN), MPO release, beta‐glucuronidase (BGL) release, superoxide anion (O2−) production from the neutrophils of patients with MPO‐ANCA related GN were measured. the effect of plasma on MPO release from neutrophils was also studied in patients with MPO‐ANCA related GN. Neutrophils and plasma were obtained from patients with MPO‐ANCA related GN, GN unrelated to MPO‐ANCA and healthy controls. MPO release from the neutrophils of patients with MPO‐ANCA related GN was higher than that of controls significantly. This was also higher than that in patients with GN unrelated to MPG‐ANCA, but this was not statistically significant. Superoxide anion production from neutrophils of patients with MPO‐ANCA related GN was significantly higher than that in patients with GN unrelated MPO‐ANCA, However, BGL release was not significantly different among three groups. Furthermore, MPO release and O2− production increased in parallel with clinical activity of MPO‐ANCA related GN. Neutrophils of patients with MPO‐ANCA related GN showed to be significantly more sensitive to FMLP on MPO release than those in the other two groups. However, plasma from MPO‐ANCA related GN increased the sensitivity to FMLP on MPO release, but not BGL release, in neutrophils obtained from healthy controls, whereas it suppressed MPO release from neutrophils with MPO‐ANCA related GN. This suggests that in patients with MPO‐ANCA related GN MPO can be highly released from activated neutrophils and that the plasma of patients with MPO‐ANCA contains factor(s) which modulate reactivity of neutrophils.

Tubulointerstitial immune complex nephritis in a patient with systemic lupus erythematosus: role of peritubular capillaritis with immune complex deposits in the pathogenesis of the tubulointerstitial nephritis

The correct name of the fourth author should be given as Yoshihiro Arimura, not Yoshiro Arimura.

Protective Role of HLA-DRB1*13:02 against Microscopic Polyangiitis and MPO-ANCA-Positive Vasculitides in a Japanese Population: A Case-Control Study

Among antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), granulomatosis with polyangiitis (GPA) and proteinase 3-ANCA-positive AAV (PR3-AAV) are prevalent in European populations, while microscopic polyangiitis (MPA) and myeloperoxidase-ANCA-positive AAV (MPO-AAV) are predominant in the Japanese. We previously demonstrated association of DRB1*09:01-DQB1*03:03 haplotype, a haplotype common in East Asians but rare in the European populations, with MPA/MPO-AAV, suggesting that a population difference in HLA-class II plays a role in the epidemiology of this disease. To gain further insights, we increased the sample size and performed an extended association study of DRB1 and DPB1 with AAV subsets in 468 Japanese patients with AAV classified according to the European Medicines Agency algorithm (MPA: 285, GPA: 92, eosinophilic GPA [EGPA]: 56, unclassifiable: 35) and 596 healthy controls. Among these patients, 377 were positive for MPO-ANCA and 62 for PR3-ANCA. The significance level was set at α = 3.3x10-4 by applying Bonferroni correction. The association of DRB1*09:01 with MPO-AAV was confirmed (allele model, P = 2.1x10-4, odds ratio [OR] = 1.57). Protective association of DRB1*13:02 was detected against MPO-AAV (allele model, P = 2.3x10-5, OR = 0.42) and MPA (dominant model, P = 2.7x10-4, OR = 0.43). A trend toward increased frequency of DPB1*04:01, the risk allele for GPA in European populations, was observed among Japanese patients with PR3-AAV when conditioned on DRB1*13:02 (Padjusted = 0.0021, ORadjusted = 3.48). In contrast, the frequency of DPB1*04:01 was decreased among Japanese patients with MPO-AAV, and this effect lost significance when conditioned on DRB1*13:02 (Padjusted = 0.16), suggesting that DRB1*13:02 or other allele(s) in linkage disequilibrium may be responsible for the protection. The differential association of DPB1*04:01 with PR3-AAV and MPO-AAV and difference in DPB1*04:01 allele frequencies between populations supported the hypothesis that the HLA-class II population difference may account in part for these epidemiologic characteristics. Furthermore, taken together with our previous observations, the haplotype carrying DRB1*13:02 was suggested to be a shared protective factor against multiple autoimmune diseases.