Publications by authors named "Haruki Shida"

28 Publications

  • Page 1 of 1

Elevated Myeloperoxidase-DNA Complex Levels in Sera of Patients with IgA Vasculitis.

Pathobiology 2021 Nov 23:1-6. Epub 2021 Nov 23.

Department of Dermatology, St. Marianna University School of Medicine, Kawasaki, Japan.

Introduction: IgA vasculitis is a systemic disease that results from the entrapment of circulating IgA-containing immune complexes in small-vessel walls in the skin, kidneys, and gastrointestinal tract. An excessive formation of neutrophil extracellular traps (NETs) is involved in the pathogenesis of vasculitis, especially in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. This study aimed to clarify whether NETs are implicated in IgA vasculitis.

Methods: Twenty-two patients with IgA vasculitis and 4 healthy volunteers were enrolled in this study. Serum levels of myeloperoxidase (MPO)-DNA complex, a fragment derived from NETs, were determined by enzyme-linked immunosorbent assay (ELISA), and the association between MPO-DNA complex levels and clinical parameters was examined. The presence of the ANCA was also assessed by ELISA specific for MPO and proteinase 3 (PR3) and indirect immunofluorescence (IIF), followed by assessing the differences in clinical parameters with and without the ANCA.

Results: Serum MPO-DNA complex levels were significantly higher in patients with IgA vasculitis than those in healthy controls. A significant positive correlation between the serum MPO-DNA complex and IgA levels was noted. Interestingly, 63.6% of IgA vasculitis patients were ANCA-positive in IIF with an atypical pattern, whereas neither MPO-ANCA nor PR3-ANCA was detected by ELISA. These findings indicated that some IgA vasculitis patients possessed the so called minor ANCA. Serum IgA and MPO-DNA complex levels and the frequency of hematuria in the minor ANCA-positive group were significantly higher than in the minor ANCA-negative group.

Conclusion: The collective findings suggested that NETs are certainly involved in the pathogenesis of IgA vasculitis.
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http://dx.doi.org/10.1159/000519869DOI Listing
November 2021

Macrolide treatment for COVID-19: Will this be the way forward?

Biosci Trends 2020 May 5;14(2):159-160. Epub 2020 Apr 5.

Department of Respiratory Medicine, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic that has developed in late 2019 and 2020 is a serious threat to human health. With no vaccines or drugs approved for prevention and treatment until now, all efforts at drug design and/or clinical trials of already approved drugs are worthy and creditable. Using structure-based drug selection for identification of SARS-CoV-2 protease inhibitors, old drugs such as macrolides (MAC) were predicted to be effective for COVID-19. Lately, the anti-viral effects of macrolides have attracted considerable attention. Very recently, hydroxychloroquine in combination with azithromycin treatment was reported to be effective for COVID-19. We believe that treatments with macrolides alone or in combination with other drugs are promising and open the possibility of an international strategy to fight this emerging viral infection.
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http://dx.doi.org/10.5582/bst.2020.03058DOI Listing
May 2020

A case of follicular lymphoma accompanied with chylous ascites.

Blood Res 2019 Sep 25;54(3):163. Epub 2019 Sep 25.

Department of Hematology, Sapporo Kyoritsu Gorinbashi Hospital, Sapporo, Japan.

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http://dx.doi.org/10.5045/br.2019.54.3.163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779936PMC
September 2019

Genome Sequencing Verifies Relapsed Infection of .

Open Forum Infect Dis 2019 May 25;6(5):ofz200. Epub 2019 Apr 25.

Center for Animal Disease Control, University of Miyazaki, Japan.

Background: Recurrent infections of are often reported, and long-term antimicrobial treatment is empirically recommended to prevent such infections. However, there have been no studies examining whether recurrent infections are relapses of former infections or reinfections with different clones.

Methods: A 69-year-old woman presented with recurrent bacteremia-associated cellulitis after a 51-day interval. We isolated 10 colonies from the blood cultures obtained during each of the 2 episodes and subjected them to whole-genome sequencing (WGS). High-confidence single-nucleotide polymorphisms (SNPs) were identified by an assembly based method. Heterogeneous SNP sites were identified by read mapping. The susceptibility of a representative isolate to 14 antimicrobials was also examined.

Results: Whole-genome sequence analysis revealed only 6 SNP sites among the 20 isolates at the whole-genome level. Based on the 6 SNPs, 5 within-host variants (referred to as genotypes) were identified. All 5 genotypes were detected in the first infection; however, only 2 genotypes were detected in the second infection. Although the clone showed a higher minimum inhibitory concentration to fluoroquinolones and macrolides and responsible mutations were identified, none of the 6 SNPs appeared related to additional resistance.

Conclusions: The second infection analyzed here was a relapse of the first infection. A certain level of within-host genomic heterogeneity of the clone was already present in the first infection. Our results suggest the importance of longer treatment courses to eradicate for preventing the relapse of its infection.
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http://dx.doi.org/10.1093/ofid/ofz200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6527084PMC
May 2019

The presence of anti-neutrophil extracellular trap antibody in patients with microscopic polyangiitis.

Rheumatology (Oxford) 2019 07;58(7):1293-1298

Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Sapporo, Japan.

Objective: Although ANCA is the major autoantibody in patients with ANCA-associated vasculitis, previous studies have suggested the presence of anti-neutrophil extracellular trap (NET) antibody in patients with microscopic polyangiitis (MPA), one type of ANCA-associated vasculitis. In this study, we aimed to determine the prevalence and pathogenic role of anti-NET antibody (ANETA) in MPA.

Methods: We examined the presence or absence of ANETA in sera obtained from 19 MPA patients by indirect immunofluorescence. We compared the clinical parameters, including age, sex, MPO-ANCA, creatinine, CRP, MPO-DNA complexes and vasculitis activity, in ANETA-positive and ANETA-negative MPA patients. We investigated the serum NET induction and degradation abilities of ANETA-positive and ANETA-negative MPA patients with reference to healthy controls (n = 8). Furthermore, we assessed the relationship between ANETA and the effect of IgG depletion on the serum NET degradation ability.

Results: ANETA was present in 10 of the 19 MPA patients. There was no significant difference in the clinical parameters in ANCA-positive and ANCA-negative MPA patients. Although the NET induction ability was higher and the NET degradation ability was lower in MPA sera than those in healthy controls, these abilities were not different between ANETA-positive and ANETA-negative MPA sera. Interestingly, the NET degradation ability in some sera with ANETA was markedly increased by IgG depletion.

Conclusion: Some MPA patients produce ANETA and some ANETA possess an inhibitory function against the serum NET degradation ability. Although further studies are needed, ANETA is worthy of attention in order to understand the pathophysiology of MPA.
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http://dx.doi.org/10.1093/rheumatology/kez089DOI Listing
July 2019

Methotrexate-induced myelodysplasia mimicking myelodysplastic syndrome.

Blood Res 2018 Dec 17;53(4):268. Epub 2018 Dec 17.

Health Care Center, Hokkaido University, Sapporo, Japan.

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http://dx.doi.org/10.5045/br.2018.53.4.268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6300683PMC
December 2018

Detection of Autoreactive Type II NKT Cells: A Pilot Study of Comparison Between Healthy Individuals and Patients with Vasculitis.

Cytometry A 2018 11 25;93(11):1157-1164. Epub 2018 Sep 25.

Faculty of Health Sciences, Hokkaido University, Sapporo, Japan.

NKT cells are defined as T cells that recognize hydrophobic antigens presented by class I MHC-like molecules, including CD1d. Among CD1d-restricted NKT cells, type I and type II subsets have been noted. CD1d-restricted type I NKT cells are regarded as pro-inflammatory cells in general. On the contrary, accumulated evidence has demonstrated an anti-inflammatory property of CD1d-restricted type II NKT cells. In our earlier study using a rat model with vasculitis, we demonstrated the pro-inflammatory function of CD1d-restricted type II NKT cells and identified that one such cell recognized P of rat sterol carrier protein 2 (rSCP2 ), which appeared on vascular endothelial cells presented by CD1d. Based on this evidence, we attempted to detect human CD1d-restricted type II NKT cells in peripheral blood using hSCP2 , the human counterpart of rSCP2 together with a CD1d tetramer in flow cytometry. First, we determined the binding of hSCP2 to CD1d. Next, we detected CD3-positive hSCP2 -loaded CD1d (hSCP2 /CD1d) tetramer-binding cells in peripheral blood of healthy donors. The abundance of TGF-β-producing cells rather than TNF-α-producing cells in CD3-positive hSCP2 /CD1d tetramer-binding cells suggests the anti-inflammatory property of SCP2-loaded CD1d (SCP2/CD1d) tetramer-binding type II NKT cells in healthy individuals. Furthermore, we compared cytokine profile between healthy individuals and patients with vasculitis in a pilot study. Interestingly, the percentage of TGF-β-producing cells in SCP2/CD1d tetramer-binding type II NKT cells in vasculitic patients was significantly lower than that in healthy controls despite the greater number of these cells. Although further studies to clarify the mechanism and significance of this phenomenon are needed, SCP2/CD1d tetramer-binding type II NKT cells in peripheral blood should be examined in more detail to understand the pathophysiology of vasculitides in humans. © 2018 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23618DOI Listing
November 2018

Anti-neutrophil extracellular trap antibody in a patient with relapse of anti-neutrophil cytoplasmic antibody-associated vasculitis: a case report.

BMC Nephrol 2018 06 22;19(1):145. Epub 2018 Jun 22.

Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo, 0600812, Japan.

Background: Neutrophil extracellular traps (NETs) are web-like DNA decorated with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. Although NETs are essential in innate immunity, an excessive formation of NETs has adverse effects, e.g., induction of anti-neutrophil cytoplasmic antibody (ANCA), to the hosts. Since ANCA can induce NET formation in the primed neutrophils, a positive feedback loop can be formed between NETs and ANCA, which is called "ANCA-NETs vicious cycle."

Case Presentation: A 79-year-old Japanese woman developed hydralazine-induced pauci-immune necrotizing crescentic glomerulonephritis with MPO-ANCA. Although the illness improved after cessation of hydralazine, MPO-ANCA-associated vasculitis relapsed 16 months later. Remission was achieved 5 months after beginning of administration of prednisone. In order to determine the involvement of ANCA-NETs vicious cycle in this patient, we examined NET degradation and induction activities in sera obtained at the disease onset (Serum A; MPO-ANCA, 107 IU/ml), at relapse (Serum B; MPO-ANCA, 195 IU/ml), at 3 months after treatment (Serum C; MPO-ANCA, 4.5 IU/ml), and at remission (Serum D; MPO-ANCA, 2.4 IU/ml). NET degradation activity was low in the all sera. NET induction activity was high in Sera A, B, and C but not in D. Additionally, we demonstrated the presence of anti-NET antibody (ANETA) in Sera B and C but not in A or D.

Conclusions: The collective findings suggest NET induction potential of ANETA in the present patient and that the ANETA could contribute to the enhancement of NETs resulting in amplification of the ANCA-NETs vicious cycle.
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http://dx.doi.org/10.1186/s12882-018-0953-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013953PMC
June 2018

Successful additional clarithromycin and tacrolimus treatment for hypereosinophilia associated with eosinophilic granulomatosis with polyangiitis.

Transl Clin Pharmacol 2018 Jun 18;26(2):60-63. Epub 2018 Jun 18.

Department of Internal Medicine, JCHO Hokkaido Hospital, Sapporo 062-8618, Japan.

A 41-year-old man suffering from eosinophilic granulomatosis with polyangiitis (EPGA), diagnosed at another clinic on the basis of American College of Rheumatology Criteria, with a history of bronchial asthma, eosinophilia, mononeuritis multiplex, and non-fixed pulmonary infiltrates, was admitted to our department for further treatment. The patient complained of chest pain that started recently. An echocardiogram identified myocardial thickening and decreased wall motion, based on which the patient was diagnosed as having EPGA with myocarditis. The patient was successfully treated using glucocorticoids, such as methyl prednisolone (PSL) and PSL in combination with cyclophosphamide (CPM). However, CPM administration was discontinued afterwards because of the risk of bone marrow toxicity, the increased eosinophilic count (EOC) that we considered as an index of disease activity. Subsequently, the patient received additional clarithromycin (CAM) and tacrolimus (TAC) treatment considering their immunomodulatory effects. As a result, the EOC decreased and the PSL dosage could be reduced. This case shows that additional CAM and TAC treatment may be beneficial in some cases of EPGA.
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http://dx.doi.org/10.12793/tcp.2018.26.2.60DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989262PMC
June 2018

Successful treatment of primary immune thrombocytopenia accompanied by diabetes mellitus treated using clarithromycin followed by prednisolone.

Drug Discov Ther 2018 May 23;12(2):101-103. Epub 2018 Apr 23.

Health Care Center, Hokkaido University.

Macrolides have immunomodulatory effects including anti-inflammatory effects as well as antibacterial activity. In consideration of these immunomodulatory effects, we report a patient with primary immune thrombocytopenia (ITP) treated using clarithromycin (CAM), a macrolide, followed by prednisolone (PSL). A 78-year-old man with thrombocytopenia was admitted to our hospital for further examination. Initial laboratory data showed reduced platelet counts (1.7 × 10/μL). Finally, we diagnosed the patient as having primary ITP. Because the patient was suffering from diabetes mellitus (DM), he was treated with CAM as an alternative to PSL. The platelet count increased to 6.1 × 10/μL. The CAM treatment was terminated owing to gradual nausea and palpitation. During the CAM treatment, the DM was under control. We reinitiated treatment for ITP. The patient was successfully treated using PSL without severe hyperglycemia. This case shows that CAM treatment may represent a useful option for ITP patients who cannot receive PSL due to DM.
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http://dx.doi.org/10.5582/ddt.2018.01008DOI Listing
May 2018

Elevated Level of Myeloperoxidase-Deoxyribonucleic Acid Complex in the Middle Ear Fluid Obtained From Patients With Otitis Media Associated With Antineutrophil Cytoplasmic Antibody-associated Vasculitis.

Otol Neurotol 2018 04;39(4):e257-e262

Faculty of Health Sciences.

Objective: The purpose was to explore the presence of myeloperoxidase (MPO)-deoxyribonucleic acid (DNA) complex as a surrogate marker of neutrophil extracellular traps (NETs) in the middle ear fluid, and to clarify the correlation between its quantifiable level and hearing outcome in patients with otitis media associated with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).

Study Design: Prospective study.

Setting: Tertiary referral center.

Patients: Nine AAV patients presenting with otitis media.

Intervention: Collection of the fluid samples from middle ear.

Main Outcome Measure: The quantifiable levels of MPO-DNA complex using an enzyme-linked immunosorbent assay.

Results: The quantifiable levels of MPO-DNA complex in patients with AAV were significantly higher than those in controls (p < 0.001). In particular, both ANCA-positive and -negative cases indicated higher levels of MPO-DNA complex compared with the controls (p = 0.004 and p = 0.006, respectively). The significant negative correlations were observed between the level of MPO-DNA complex and the functional hearing values for air (r = -0.82, p = 0.009) and bone conduction (r = -0.73, p = 0.028), respectively.

Conclusion: This analysis is the first to reveal the presence of elevated levels of MPO-DNA complex in the middle ear fluid, suggesting the pathogenic role of NETs in otitis media associated with AAV. NETs may be a valuable biomarker for use in clinical decision-making and predicting hearing outcome, regardless of ANCA status.
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http://dx.doi.org/10.1097/MAO.0000000000001708DOI Listing
April 2018

Clinical significance of plasma presepsin levels in patients with systemic lupus erythematosus.

Mod Rheumatol 2018 Sep 14;28(5):865-871. Epub 2017 Dec 14.

a Department of Rheumatology, Endocrinology and Nephrology, Graduate School of Medicine and Faculty of Medicine , Hokkaido University , Sapporo , Japan.

Objectives: Presepsin (PSEP: soluble CD14 subtype) is produced from bacteria-stimulated monocytes or neutrophils, thus recognized as a biomarker of sepsis. Aberrant functions in monocyte or neutrophils are increasingly recognized in systemic lupus erythematosus (SLE). We investigated whether plasma PSEP reflects disease activity in patients with SLE.

Methods: This retrospective study comprised 35 patients with SLE and 72 with non-SLE autoimmune diseases who visited our facility during the period from August 2012 to September 2015. Plasma PSEP levels and laboratory data were compared between SLE and non-SLE. Clinical markers of SLE disease activity, including SLE disease activity index 2000 (SLEDAI-2K), serum complement concentrations and serum anti-ds-DNA antibodies were assessed in correlation with plasma PSEP levels.

Results: Plasma PSEP levels in SLE were higher than those in non-SLE. This phenomenon holds true when comparing SLE and non-SLE patients in the absence of infection (p = .0008). Plasma PSEP levels in SLE patients negatively correlated with C3 (r = -0.4454, p = .0430), CH50 (r = -0.4502, p = .0406) and positively with SLEDAI-2K (r = 0.4801, p = .0237).

Conclusion: Elevated plasma PSEP levels were correlated with disease activity of SLE, suggesting inappropriate monocyte or neutrophil activation in the pathophysiology of SLE exacerbation.
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http://dx.doi.org/10.1080/14397595.2017.1408755DOI Listing
September 2018

Successful treatment with clarithromycin and/or tacrolimus for two patients with polymyalgia rheumatica.

Drug Discov Ther 2017;11(4):223-225

Department of Medicine Ⅱ, Hokkaido University Graduate School of Medicine.

Polymyalgia rheumatica (PMR) is an inflammatory rheumatic disease in the elderly. Glucocorticoids (GCs) remain the mainstay of treatment. GC therapy usually dramatically improves the clinical picture, but approximately one-third of patients experience disease recurrence when the dose is reduced. Moreover, long-term use of GCs causes adverse reactions. Macrolide antibiotics have anti-inflammatory action. Several recent studies have reported the successful treatment of rheumatoid arthritis (RA) and PMR treated using clarithromycin (CAM), a macrolide, because of its anti-inflammatory action. Tacrolimus (TAC) has been indicated as a treatment for RA in patients who failed to respond to methotrexate. Recently, a case of RA was successfully treated using CAM and TAC according to one report. Reported here are two cases of PMR treated using CAM and/or TAC. Case 1: A 73-year-old man suffering from PMR was successfully treated with prednisolone (PSL) and CAM. Because his muscle pain disappeared, CAM was discontinued. However, the pain returned after that discontinuation, so CAM was successfully administered again. Case 2: An 83-year-old man suffering from PMR was successfully treated with PSL and CAM. Because muscle pain disappeared, the CAM dosage was halved. The pain returned after the dosage was reduced, so the CAM dosage was successfully resumed and the PSL dosage was reduced. When the PSL dosage was reduced, muscle pain recurred. Because the PSL and CAM dosages were not successfully increased, TAC was also administered and was found to be effective at treating muscle pain. These two cases suggest that CAM and/or TAC are effective at treating PMR.
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http://dx.doi.org/10.5582/ddt.2017.01040DOI Listing
July 2019

Successful treatment of three patients with organizing pneumonia associated with rheumatoid arthritis using clarithromycin and prednisolone.

Drug Discov Ther 2017;11(4):218-222

Department of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine.

Macrolides have anti-inflammatory effects and have been used to treat diffuse panbronchiolitis, bronchiectasis, and cystic fibrosis. Lately, several cases of cryptogenic organizing pneumonia (COP) and radiotherapy-related organizing pneumonia (OP) that were successfully treated with macrolides considering their anti-inflammatory effects were reported. We report three cases of OP associated with rheumatoid arthritis (RA) successfully treated with clarithromycin (CAM) and prednisolone (PSL). Case 1: A 70-year-old woman suffering from RA was admitted with cough and severe dyspnea. She was diagnosed with OP associated with RA on the basis of computed tomography (CT) findings and transbronchial lung biopsy results. She was successfully treated with PSL and cyclosporine A. At the exacerbation of OP, she was successfully treated with CAM and PSL. Case 2: A 74-year-old man suffering from COP visited our department with arthralgia and articular swellings. He was diagnosed with RA, which was thought to be associated with OP. He was successfully treated with CAM and PSL. Case 3: A 54-year-old man suffering from RA presented with an exacerbation of arthralgia and articular swellings and cough. He was diagnosed with OP associated with RA on the basis of CT findings. He was successfully treated with CAM and PSL. The present cases suggest that CAM and PSL treatment may be effective in some cases of OP associated with RA.
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http://dx.doi.org/10.5582/ddt.2017.01043DOI Listing
July 2019

A case of Waldenström's macroglobulinemia treated using clarithromycin and prednisolone.

Transl Clin Pharmacol 2017 Sep 15;25(3):134-137. Epub 2017 Sep 15.

Department of Diagnostic Radiology, JCHO Hokkaido Hospital, Sapporo, Japan.

We report a case of Waldenström's macroglobulinemia (WM) treated using clarithromycin (CAM) and prednisolone (PSL). An 84-year-old woman was admitted to our hospital for bleeding after a tooth extraction and hematuria. Computed tomography showed multiple ill-defined nodules in the omentum (omental cake). Although the cause of the omental cake remained unclear, the patient was diagnosed with WM, based on the detection of M-protein of immunoglobulin (Ig) M in serum and lymphoplasmacytes in bone marrow. The bleeding tendency in the patient may have been due to acquired hemophilia and/or hyper IgM-induced platelet dysfunction. The patient was treated using CAM (800 mg/day) and PSL (10 mg/day). As a result, IgM levels gradually decreased. Because the omental cake contracted along with improvement in IgM, it was thought to be lymphoplasmacytic lymphoma-like lymphoma. This case shows that treatment using CAM and PSL may be effective in some cases of WM.
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http://dx.doi.org/10.12793/tcp.2017.25.3.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033378PMC
September 2017

Measurement of NET formation in vitro and in vivo by flow cytometry.

Cytometry A 2017 08 17;91(8):822-829. Epub 2017 Jul 17.

Faculty of Health Sciences, Hokkaido University, Sapporo, Japan.

Neutrophil extracellular traps (NETs) are extracellular chromatin fibers adorned with antimicrobial proteins, such as myeloperoxidase (MPO), which are extruded from activated neutrophils. NETosis is the metamorphosis of neutrophils with NET formation that follows decondensation of DNA and rupture of the plasma membrane. Although NETs play important roles in innate immunity, excessive formation of NETs can be harmful to the hosts. Until now, various methods for evaluation of NETs have been reported. Although each has a virtue, the gold standard has not been established. Here we demonstrate a simple, objective, and quantitative method to detect NETs using flow cytometry. This method uses a plasma membrane-impermeable DNA-binding dye, SYTOX Green. SYTOX Green-positive cells were detected in human peripheral polymorphonuclear cells exposed to a NET inducer, phorbol 12-myristate 13-acetate (PMA). The number of SYTOX Green-positive cells was increased depending on the exposure duration and concentrations of PMA. Furthermore, co-localization of MPO and plasma membrane-appendant DNA of SYTOX Green-positive cells was demonstrated. Moreover, a NET inhibitor, diphenylene iodonium, could significantly reduce the number of SYTOX Green-positive cells induced by PMA. The collective evidence suggests that SYTOX Green-positive cells include neutrophils that formed NETs. The established method could detect neutrophils that underwent NETosis but not early apoptosis with equivalence in quantification to another well-used image analysis, which is based on fluorescent staining. Additionally, NETs that were formed in vivo were also detectable by this method. It is conceivable that the established method will bring us better understanding of the relation between NETosis and human diseases. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.
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http://dx.doi.org/10.1002/cyto.a.23169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601186PMC
August 2017

The Presence of Anti-Lactoferrin Antibodies in a Subgroup of Eosinophilic Granulomatosis with Polyangiitis Patients and Their Possible Contribution to Enhancement of Neutrophil Extracellular Trap Formation.

Front Immunol 2016 23;7:636. Epub 2016 Dec 23.

Department of Pathology, Hokkaido University Graduate School of Medicine , Sapporo , Japan.

Lactoferrin (Lf) is one of the antigens of antineutrophil cytoplasmic antibodies (ANCA) and functions as an endogenous suppressor of neutrophil extracellular trap (NET) formation. However, the prevalence and pathogenicity of anti-lactoferrin antibodies (aLf) in ANCA-associated vasculitis (AAV) remain unrevealed. This study aimed to examine the significance of aLf in AAV, initially. Sixty-five sera from AAV patients, including 41 microscopic polyangiitis, 5 granulomatosis with polyangiitis, and 19 eosinophilic granulomatosis with polyangiitis (EGPA) patients, were subjected to aLf detection using enzyme-linked immunosorbent assay. Clinical characteristics were compared between aLf-positive and aLf-negative patients. Neutrophils from healthy donors were exposed to suboptimal dose (10 nM) of phorbol myristate acetate (PMA) with aLf followed by evaluation of NET formation. Results demonstrated that 4 out of 65 AAV sera (6.2%) were positive for aLf. All of them were EGPA sera (4/19, 21.1%). In EGPA, the frequency of renal involvement, serum CRP levels, and Birmingham Vasculitis Activity Score (BVAS) in the aLf-positive patients was significantly higher than those in the aLf-negative patients, and the aLf titer correlated positively with the serum CRP level and BVAS. The NET formation was particularly enhanced by combined stimulation of 10 nM PMA and 1 µg/mL aLf. IgG isolated from sera of the aLf-positive EGPA patients (250 µg/mL) enhanced NET formation induced by 10 nM of PMA, and the effect was abolished completely by absorption of the aLf. This pilot study suggests that aLf enhance NET formation induced by PMA and are associated with disease activity of EGPA.
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http://dx.doi.org/10.3389/fimmu.2016.00636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179553PMC
December 2016

Peptidylarginine Deiminase Inhibitor Suppresses Neutrophil Extracellular Trap Formation and MPO-ANCA Production.

Front Immunol 2016 8;7:227. Epub 2016 Jun 8.

Faculty of Health Sciences, Hokkaido University , Sapporo , Japan.

Myeloperoxidase-antineutrophil cytoplasmic antibody (MPO-ANCA)-associated vasculitis is a systemic small-vessel vasculitis, wherein, MPO-ANCA plays a critical role in the pathogenesis. Neutrophil extracellular traps (NETs) released from activated neutrophils are composed of extracellular web-like DNA and antimicrobial proteins, including MPO. Diverse stimuli, such as phorbol myristate acetate (PMA) and ligands of toll-like receptors (TLR), induce NETs. Although TLR-mediated NET formation can occur with preservation of living neutrophilic functions (called vital NETosis), PMA-stimulated neutrophils undergo cell death with NET formation (called suicidal NETosis). In the process of suicidal NETosis, histones are citrullinated by peptidylarginine deiminase 4 (PAD4). Since this step is necessary for decondensation of DNA, PAD4 plays a pivotal role in suicidal NETosis. Although NETs are essential for elimination of microorganisms, excessive formation of NETs has been suggested to be implicated in MPO-ANCA production. This study aimed to determine if pan-PAD inhibitors could suppress MPO-ANCA production in vivo. At first, NETs were induced in peripheral blood neutrophils derived from healthy donors (1 × 10(6)/ml) by stimulation with 20 nM PMA with or without 20 μM propylthiouracil (PTU), an anti-thyroid drug. We then determined that the in vitro NET formation was inhibited completely by 200 μM Cl-amidine, a pan-PAD inhibitor. Next, we established mouse models with MPO-ANCA production. BALB/c mice were given intraperitoneal (i.p.) injection of PMA (50 ng at days 0 and 7) and oral PTU (2.5 mg/day) for 2 weeks. These mice were divided into two groups; the first group was given daily i.p. injection of PBS (200 μl/day) (n = 13) and the other group with daily i.p. injection of Cl-amidine (0.3 mg/200 μl PBS/day) (n = 7). Two weeks later, citrullination as an indicator of NET formation in the peritoneum and serum MPO-ANCA titer was compared between the two groups. Results demonstrated that citrullination in the peritoneum was significantly reduced in the Cl-amidine-treated mice compared with the vehicle-injected control mice (38% reduction). Additionally, the serum MPO-ANCA titer of the Cl-amidine-treated mice (32.3 ± 31.0 ng/ml) was significantly lower than that in the vehicle-injected mice (132.1 ± 41.6 ng/ml). The collective findings indicate that excessive formation of NETs may be implicated in MPO-ANCA production in vivo.
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http://dx.doi.org/10.3389/fimmu.2016.00227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4896908PMC
July 2016

NETosis markers: Quest for specific, objective, and quantitative markers.

Clin Chim Acta 2016 Aug 31;459:89-93. Epub 2016 May 31.

Faculty of Health Sciences, Hokkaido University, Sapporo, Japan. Electronic address:

More than 10years have passed since the discovery of neutrophil extracellular traps (NETs) in 2004. NETs are extracellular web-like DNA decorated with antimicrobial proteins, which are released from activated neutrophils. The state of neutrophils with NET formation is called NETosis. It has been realized that NETosis includes suicidal NETosis and vital NETosis. The former state means cell death of neutrophils, whereas the latter state preserves living neutrophilic functions. Although both suicidal and vital NETosis play essential roles in elimination of microorganisms, excessive formation of NETs, especially the ones derived from suicidal NETosis, can harm the hosts. Therefore, the discovery of NETosis markers and development of evaluation methods are important. In this review, we compare the methods for evaluating NETosis, including immunocytological and immunohistological detection of co-localized neutrophil-derived proteins and extracellular DNA, and citrullinated histones, detection of NET remnants in fluid samples, and flow cytometric detection of cell-appendant NET components, with focus on the specificity, objectivity, and quantitativity. Since the gold standard marker of NETosis or method of NET detection has not been established yet, researchers should choose the most appropriate marker or method in each situation based on the knowledge of the respective virtues and faults.
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http://dx.doi.org/10.1016/j.cca.2016.05.029DOI Listing
August 2016

Circulating Neutrophil Extracellular Trap Levels in Well-Controlled Type 2 Diabetes and Pathway Involved in Their Formation Induced by High-Dose Glucose.

Pathobiology 2016 18;83(5):243-51. Epub 2016 May 18.

Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

Objectives: Although intensive therapy for type 2 diabetes (T2D) prevents microvascular complications, 10% of well-controlled T2D patients develop microangiopathy. Therefore, the identification of risk markers for microvascular complications in well-controlled T2D patients is important. Recent studies have demonstrated that high-dose glucose induces neutrophil extracellular trap (NET) formation, which can be a risk for microvascular disorders. Thus, we attempted to determine the correlation of circulating NET levels with clinical/laboratory parameters in well-controlled T2D patients and to reveal the mechanism of NET formation induced by high-dose glucose.

Methods: Circulating NET levels represented by myeloperoxidase (MPO)-DNA complexes in the serum of 11 well-controlled T2D patients and 13 healthy volunteers were determined by enzyme-linked immunosorbent assay. The pathway involved in the NET formation induced by high-dose glucose was determined using specific inhibitors.

Results: Serum MPO-DNA complex levels were significantly higher in some well-controlled T2D patients in correlation with the clinical/laboratory parameters which have been regarded as risk markers for microvascular complications. The aldose reductase inhibitor, ranirestat, could inhibit the NET formation induced by high-dose glucose.

Conclusions: Elevated levels of circulating NETs can be a risk marker for microvascular complications in well-controlled T2D patients. The polyol pathway is involved in the NET formation induced by high-dose glucose.
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http://dx.doi.org/10.1159/000444881DOI Listing
January 2017

Post-steroid neuropsychiatric manifestations are significantly more frequent in SLE compared with other systemic autoimmune diseases and predict better prognosis compared with de novo neuropsychiatric SLE.

Autoimmun Rev 2016 Aug 22;15(8):786-94. Epub 2016 Mar 22.

Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.

In patients with systemic lupus erythematosus (SLE), neuropsychiatric (NP) symptoms sometimes occur after administration of corticosteroids, making differential diagnosis between NPSLE and steroid-induced psychosis challenging for clinicians. The aim of this study was to clarify the characteristics of post-steroid NP disease (PSNP) in patients with SLE. Clinical courses of 146 patients with SLE and 162 with other systemic autoimmune diseases, all in the absence of NP manifestations on admission, were retrospectively analyzed. Forty-three NPSLE patients on admission (de novo NPSLE) were also investigated. All patients were consecutively recruited and treated with 40mg/day or more of prednisolone in Hokkaido University Hospital between April 2002 and March 2015. The prevalence of PSNP was strikingly higher in SLE patients than other systemic autoimmune diseases (24.7% vs. 7.4%, OR 4.09, 95% CI 2.04-8.22). As independent risk factors to develop PSNP in SLE patients, past history of mental disorder and the presence of antiphospholipid syndrome were identified using multiple logistic regression analysis. In patients with PSNP-SLE, mood disorder was significantly more frequent than in de novo NPSLE (47.2% vs. 20.9%, OR 3.38, 95% CI 1.26-9.04). Of PSNP-SLE patients, two-thirds were with one or more abnormal findings in cerebrospinal fluid, electroencephalogram, MRI or SPECT. Majority of our PSNP-SLE patients received intensified immunosuppressive treatments and experienced improvement in most cases. PSNP-SLE had better relapse-free survival than de novo NPSLE (p<0.05, log rank test). In conclusion, PSNP frequently occurred in patients with SLE and treated successfully with immunosuppressive therapy, indicating that NPSLE is likely to harbor patients with PSNP-SLE.
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http://dx.doi.org/10.1016/j.autrev.2016.03.017DOI Listing
August 2016

The responses of macrophages in interaction with neutrophils that undergo NETosis.

J Autoimmun 2016 Feb 4;67:19-28. Epub 2015 Sep 4.

Faculty of Health Sciences, Hokkaido University, Sapporo 0600812, Japan. Electronic address:

Neutrophil extracellular traps (NETs) are net-like chromatin fibers decorated with antimicrobial proteins, which are released from dying neutrophils. The death of neutrophils with NET formation is called NETosis. Although NETs play important roles in the innate immunity, especially in the elimination of microbes, the extracellular release of DNA and intra-cytoplasmic/nuclear proteins can, on the other hand, result in diverse adversities to the hosts. Therefore, NETosis is adequately regulated in vivo. Currently, two mechanisms, namely DNase I-dependent digestion and phagocytosis by macrophages, have been shown as such regulatory mechanisms. In this study, we focused on the interaction of macrophages and neutrophils that underwent NETosis. Results demonstrated that macrophages displayed a phenotype-dependent response after degradation of NETs. Several hours after the interaction, M2 macrophages induced a pro-inflammatory response, while M1 macrophages underwent cell death with nuclear decondensation. This nuclear decondensation of M1 macrophages occurred in a peptidylarginine deiminase 4-dependent manner and resulted in a local release of extracellular DNA. Thereafter, M1 macrophages degraded DNA derived from themselves in a caspase-activated DNase-dependent manner resulting in the clearance of extracellular DNA within 24 h. This transient increase and subsequent clearance mechanism of extracellular DNA seems very reasonable in terms of the double-edged sword-like property of NETs. The collective findings demonstrate a novel phenotype- and time-dependent regulation of NETosis by macrophages.
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http://dx.doi.org/10.1016/j.jaut.2015.08.018DOI Listing
February 2016

Enhanced formation and disordered regulation of NETs in myeloperoxidase-ANCA-associated microscopic polyangiitis.

J Am Soc Nephrol 2014 May 2;25(5):990-7. Epub 2014 Jan 2.

Faculty of Health Sciences, Hokkaido University, Sapporo, Japan

Microscopic polyangiitis (MPA) is an ANCA-associated vasculitis that affects small vessels, especially renal glomeruli. We recently demonstrated that the abnormal formation and impaired degradation of neutrophil extracellular traps (NETs) may be crucially involved in the generation of myeloperoxidase (MPO)-ANCA and subsequent development of MPA. This study assessed the formation and regulation of NETs in patients with MPO-ANCA-associated MPA. Peripheral blood samples were obtained from 38 patients with MPO-ANCA-associated MPA, 23 patients with systemic lupus erythematosus (SLE), and 8 healthy controls. IgG eluted from MPO-ANCA-associated MPA sera demonstrated the highest ability to induce NETs, and this ability correlated with disease activity and paralleled ANCA affinity for MPO. Moreover, addition of recombinant human MPO to these IgG samples reduced NET induction. Additionally, MPO-ANCA-associated MPA sera exhibited lower rates of NET degradation that recovered partially upon depletion of IgG. The activity of DNase I, an important regulator of NETs, was also lower in MPO-ANCA-associated MPA and SLE sera. IgG depletion from MPO-ANCA-associated MPA sera partially restored the rate of NET degradation, and addition of DNase I synergistically enhanced this restoration. Addition of anti-MPO antibodies did not inhibit DNase I activity, and some MPO-ANCA-associated MPA sera contained anti-NET antibodies at levels not correlated with MPO-ANCA titers, suggesting the involvement of unidentified autoantibodies as well. The collective evidence suggests a vicious cycle involving MPO-ANCA and the regulation of NETs could be critically involved in the pathogenesis of MPO-ANCA-associated MPA.
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http://dx.doi.org/10.1681/ASN.2013060606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4005303PMC
May 2014

[Clinical significance of antiphospholipid antibody measured by EliA anticardiolipin antibodies and anti-β2Glycoprotein I antibodies in antiphospholipid syndrome].

Nihon Rinsho Meneki Gakkai Kaishi 2014 ;37(5):430-6

Division of Rheumatology, Endocrinology and Nephrology, Graduate School of Medicine, Hokkaido University.

Anticardiolipin antibodies (aCL-IgG/IgM) and anti-β2-glycoprotein I antibodies (aβ2GPI-IgG/IgM) are laboratory tests included in the current classification criteria for definite antiphospholipid syndrome (APS). However, not all of these assays have been commercially available in Japan. We investigated the efficacy of aCL-IgG/IgM and aβ2GPI-IgG/IgM assays using fluorescence enzyme immunoassay (Phadia:EliA(TM)) for the diagnosis of APS in Japan. This study comprised 229 sera from patients (100 with APS and 129 without APS). The diagnosis of APS was made according to Sydney revised Sapporo criteria. EliA(TM)Cardiolipin and EliA(TM)β2-Glycoprotein (Phadia AB. Uppsala Sweden) were used to detect aCL IgG/M and aβ2GPI IgG/M, respectively. Sensitivity, specificity and area under the receiver operating characteristic curve (AUC) were as follows; aCL-IgG (45%, 94%, 0.80), aCL-IgM (20%, 94%, 0.54), aβ2GPI-IgG (33%, 98%, 0.88) and aβ2GPI-IgM (16%, 99%, 0.64) respectively. aCL-IgM, aβ2GPI-IgG or aβ2GPI-IgM were detected in 10 patients (18%) with aCL-IgG negative. The use of Phadia:EliA(TM)antiphospholipid antibodies assays improve the diagnostic yield of thrombotic risk in APS patients.
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http://dx.doi.org/10.2177/jsci.37.430DOI Listing
October 2015

Possible linkage between microscopic polyangiitis and thrombosis via neutrophil extracellular traps.

Clin Exp Rheumatol 2014 Jan-Feb;32(1):149-50. Epub 2013 Dec 9.

Undergraduate Student, Hokkaido University School of Medicine, Sapporo, Japan.

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October 2014

Immunity-based diagnosis for a motherboard.

Sensors (Basel) 2011 18;11(4):4462-73. Epub 2011 Apr 18.

Department of Information Network and Communication, Kanagawa Institute of Technology, Atsugi, Kanagawa, Japan.

We have utilized immunity-based diagnosis to detect abnormal behavior of components on a motherboard. The immunity-based diagnostic model monitors voltages of some components, CPU temperatures, and fan speeds. We simulated abnormal behaviors of some components on the motherboard, and we utilized the immunity-based diagnostic model to evaluate motherboard sensors in two experiments. These experiments showed that the immunity-based diagnostic model was an effective method for detecting abnormal behavior of components on the motherboard.
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http://dx.doi.org/10.3390/s110404462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3231323PMC
August 2012
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