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B lineage cells are critically involved in ANCA-associated vasculitis (AAV), evidenced by alterations in circulating B cell subsets and beneficial clinical effects of rituximab (anti-CD20) therapy. This treatment renders a long-term, peripheral B cell depletion, but allows for the survival of long-lived plasma cells. Therefore, there is an unmet need for more reversible and full B lineage cell targeting approaches. To find potential novel therapeutic targets, RNA sequencing of CD27 + memory B cells of patients with active AAV was performed, revealing an upregulated NF-κB-associated gene signature. NF-κB signaling pathways act downstream of various B cell surface receptors, including the BCR, CD40, BAFFR and TLRs, and are essential for B cell responses. Here we demonstrate that novel pharmacological inhibitors of NF-κB inducing kinase (NIK, non-canonical NF-κB signaling) and inhibitor-of-κB-kinase-β (IKKβ, canonical NF-κB signaling) can effectively inhibit NF-κB signaling in B cells, whereas T cell responses were largely unaffected. Moreover, both inhibitors significantly reduced B cell proliferation, differentiation and production of antibodies, including proteinase-3 (PR3) autoantibodies, in B lineage cells of AAV patients. These findings indicate that targeting NF-κB, particularly NIK, may be an effective, novel B lineage cell targeted therapy for AAV and other autoimmune diseases with prominent B cell involvement., Competing Interests: Declaration of competing interest “The authors have declared that no conflict of interest exists”., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Immunoglobulin G (IgG) contains a conserved N-glycan in the fragment crystallizable (Fc), modulating its structure and effector functions. In anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) alterations of IgG Fc-glycosylation have been observed to correlate with the disease course. Here, we examined longitudinal changes in N - linked Fc glycans of IgG in an AAV patient cohort and their relationship with disease flares., Methods: Using liquid chromatography coupled with mass spectrometry, we analysed IgG Fc-glycosylation in 410 longitudinal samples from 96 individuals with AAV., Results: Analysis of the cross-sectional differences as well as longitudinal changes demonstrated that IgGs of relapsing PR3-ANCA patients have higher ΔFc-bisection at diagnosis ( P = 0.004) and exhibit a decrease in Fc-sialylation prior to the relapse ( P = 0.0004), discriminating them from non-relapsing patients. Most importantly, PR3-ANCA patients who experienced an ANCA rise and relapsed shortly thereafter, exhibit lower IgG Fc-fucosylation levels compared to non-relapsing patients already 9 months before relapse ( P = 0.02)., Discussion: Our data indicate that IgG Fc-bisection correlates with long-term treatment outcome, while lower IgG Fc-fucosylation and sialylation associate with impending relapse. Overall, our study replicated the previously published reduction in total IgG Fc-sialylation at the time of relapse, but showed additionally that its onset precedes relapse. Furthermore, our findings on IgG fucosylation and bisection are entirely new. All these IgG Fc-glycosylation features may have the potential to predict a relapse either independently or in combination with known risk factors, such as a rise in ANCA titre., Competing Interests: IW was employed by Genos Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wojcik, Wuhrer, Heeringa, Stegeman, Rutgers and Falck.)

The introduction of immunosuppressive therapy for ANCA-associated vasculitis (AAV) has greatly improved outcomes, though patients now accumulate damage from vasculitis activity and adverse effects of treatment. Prediction of treatment outcomes using gene variants might help reduce this damage by allowing for personalized treatment. Several studies have studied genetic polymorphisms in relation to treatment outcomes of AAV. This review gives an overview of these studies, discussing both gene polymorphisms associated with inflammatory pathways (potentially influencing disease outcomes such as activity, severity, and relapse risk) and pharmacogenetics (potentially influencing drug metabolism and/or drug response). Subsequently, potential benefits of testing genetic variants for AAV and the steps needed for its implementation in clinical practice are discussed. The conclusion of this review is that measurement of most polymorphisms is currently not indicated in clinical practice.

Background and Objectives: Cyclophosphamide has been the mainstay of treatment of ANCA-associated vasculitis. However, cyclophosphamide has unfavorable side effects and alternatives are needed. Evidence suggests that mycophenolate mofetil can induce sustained remission in nonlife-threatening disease. The purpose of this study was to compare the efficacy and safety of mycophenolate mofetil versus cyclophosphamide for the induction treatment of nonlife-threatening relapses of proteinase 3-ANCA- and myeloperoxidase-ANCA-associated vasculitis., Design, Setting, Participants, & Measurements: We conducted a multicenter randomized, controlled trial. Participants with a first or second relapse of ANCA-associated vasculitis were randomized to induction treatment with cyclophosphamide or mycophenolate mofetil both in combination with glucocorticoids. Maintenance therapy consisted of azathioprine in both arms. Primary outcome was remission at 6 months, and secondary outcomes included disease-free survival at 2 and 4 years., Results: Eighty-four participants were enrolled, of whom 41 received mycophenolate mofetil and 43 received cyclophosphamide. Eighty-nine percent of participants were proteinase 3-ANCA positive. At 6 months, 27 (66%) mycophenolate mofetil-treated participants versus 35 (81%) cyclophosphamide-treated participants were in remission ( P =0.11). Disease-free survival rates at 2 and 4 years were 61% and 39% for cyclophosphamide, respectively, and 43% and 32% for mycophenolate mofetil, respectively (at 4 years, log rank test, P =0.17)., Conclusions: We did not demonstrate mycophenolate mofetil to be similarly effective as cyclophosphamide in inducing remission of relapsed ANCA-associated vasculitis. However, mycophenolate mofetil might be an alternative to cyclophosphamide for the treatment of selected patients with nonlife-threatening relapses., (Copyright © 2019 by the American Society of Nephrology.)

Objectives: We aimed to investigate whether five potential functional haplotypes of the glucocorticoid receptor (GR) gene and a single-nucleotide polymorphism of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) are associated with clinical outcome in ANCA-associated vasculitis., Methods: Patients diagnosed with ANCA-associated vasculitis (n = 241) were genotyped for five polymorphisms of the GR gene and one polymorphism of the HSD11B1 gene. GR gene haplotypes were predicted based on genotyping results. Relapse-free survival, mortality, renal survival, metabolic adverse events and infections were compared between carriers and non-carriers of GR haplotypes and the HSD11B1 genotype., Results: Carriers of haplotype 4 (ER22/23EK + 9β+TthIII1) of GR had a significantly higher 5-year mortality risk [hazard ratio (HR) 4.5 (95% CI 1.6, 12.8)] and had a higher risk of developing end-stage renal disease [HR 7.4 (95% CI 1.9, 28.7)]. Carriers of a minor variant of HSD11B1 more frequently experienced relapse [HR 2.5 (95% CI 1.5, 4.1)] except if they also carried haplotype 1 (BclI) of GR. Homozygous carriers of haplotype 1 had a higher risk of developing dyslipidaemia [HR 4.1 (95% CI 1.8, 9.6)]. The occurrence of infections did not differ between GR haplotypes and HSD11B1 genotypes., Conclusion: Haplotypes 1 and 4 of GR and a polymorphism of the HSD11B1 gene were associated with clinically relevant inflammatory and metabolic outcomes in ANCA-associated vasculitis., (© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Azathioprine hypersensitivity syndrome is a rare complication of azathioprine therapy. Its symptoms resemble infection or relapse of inflammatory disease, hindering correct diagnosis. Current literature is limited to sporadic case reports and reviews., Objective: To estimate the incidence of azathioprine hypersensitivity syndrome and describe its characteristics in the context of an observational cohort of patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Also, to facilitate early recognition and awareness among clinicians., Methods: Within a cohort of 290 patients with ANCA-associated vasculitis receiving azathioprine maintenance therapy, frequency of azathioprine hypersensitivity was described and characteristics were compared between hypersensitive and nonhypersensitive patients. Clinical picture, laboratory abnormalities, and concurrent medication of patients with azathioprine hypersensitivity were described., Results: Of 290 patients, 25 (9%) experienced azathioprine hypersensitivity after a median of 14 (interquartile range [IQR] 12-18) days. Frequent symptoms were fever (100%), malaise (60%), arthralgia (36%), and rash (32%). All patients used prednisolone (median 10 mg/day, IQR 9.4-16.3 mg/day) at the time of the hypersensitivity reaction. Most patients had a rise in C-reactive protein (CRP), leukocyte counts, and neutrophil counts, but no eosinophilia. Thiopurine S-methyltransferase (TPMT) activity was significantly lower in hypersensitive patients (median 74.4 [IQR 58.0-80.1] nmol/gHb/L) compared with controls (median 81.4 [71.9-90.5] nmol/gHb/L), P = .01. Hypersensitive patients had a higher risk of relapse (hazard ratio 2.2, 95% confidence interval 1.2-4.2; P = .01)., Conclusions: Azathioprine hypersensitivity syndrome is strikingly common in ANCA-associated vasculitis, might be associated with reduced TPMT activity, is accompanied by an increase in neutrophil counts, and may occur even during concomitant prednisolone therapy. Proper recognition may prevent unnecessary hospital procedures and damage to the patient., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Objective: Physical quality of life is reduced in ANCA-associated vasculitis (AAV). This study aims to investigate whether this may be explained by reduced muscle strength and physical activity resulting from disease damage and steroid myopathy., Methods: Forty-eight AAV patients were sequentially included from the outpatient clinic. Patients in different stages of disease and treatment underwent measurements of muscle strength and anthropometric parameters. Patients filled in physical activity (Baecke) and quality of life questionnaires (RAND-36) and carried an accelerometer for a week. Muscle strength and physical activity were compared to quality of life, prednisolone use and disease duration., Results: Most AAV patients had lower knee extension (76%) and elbow flexion (67%) forces than expected based on healthy norms. Also, physical (P<0.001) and mental (P = 0.01) quality of life were significantly reduced compared to healthy norm values. Lower knee extension force (P = 0.009), younger age <70 (P<0.001) and relapse of vasculitis (P = 0.003) were associated with lower age-adjusted physical quality of life. Lower Baecke index (P = 0.006), higher prednisolone dose (P = 0.005) and ENT involvement (P = 0.006) were associated with lower age-adjusted mental quality of life. Leg muscle strength showed no association with current or cumulative prednisolone use. Disease duration was longer in patients with knee extension force below healthy norms (P = 0.006)., Conclusion: Knee extension force and physical activity are positively associated with quality of life in AAV. Knee extension force decreases with longer disease duration, suggesting that disease- and treatment-related damage have a cumulative negative effect on muscle strength., Competing Interests: The authors have declared that no competing interests exist.

Background: Early detection of renal involvement in anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is of major clinical importance to allow prompt initiation of treatment and limit renal damage. Urinary soluble cluster of differentiation 163 (usCD163) has recently been identified as a potential biomarker for active renal vasculitis. However, a significant number of patients with active renal vasculitis test negative using usCD163. We therefore studied whether soluble CD25 (sCD25), a T cell activation marker, could improve the detection of renal flares in AAV., Methods: sCD25 and sCD163 levels in serum and urine were measured by enzyme-linked immunosorbent assay in 72 patients with active renal AAV, 20 with active extrarenal disease, 62 patients in remission and 18 healthy controls. Urinary and blood CD4+ T and CD4+ T effector memory (TEM) cell counts were measured in 22 patients with active renal vasculitis. Receiver operating characteristics (ROC) curves were generated and recursive partitioning was used to calculate whether usCD25 and serum soluble CD25 (ssCD25) add utility to usCD163., Results: usCD25, ssCD25 and usCD163 levels were significantly higher during active renal disease and significantly decreased after induction of remission. A combination of usCD25, usCD163 and ssCD25 outperformed all individual markers (sensitivity 84.7%, specificity 95.1%). Patients positive for sCD25 but negative for usCD163 (n = 10) had significantly higher C-reactive protein levels and significantly lower serum creatinine and proteinuria levels compared with the usCD163-positive patients. usCD25 correlated positively with urinary CD4+ T and CD4+ TEM cell numbers, whereas ssCD25 correlated negatively with circulating CD4+ T and CD4+ TEM cells., Conclusion: Measurement of usCD25 and ssCD25 complements usCD163 in the detection of active renal vasculitis.

ANCA-associated vasculitis (AAV) is characterized by inflammation and destruction of small and medium-sized vessels. Current management strategies for AAV have been validated in large groups of patients. However, recent insights indicate that distinct patient subsets may actually exist within AAV, thereby justifying the development of more personalized treatment strategies. In this review, we discuss current evidence for a better classification of AAV based on ANCA type. We describe how thus defined categories of AAV patients may differ in genetic background, clinical presentation, immune pathology, response to treatment and disease outcome. We also explore how these insights may provide a rationale for targeted treatments in different categories of AAV patients. Finally, we provide recommendations on how to further establish precision medicine in AAV.

Objective: Azathioprine is a widely used immunosuppressive drug. Genetic polymorphisms and activity of the enzyme thiopurine methyltransferase (TPMT) have been associated with azathioprine efficacy and toxicity in several populations. We investigated whether these associations also exist for ANCA associated vasculitis (AAV) patients, who receive azathioprine maintenance therapy after remission induction with cyclophosphamide., Methods: 207 AAV patients treated with cyclophosphamide induction and azathioprine maintenance therapy were included and followed for 60 months. TPMT genotype and tertiles of TPMT activity were compared to relapse free survival and occurrence of adverse events, particularly leukopenia. Multivariable regression was performed to account for confounders., Results: In univariable analysis, relapse free survival was not significantly associated with TPMT genotype (P = 0.41) or TPMT activity (P = 0.07), although it tended to be longer in lower tertiles of TPMT activity. There was no significant association of TPMT genotype and activity with occurrence of any adverse event. In multiple regression, leukocyte counts at the end of cyclophosphamide induction were related to risk of leukopenia during azathioprine therapy [P<0.001; OR 0.54 (95% CI 0.43-0.68)] and risk of relapse during follow-up [P = 0.001; HR 1.17 (95% CI 1.07-1.29)] irrespective of TMPT genotype or activity., Conclusion: TPMT genotype and activity were not independent predictors of relapse, and could not predict leukopenia or other adverse effects from azathioprine. Leukocyte counts after cyclophosphamide induction were related to both outcomes, implying a greater influence of cyclophosphamide response compared to azathioprine and TPMT in AAV patients.

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases characterized by necrotizing inflammation of small- to medium-sized blood vessels, affecting primarily the lungs and kidneys. Both animal and human studies show that the balance between inflammatory- and regulatory T- and B cells determines the AAV disease pathogenesis. Recent evidence shows malfunctioning of the regulatory lymphocyte compartment in AAV. In this review we summarize the immune regulatory properties of both T- and B cells in patients with AAV and discuss how aberrations herein might contribute to the disease pathogenesis., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: We studied whether in ANCA-associated vasculitis patients, duration of AZA maintenance influenced relapse rate during long-term follow-up., Methods: Three hundred and eighty newly diagnosed ANCA-associated vasculitis patients from six European multicentre studies treated with AZA maintenance were included; 58% were male, median age at diagnosis 59.4 years (interquartile range: 48.3-68.2 years); granulomatosis with polyangiitis, n = 236; microscopic polyangiitis, n = 132; or renal limited vasculitis, n = 12. Patients were grouped according to the duration of AZA maintenance after remission induction: ⩽18 months, ⩽24 months, ⩽36 months, ⩽48 months or > 48 months. Primary outcome was relapse-free survival at 60 months., Results: During follow-up, 84 first relapses occurred during AZA-maintenance therapy (1 relapse per 117 patient months) and 71 after withdrawal of AZA (1 relapse/113 months). During the first 12 months after withdrawal, 20 relapses occurred (1 relapse/119 months) and 29 relapses >12 months after withdrawal (1 relapse/186 months). Relapse-free survival at 60 months was 65.3% for patients receiving AZA maintenance >18 months after diagnosis vs 55% for those who discontinued maintenance ⩽18 months (P = 0.11). Relapse-free survival was associated with induction therapy (i.v. vs oral) and ANCA specificity (PR3-ANCA vs MPO-ANCA/negative)., Conclusion: Post hoc analysis of combined trial data suggest that stopping AZA maintenance therapy does not lead to a significant increase in relapse rate and AZA maintenance for more than 18 months after diagnosis does not significantly influence relapse-free survival. ANCA specificity has more effect on relapse-free survival than duration of maintenance therapy and should be used to tailor therapy individually., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com)

Background: Cytoplasmic anti-neutrophil cytoplasmic antibody (C-ANCA) positivity at remission has been associated with an increased relapse rate in patients with proteinase 3 anti-neutrophil cytoplasmic antibody-associated vasculitis (PR3-AAV) after a switch to azathioprine maintenance therapy. We therefore hypothesized that extended azathioprine maintenance therapy could reduce the incidence of relapse in this setting., Methods: Patients newly diagnosed with PR3-AAV at 12 centres in The Netherlands during 2003-11 who received a standardized induction regimen consisting of oral cyclophosphamide and corticosteroids were enrolled (n = 131). Patients were randomized to standard or extended azathioprine maintenance therapy when C-ANCA was positive at the time of stable remission. Standard maintenance treatment consisted of azathioprine (1.5-2.0 mg/kg) until 1 year after diagnosis and subsequent tapering to 25 mg every 3 months. Extended azathioprine maintenance therapy (1.5-2.0 mg/kg) was continued until 4 years after diagnosis and tapered thereafter. The primary endpoint was relapse-free survival at 4 years after diagnosis., Results: In patients with PR3-AAV who were C-ANCA positive at the time of stable remission, relapse-free survival at 4 years after diagnosis did not differ significantly between standard azathioprine (n = 24) and extended azathioprine (n = 21) maintenance therapy (P = 0.40). There was also no significant difference in relapse-free survival between patients receiving standard azathioprine (n = 106) versus extended azathioprine maintenance therapy (n = 21; P = 0.94). In addition, there was no difference in the relapse rate between patients with PR3-AAV who were C-ANCA positive (n = 45) at the time of remission versus patients who became C-ANCA negative at the time of remission (n = 82; P = 0.62)., Conclusions: This randomized trial suggests that extended azathioprine maintenance therapy has only a limited effect on the prevention of relapse in patients with PR3-AAV at 4 years after diagnosis. Moreover, positive C-ANCA status at stable remission was not associated with an increased rate of relapse., Trial Registration: ClinicalTrials.gov NCT 00128895., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objective: One of the side effects of cyclophosphamide is earlier menopause and primary ovarian insufficiency. This study was undertaken to investigate the onset of menopause and the incidence of primary ovarian insufficiency in women with antineutrophil cytoplasmic antibody-associated vasculitis (AAV), especially after treatment with orally administered cyclophosphamide., Methods: We retrospectively studied the onset of menopause and the influence of cyclophosphamide in women diagnosed as having AAV in our center between 1970 and 2012., Results: Ninety-four premenopausal women diagnosed as having AAV were included. Sixty-seven patients received cyclophosphamide, and 27 received other, mostly immunosuppressive, medication. Forty-six cyclophosphamide-treated women developed menopause, 22 of whom were considered to have primary ovarian insufficiency. None of the patients who were not treated with cyclophosphamide developed primary ovarian insufficiency. There was a significant association between a cumulative cyclophosphamide dose of >16.6 gm, versus a cumulative dose of <16.6 gm, and menopause (χ(2)  = 8.72, P = 0.003; odds ratio [OR] 2.60 [95% confidence interval 1.38-4.90]). In addition, there was a significant association between a cumulative cyclophosphamide dose of <16.6 gm, versus no cyclophosphamide exposure, and menopause (χ(2)  = 16.37, P < 0.001; OR 7.32 [95% confidence interval 2.79-19.20]). Both women who received cyclophosphamide and those who did not experienced involuntary childlessness., Conclusion: Earlier menopause and primary ovarian insufficiency frequently develop after oral cyclophosphamide therapy in premenopausal women with AAV. Involuntary childlessness is common after the development of primary ovarian insufficiency, but it also occurs in women not treated with cyclophosphamide. These findings emphasize the importance of the use of drugs that are not toxic to gonadal function in women of childbearing age., (© 2016, American College of Rheumatology.)

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are severe chronic auto-immune diseases in which the small vessels are inflamed. Nowadays, in the majority of patients disease can be brought into remission with cyclophosphamide and corticosteroids. However, depending upon disease characteristics patients with AAV have a risk of 29-60% to experience relapses of disease within 5 years despite maintenance therapy after induction of remission with less toxic agents, such as azathioprine, methotrexate or mycophenolate mofetil. More recently, rituximab has been found effective in both induction and maintenance of remission in AAV. This review discusses the different aspects of maintenance therapy in AAV based on reported cohorts and studies, including the different agents, therapy duration, efficacy or lack thereof and future directions. Finally, recommendations are made who to treat and for how long., (© The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Objective: We evaluated 26 patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) with progressive disease despite treatment with cyclophosphamide and steroids treated with additional plasmapheresis and compared outcome with 50 matched-disease controls., Methods: Patients diagnosed with AAV and treated with cyclophosphamide from January 1990 to December 2009 (n = 272) were included when plasmapheresis was not started at diagnosis but added for progressive disease during initial standard therapy (n = 26). We selected controls equal for age, Birmingham vasculitis activity score, and creatinine at diagnosis. Primary endpoint was estimated glomerular filtration rate (eGFR) or death., Results: Plasmapheresis was added 18 days (range 5-41) after start of therapy. In 11 patients, a rise in serum creatinine >30% led to plasmapheresis; insufficient response to induction (n = 11), progressive pulmonary disease (n = 3), or progressive necrotic lesions (n = 1) were other indications.In the plasmapheresis group, six patients were in need of renal replacement therapy (RRT), and three controls. Five years after diagnosis, four patients had died in the plasmapheresis against eight controls (P = 0.94). At baseline, mean eGFR was 44 ml/min/1.73 m(2) in plasmapheresis group versus 43 ml/min/1.73 m(2) in controls. At start of plasmapheresis, eGFR was 26 ml/min/1.73 m(2) (P = 0.003), at 6 months mean eGFR had significantly improved to 44 ml/min/1.73 m(2) (P = 0.0003), comparable to eGFR in controls, 48 ml/min/1.73 m(2). During long-term follow-up, there was no difference in renal function between the groups., Conclusion: AAV patients with progressive disease despite standard induction therapy in whom plasmapheresis was added had significant improvement in renal function and similar long-term outcome in both renal and patient survival as matched disease controls., (© 2014 Wiley Periodicals, Inc.)

Objective: Regulatory B cells (Bregs) constitute a subset of B cells with immunomodulatory properties. Numerical and functional alterations in the Breg compartment have been associated with autoimmunity. The aim of this study was to assess the frequency and function of Bregs in patients with ANCA-associated vasculitis (AAV)., Methods: B cell subsets were determined in the peripheral blood of 48 AAV patients (12 active, 36 in remission) and 41 healthy controls (HCs) by flow cytometry. Bregs were defined within the CD19(+) population as CD24(hi)CD38(hi) or CD24(hi)CD27(+) cells. The percentage of IL-10-positive B cells in circulation was analysed by flow cytometry. Sorted CD19(+) B cells were co-cultured with monocytes to evaluate their capacity to inhibit monocyte TNF-α production upon lipopolysaccharide stimulation., Results: The frequency of circulating CD19(+)CD24(hi)CD38(hi) cells was not different in AAV patients in remission compared with HCs, but was decreased in patients with active disease [mean in HCs 5.5% (s.d. 1.6) vs active 3.8% (s.d. 2.8), P = 0.0104]. Furthermore, the percentage of CD19(+)CD24(hi)CD27(+) cells was significantly decreased in both remission and active patients when compared with HCs [HCs 15.0% (s.d. 9.3) vs remission 6.6% (s.d. 4.4) (P < 0.0001) vs active 6.4% (s.d. 6.2) (P = 0.0006)]. The frequency of IL-10-positive B cells was comparable between patients and HCs. B cells from AAV patients suppressed monocyte TNF-α production to a similar extent to cells from HCs., Conclusion: Based on immunophenotypic classification, Bregs are numerically diminished in AAV patients. However, B cell function in terms of IL-10 production and their capacity to suppress monocyte activation is not compromised in AAV patients in remission., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objective: Differential gene expression in CD177+ and CD177- neutrophils was investigated, in order to detect possible differences in neutrophil function which could be related to the pathogenesis of ANCA-associated Vasculitides (AAV)., Methods: Neutrophils were isolated from healthy controls (HC) with high, negative or bimodal CD177 expression, and sorted into CD177+ and CD177- subpopulations. Total RNA was screened for expression of 24,000 probes with Illumina Ref-8 Beadchips. Genes showing differential expression between CD177+ and CD177- subsets in microarray analysis were re-assessed using quantitative-PCR. CD177 expression on neutrophil precursors in bone marrow was analyzed using quantitative PCR and flowcytometry., Results: The proportion of CD177+ cells increased during neutrophil maturation in bone marrow. Fold change analysis of gene expression profile of sorted CD177+ and CD177- neutrophils resulted in 14 genes with fold change (fc) >3 difference in expression. Interestingly, 10 of these genes have been reported to change significantly in expression during neutrophil maturation, and most of these genes were granule protein (GP) coding genes. mRNA expression levels measured by RT-PCR of a number of these GP, and of PR3 and MPO were higher in the CD177- neutrophil subset in HC, however, particular granule protein amounts were comparable between CD177+ and CD177- neutrophil subsets. AAV patients had higher amounts of CD177+ neutrophils, but contrary to neutrophils from HC expression of GP-genes was increased, possibly due to activation., Conclusion: The neutrophil population can be distinguished by membrane expression of CD177 into subsets that are different in expression of GP mRNA but not in GP protein production. GP gene expression is also elevated in AAV patients, which is not explained by skewed distribution of CD177+ and CD177- subsets but may be associated with neutrophil activation during on-going inflammation.

Background: In anti-neutrophil cytoplasmic antibodies (ANCA) associated small vessel vasculitis (AAV), rapid testing for ANCA and anti-glomerular basement membrane (GBM) antibodies may be beneficial for therapeutic purpose., Objective: We analysed the diagnostic performance of two rapid ANCA and anti-GBM test methods in 260 patients with suspected AAV., Methods: Between January 2004 and November 2010, we analysed 260 samples by qualitative Dotblot (Biomedical Diagnostics); retrospective analysis followed with directly coated highly sensitive automated Phadia ELiA and ELiA anti-GBM. Results were related to the final clinical diagnosis and compared with routine capture ELISA., Results: Seventy-four patients had a final diagnosis of AAV (n=62) or anti-GBM disease (n=12). Both Dotblot and ELiA detected all 12 cases of anti-GBM disease; 2 false positive results were found. Dotblot detected ANCA in 56 of 62 AAV patients (sensitivity 90%, NPV 97%), and showed 5 false positives (specificity 97%, PPV 90%). The Phadia ELiA anti-PR3(s) or anti-MPO(s) was positive in 57 of 62 AAV patients (sensitivity 92%, NPV 97%), and had 5 false positives (specificity 97%, PPV 88%). Routine capture ELISA was equally accurate (sensitivity 94%, specificity 97%, PPV 88%, NPV 98%)., Conclusion: The Dotblot and Phadia ELiA on anti-GBM, anti-PR3(s) and anti-MPO(s) performed excellently; results were almost identical to routine ELISA. When suspicion of AAV or anti-GBM disease is high and diagnosis is urgently needed, both tests are very powerful for rapid serological diagnosis. Further studies have to confirm the test performances in samples routinely presented for ANCA testing and in follow-up of positive patients., (Copyright © 2013 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.)

The antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAVs) comprise granulomatosis with polyangiitis (GPA), primarily associated with antibodies to proteinase 3 (PR3-ANCA); microscopic polyangiitis (MPA); and eosinophilic granulomatosis with polyangiitis (EGPA), both principally associated with antibodies to myeloperoxidase (MPO-ANCA). Genetic and environmental factors are involved in their etiopathogenesis, with a possible role for silica exposure in AAVs and Staphylococcus aureus infection in GPA. The distinct associations of PR3-ANCA and MPO-ANCA with different HLA class II antigens, which are stronger than those with the associated diseases, suggest a pathogenic role for those ANCAs and indicate that GPA and MPA are different diseases. Both in vitro and in vivo experimental data have shown that MPO-ANCA can induce necrotizing small-vessel vasculitis and glomerulonephritis. The additional role of the alternative pathway of complement activation has been demonstrated in human and experimental pathology. Also, T cells seem to be involved in lesion development, particularly in the kidney. Granuloma formation, as seen in PR3-ANCA-associated GPA, is not well explained by the presence of autoantibodies in experimental models. Here, T cells seem crucial. Recently obtained insights into the pathogenesis of AAVs have led to more targeted treatment of these life-threatening diseases., (Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Background and Objectives: This study evaluated predictors for patient and renal survival in patients with ANCA-associated vasculitis (AAV) with and without renal involvement., Design, Setting, Participants, & Measurements: There were 273 consecutive AAV patients from January 1990 until December 2007 who were followed until death, loss to follow-up, or December 2010. Based on organ involvement, patients were divided into renal (n=212) and nonrenal groups (n=61). The primary end point was ESRD requiring renal replacement therapy (RRT) or renal transplantation or death., Results: Patient survival was significantly better in the nonrenal group compared with the renal group (hazard ratio, 0.55; 95% confidence interval, 0.33 to 0.92; P=0.02). In the renal group, renal survival was significantly worse in MPO-ANCA-positive patients (n=65) compared with PR3-ANCA-positive patients (n=138) (hazard ratio, 2.1; 95% confidence interval, 1.11 to 3.8; P=0.01). Of 48 patients who needed RRT at diagnosis, 11 patients (23%) died within 6 months and 14 patients (29%) did not regain renal function. Of all 23 patients who regained renal function after RRT, 7 patients (30%) were temporarily dialysis independent and needed dialysis later (range, 13-63 months). Five patients had a renal relapse in the 6 months before restart of RRT. Of all 203 PR3-ANCA-positive and MPO-ANCA-positive patients with renal involvement, 12 patients (6%) developed ESRD during follow-up. These patients were classified as CKD stage 4 or 5 after initial treatment and eight patients had a renal relapse before becoming dialysis dependent., Conclusions: AAV patients with renal involvement who needed RRT had the worst survival probability. In multivariate analysis, the only major determinants for long-term renal survival were renal function at 6 months and renal relapses.

Anti-neutrophil cytoplasmic antibody-associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases.

Introduction: Low testosterone levels in men are associated with fatigue, limited physical performance and reduced health-related quality of life (HRQOL); however, this relationship has never been assessed in patients with anti-neutrophil cytoplasmic antibodies (ANCA) -associated vasculitides (AAV). The aim of this study was to assess the prevalence of androgen deficiency and to investigate the role of testosterone in fatigue, limited physical condition and reduced HRQOL in men with AAV., Methods: Male patients with AAV in remission were included in this study. Fatigue and HRQOL were assessed by the multi-dimensional fatigue inventory (MFI)-20 and RAND-36 questionnaires., Results: Seventy male patients with a mean age of 59 years (SD 12) were included. Scores of almost all subscales of both questionnaires were significantly worse in patients compared to controls. Mean total testosterone and free testosterone levels were 13.8 nmol/L (SD 5.6) and 256 pmol/L (SD 102), respectively. Androgen deficiency (defined according to Endocrine Society Clinical Practice Guidelines) was present in 47% of patients. Scores in the subscales of general health perception, physical functioning and reduced activity were significantly worse in patients with androgen deficiency compared to patients with normal androgen levels. Testosterone and age were predictors for the RAND-36 physical component summary in multiple linear regression analysis. Testosterone, age, vasculitis damage index (VDI) and C-reactive protein (CRP) were associated with the MFI-20 subscale of general fatigue., Conclusions: This study showed that androgen deficiency was present in a substantial number of patients with AAV. Testosterone was one of the predictors for physical functioning and fatigue. Testosterone may play a role in fatigue, reduced physical performance and HRQOL in male patients with AAV.

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic inflammatory disorders that include granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), Churg-Strauss syndrome and renal limited vasculitis (RLV). Extracellular high-mobility group box 1 (HMGB1) acts as an alarmin and has been shown to be a biomarker of disease activity as well as an autoantigen in systemic lupus erythematosus (SLE) and, possibly, in AAV. This study aims to assess antibodies against HMGB1 and HMGB1 levels as biomarkers for AAV disease activity and predictors of relapsing disease., Methods: AAV patients with active disease and healthy controls (HC) were evaluated for anti-HMGB1 antibodies while serum HMGB1 levels were measured longitudinally in AAV patients at presentation, during remission, prior to and at relapses., Results: HMGB1 levels were similar between AAV patients at presentation (n = 52) and HC (n = 35) (2.64 ± 1.80 ng/ml vs. 2.39 ± 1.09 ng/ml; P = 0.422) and no difference regarding HMGB1 levels could be found among AAV disease subsets (GPA: 2.66 ± 1.83 ng/ml vs. MPA: 3.11 ± 1.91 ng/ml vs. RLV: 1.92 ± 1.48 ng/ml; P = 0.369). AAV patients with renal involvement had lower HMGB1 levels than patients without renal involvement at presentation (2.35 ± 1.48 ng/ml vs. 3.52 ± 2.41 ng/ml; P = 0.042). A negative correlation was observed between HMGB1 levels and 24-hour proteinuria (ρ = -0.361, P = 0.028). Forty-nine AAV patients were evaluated for HMGB1 levels during follow-up and no differences were observed between relapsing and nonrelapsing patients (P = 0.350). No significant increase in HMGB1 levels was observed prior to a relapse compared with the remission period and changes in HMGB1 levels were not associated with an increased risk for relapse in AAV. Positivity for anti-HMGB1 antibodies was low in patients with active AAV (three out of 24 patients)., Conclusions: Serum HMGB1 levels at presentation are not increased and are lower in patients with renal involvement. Relapses are not preceded or accompanied by significant rises in HMGB1 levels and changes in HMGB1 levels are not related to ensuing relapses. Anti-HMGB1 antibodies are present in only a few patients in AAV. In contrast to SLE, HMGB1 is not a useful biomarker in AAV.

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a severe condition encompassing two major syndromes: granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis) and microscopic polyangiitis. Its cause is unknown, and there is debate about whether it is a single disease entity and what role ANCA plays in its pathogenesis. We investigated its genetic basis., Methods: A genomewide association study was performed in a discovery cohort of 1233 U.K. patients with ANCA-associated vasculitis and 5884 controls and was replicated in 1454 Northern European case patients and 1666 controls. Quality control, population stratification, and statistical analyses were performed according to standard criteria., Results: We found both major-histocompatibility-complex (MHC) and non-MHC associations with ANCA-associated vasculitis and also that granulomatosis with polyangiitis and microscopic polyangiitis were genetically distinct. The strongest genetic associations were with the antigenic specificity of ANCA, not with the clinical syndrome. Anti-proteinase 3 ANCA was associated with HLA-DP and the genes encoding α(1)-antitrypsin (SERPINA1) and proteinase 3 (PRTN3) (P=6.2×10(-89), P=5.6×10(-12,) and P=2.6×10(-7), respectively). Anti-myeloperoxidase ANCA was associated with HLA-DQ (P=2.1×10(-8))., Conclusions: This study confirms that the pathogenesis of ANCA-associated vasculitis has a genetic component, shows genetic distinctions between granulomatosis with polyangiitis and microscopic polyangiitis that are associated with ANCA specificity, and suggests that the response against the autoantigen proteinase 3 is a central pathogenic feature of proteinase 3 ANCA-associated vasculitis. These data provide preliminary support for the concept that proteinase 3 ANCA-associated vasculitis and myeloperoxidase ANCA-associated vasculitis are distinct autoimmune syndromes. (Funded by the British Heart Foundation and others.).

Objectives: CpG motifs, which are highly prevalent in bacterial DNA, have been shown to trigger the production of ANCA in vitro by B lymphocytes from patients with active ANCA-associated vasculitis (AAV). Staphylococcus aureus is associated with relapses in AAV, and CpG motifs from staphylococcal DNA may trigger ANCA production in AAV patients in remission. We investigated the presence of ANCA-producing B lymphocytes during quiescent disease and tested the capacity of these cells to produce ANCA in response to CpG., Methods: Expression of Toll-like receptor 9 (TLR9) by B lymphocytes from AAV patients and controls was assessed. Peripheral blood mononuclear cells were isolated from 23 PR3-ANCA and 15 MPO-ANCA patients (33 quiescent, 5 active disease) and 14 healthy controls, and cultured for 12 days in the presence of cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) and IL-2. B-lymphocyte activation, differentiation, immunoglobulin production and in vitro ANCA production were studied., Results: TLR9 expression by B lymphocytes was comparable in AAV patients and controls. B lymphocytes were activated and differentiated towards a plasma cell phenotype in response to CpG-ODN and IL-2. ANCA were produced in vitro by 13 out of 23 PR3-ANCA patients and 3 out of 15 MPO-ANCA patients., Conclusions: We conclude that ANCA-producing B lymphocytes can be present in the peripheral blood of AAV patients during remission. These autoreactive B lymphocytes are triggered by CpG-ODN and IL-2 to produce ANCA in vitro. CpG motifs may trigger the production of ANCA in vivo, contributing to the development of relapses in AAV.

The etiology of anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) is unknown, but the association between infections and autoimmunity has been studied extensively. In 2004, a novel theory was proposed that could link infection and autoimmunity. This 'theory of autoantigen complementarity' was based on the serendipitous finding of antibodies against complementary-PR3 (cPR3) in patients with PR3-ANCA-associated vasculitis. cPR3 demonstrated homology to several bacterial proteins, and it was hypothesized that PR3-ANCA develop in response to anti-cPR3 antibodies, as a consequence of the anti-idiotypic network. These data have not been confirmed in other patient cohorts. We investigated the presence of anti-cPR3 antibodies in a Dutch cohort of PR3-ANCA-associated vasculitis patients. Anti-cPR3 reactivity was determined in serum using ELISA. Two separate batches of cPR3 were used to determine reactivity in two separate cohorts of PR3-ANCA-associated vasculitis patients. We found that anti-cPR3-reactivity was not increased in our PR3-ANCA-associated vasculitis patients, in comparison to control groups. Further research will be necessary to prove the concept of autoantigen complementarity in autoimmune diseases.

Objective: To determine whether inhibition of p38 mitogen-activated protein kinase (p38MAPK) reduces the pathogenicity of anti-neutrophil cytoplasmic autoantibodies (ANCAs) in vitro and in vivo., Methods: The effects of the p38MAPK-specific inhibitor AR-447 were studied in vitro using neutrophil respiratory burst and degranulation assays, and in lipopolysaccharide (LPS)-stimulated human glomerular endothelial cells. In vivo, p38MAPK inhibition was investigated in a mouse anti-myeloperoxidase (MPO) IgG/LPS glomerulonephritis model. Mice were treated orally with AR-447 daily, starting before (pretreatment group) or 24 h after disease onset (treatment group), and killed after 1 or 7 day(s)., Results: In vitro, AR-447 diminished neutrophil respiratory burst and degranulation induced by patient-derived MPO-ANCA and proteinase 3 (Pr3)-ANCA. In glomerular endothelial cells, AR-447 reduced LPS-induced secretion of IL-6 and IL-8, but not of MCP-1. In mice, pretreatment with AR-447 reduced albuminuria 1 day after induction of glomerulonephritis. After 7 days, no effects on urinary abnormalities were observed upon AR-447 pretreatment or treatment. Also, glomerular neutrophil accumulation was not diminished. In contrast, glomerular macrophage accumulation and the formation of glomerular crescents was significantly reduced by AR-447 pretreatment (vehicle: 12.5 ± 5.6% crescentic glomeruli; AR-447: 7.7 ± 2.7%) and treatment (vehicle 14.6 ± 1.8%; AR-447 6.0 ± 3.4%) at 7 days., Conclusion: This study shows that p38MAPK inhibition markedly reduces ANCA-induced neutrophil activation in vitro. In vivo, p38MAPK inhibition partly reduced crescent formation when the drug was administered prior to disease induction and after disease onset, suggesting that besides p38MAPK activity other signalling pathways contribute to the disease activity.

Introduction: Toll-like receptors (TLRs) are a family of receptors that sense pathogen associated patterns such as bacterial cell wall proteins. Bacterial infections are associated with anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). Here, we assessed the expression of TLRs 2, 4, and 9 by peripheral blood leukocytes from patients with AAV, and investigated TLR mediated responses ex vivo., Methods: Expression of TLRs was determined in 38 AAV patients (32 remission, 6 active disease), and 20 healthy controls (HC). Membrane expression of TLRs 2, 4, and 9, and intracellular expression of TLR9 by B lymphocytes, T lymphocytes, NK cells, monocytes and granulocytes was assessed using 9-color flowcytometry. Whole blood from 13 patients and 7 HC was stimulated ex vivo with TLR 2, 4 and 9 ligands and production of cytokines was analyzed., Results: In patients, we observed increased proportions of TLR expressing NK cells. Furthermore, patient monocytes expressed higher levels of TLR2 compared to HC, and in a subset of patients an increased proportion of TLR4(+) monocytes was observed. Monocytes from nasal carriers of Staphylococcus aureus expressed increased levels of intracellular TLR9. Membrane expression of TLRs by B lymphocytes, T lymphocytes, and granulocytes was comparable between AAV patients and HC. Patients with active disease did not show differential TLR expression compared to patients in remission. Ex vivo responses to TLR ligands did not differ significantly between patients and HC., Conclusions: In AAV, monocytes and NK cells display increased TLR expression. Increased TLR expression by these leukocytes, probably resulting from increased activation, could play a role in disease (re)activation.

Pauci-immune necrotizing glomerulonephritis is the most frequent cause of rapidly progressive glomerulonephritis and, in most cases, is associated with antineutrophil cytoplasmic antibodies (ANCA). It is either the renal manifestation of Wegener's granulomatosis, microscopic polyangiitis of Churg-Strauss syndrome, or a renal-limited vasculitis. In this review, the histopathologic changes seen in renal biopsies of patients with pauci-immune glomerulonephritis are described. The authors also describe why the disease is sometimes limited to the kidneys, the clinical course of renal disease, treatment issues, how to deal with disease relapses, and how to prevent them from occurring. Furthermore, the necessity of renal biopsy and rebiopsy, the usefulness of rapid ANCA detection at diagnosis, and serial measurement of ANCA during follow-up are discussed. The effect of dialysis on the disease process and the possibility of renal transplantation after disease remission are also debated., (Copyright 2010 Elsevier Inc. All rights reserved.)