Publications by authors named "Amber M Hummel"

18 Publications

  • Page 1 of 1

Circulating autoreactive proteinase 3+ B cells and tolerance checkpoints in ANCA-associated vasculitis.

JCI Insight 2021 Nov 22;6(22). Epub 2021 Nov 22.

Thoracic Disease Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, Minnesota, USA.

BACKGROUNDLittle is known about the autoreactive B cells in antineutrophil cytoplasmic antibody-associated (ANCA-associated) vasculitis (AAV). We aimed to investigate tolerance checkpoints of circulating antigen-specific proteinase 3-reactive (PR3+) B cells.METHODSMulticolor flow cytometry in combination with bioinformatics and functional in vitro studies were performed on baseline samples of PBMCs from 154 well-characterized participants of the RAVE trial (NCT00104299) with severely active PR3-AAV and myeloperoxidase-AAV (MPO-AAV) and 27 healthy controls (HCs). Clinical data and outcomes from the trial were correlated with PR3+ B cells (total and subsets).RESULTSThe frequency of PR3+ B cells among circulating B cells was higher in participants with PR3-AAV (4.77% median [IQR, 3.98%-6.01%]) than in participants with MPO-AAV (3.16% median [IQR, 2.51%-5.22%]) and participants with AAV compared with HCs (1.67% median [IQR, 1.27%-2.16%], P < 0.001 for all comparisons), implying a defective central tolerance checkpoint in patients with AAV. Only PBMCs from participants with PR3-AAV contained PR3+ B cells capable of secreting PR3-ANCA IgG in vitro, proving they were functionally distinct from those of participants with MPO-AAV and HCs. Unsupervised clustering identified subtle subsets of atypical autoreactive PR3+ memory B cells accumulating through the maturation process in patients with PR3-AAV. PR3+ B cells were enriched in the memory B cell compartment of participants with PR3-AAV and were associated with higher serum CXCL13 levels, suggesting an increased germinal center activity. PR3+ B cells correlated with systemic inflammation (C-reactive protein and erythrocyte sedimentation rate, P < 0.05) and complete remission (P < 0.001).CONCLUSIONThis study suggests the presence of defective central antigen-independent and peripheral antigen-dependent checkpoints in patients with PR3-AAV, elucidating the selection process of autoreactive B cells.Trial registrationClinicalTrials.gov NCT00104299.FundingThe Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research.
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http://dx.doi.org/10.1172/jci.insight.150999DOI Listing
November 2021

Clinical Utility of Serial Measurements of Antineutrophil Cytoplasmic Antibodies Targeting Proteinase 3 in ANCA-Associated Vasculitis.

Front Immunol 2020 3;11:2053. Epub 2020 Sep 3.

Mayo Clinic and Mayo Foundation for Research and Education, Rochester, MN, United States.

The utility of ANCA testing as an indicator of disease activity in ANCA-associated vasculitis (AAV) remains controversial. This study aimed to determine the association of ANCA testing by various methods and subsequent remission and examine the utility of a widely used automated addressable laser-bead immunoassay (ALBIA) to predict disease relapses. Data from the Rituximab vs. Cyclophosphamide for ANCA-Associated Vasculitis (RAVE) trial were used. ANCA testing was performed by direct ELISA, capture ELISA, and ALBIA. Cox proportional hazards regression models were used to evaluate the association of PR3-ANCA level and subsequent remission or relapse. The ALBIA results are routinely reported as >8 when the value is high. For this study, samples were further titrated. A decrease and increase in PR3-ANCA were defined as a halving or doubling in value, respectively. A decrease in ANCA by ALBIA at 2 months was associated with shorter time to sustained remission (HR 4.52, = 0.035). A decrease in ANCA by direct ELISA at 4 months was associated with decreased time to sustained remission (HR 1.77, = 0.050). There were no other associations between ANCA decreases or negativity and time to remission. An increase in PR3-ANCA by ALBIA was found in 78 of 93 subjects (84%). Eleven (14%) had a PR3-ANCA value which required titration for detection of an increase. An increase of ANCA by ALBIA was associated with severe relapse across various subgroups. A decrease in ANCA by ALBIA at 2 months and by direct ELISA at 4 months may be predictive of subsequent remission. These results should be confirmed in a separate cohort with similarly protocolized sample and clinical data collection. A routinely used automated ALBIA for PR3-ANCA measurement is comparable to direct ELISA in predicting relapse in PR3-AAV. Without titration, 14% of the increases detected by ALBIA would have been missed. Titration is recommended when this assay is used for disease monitoring. The association of an increase in PR3-ANCA with the risk of subsequent relapse remains complex and is affected by disease phenotype and remission induction agent.
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http://dx.doi.org/10.3389/fimmu.2020.02053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495134PMC
May 2021

Remote Activation of a Latent Epitope in an Autoantigen Decoded With Simulated B-Factors.

Front Immunol 2019 25;10:2467. Epub 2019 Oct 25.

Thoracic Disease Research Unit, Mayo Clinic, Rochester, MN, United States.

Mutants of a catalytically inactive variant of Proteinase 3 (PR3)-iPR3-Val possessing a Ser195Ala mutation relative to wild-type PR3-Val-offer insights into how autoantigen PR3 interacts with antineutrophil cytoplasmic antibodies (ANCAs) in granulomatosis with polyangiitis (GPA) and whether such interactions can be interrupted. Here we report that iHm5-Val, a triple mutant of iPR3-Val, bound a monoclonal antibody (moANCA518) from a GPA patient on an epitope remote from the mutation sites, whereas the corresponding epitope of iPR3-Val was latent to moANCA518. Simulated B-factor analysis revealed that the binding of moANCA518 to iHm5-Val was due to increased main-chain flexibility of the latent epitope caused by remote mutations, suggesting rigidification of epitopes with therapeutics to alter pathogenic PR3·ANCA interactions as new GPA treatments.
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http://dx.doi.org/10.3389/fimmu.2019.02467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6823208PMC
November 2020

Exostosin 1/Exostosin 2-Associated Membranous Nephropathy.

J Am Soc Nephrol 2019 06 6;30(6):1123-1136. Epub 2019 May 6.

Sorbonne Université, Université Pierre et Marie Curie Paris 06, and Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S1155, Paris, France; and.

Background: In membranous nephropathy (MN), which is characterized by deposition of immune complexes along the glomerular basement membrane (GBM), phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A are target antigens in approximately 70% and 1%-5% of cases of primary MN, respectively. In other cases of primary MN and in secondary MN, the target antigens are unknown.

Methods: We studied 224 cases of biopsy-proven PLA2R-negative MN and 102 controls (including 47 cases of PLA2R-associated MN) in pilot and discovery cohorts. We also evaluated 48 cases of PLA2R-negative presumed primary MN and lupus MN in a validation cohort. We used laser microdissection and mass spectrometry to identify new antigens, which were localized by immunohistochemistry.

Results: Mass spectrometry detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R-negative MN, but not in PLA2R-associated MN and control cases. Immunohistochemistry staining revealed bright granular GBM staining for EXT1 and EXT2. Clinical and biopsy findings showed features of autoimmune disease, including lupus, in 80.7% of the 26 EXT1/EXT2-associated MN cases we identified. In the validation cohort, we confirmed that EXT1/EXT2 staining was detected in pure class 5 lupus nephritis (eight of 18 patients) and in presumed primary MN associated with signs of autoimmunity (three of 16 patients); only one of the 14 cases of mixed class 5 and 3/4 lupus nephritis was positive for EXT1/EXT2. Tests in seven patients with EXT1/EXT2-associated MN found no circulating anti-exostosin antibodies.

Conclusions: A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients.
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http://dx.doi.org/10.1681/ASN.2018080852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6551791PMC
June 2019

Pharmacokinetics of rituximab and clinical outcomes in patients with anti-neutrophil cytoplasmic antibody associated vasculitis.

Rheumatology (Oxford) 2018 04;57(4):639-650

Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN, USA.

Objectives: To study the determinants of the pharmacokinetics (PK) of rituximab (RTX) in patients with ANCA-associated vasculitis (AAV) and its association with clinical outcomes.

Methods: This study included data from 89 patients from the RTX in AAV trial who received the full dose of RTX (four weekly infusions of 375 mg/m2). RTX was quantified at weeks 2, 4, 8, 16 and 24, and summarized by computing the trapezoidal area under the curve. We explored potential determinants of the PK-RTX, and analysed its association with clinical outcomes: achievement of remission at 6 months, duration of B-cell depletion and time to relapse in patients who achieved complete remission.

Results: RTX serum levels were significantly lower in males and in newly diagnosed patients, and negatively correlated with body surface area, baseline B-cell count and degree of disease activity. In multivariate analyses, the main determinants of PK-RTX were sex and new diagnosis. Patients reaching complete remission at month 6 had similar RTX levels compared with patients who did not reach complete remission. Patients with higher RTX levels generally experienced longer B-cell depletion than patients with lower levels, but RTX levels at the different time points and area under the curve were not associated with time to relapse.

Conclusion: Despite the body-surface-area-based dosing protocol, PK-RTX is highly variable among patients with AAV, its main determinants being sex and newly diagnosed disease. We did not observe any relevant association between PK-RTX and clinical outcomes. The monitoring of serum RTX levels does not seem clinically useful in AAV.
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http://dx.doi.org/10.1093/rheumatology/kex484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888934PMC
April 2018

Using Mass Spectrometry to Quantify Rituximab and Perform Individualized Immunoglobulin Phenotyping in ANCA-Associated Vasculitis.

Anal Chem 2016 06 3;88(12):6317-25. Epub 2016 Jun 3.

Department of Laboratory Medicine and Pathology, Mayo Clinic , Rochester, Minnesota 55905, United States.

Therapeutic monoclonal immunoglobulins (mAbs) are used to treat patients with a wide range of disorders including autoimmune diseases. As pharmaceutical companies bring more fully humanized therapeutic mAb drugs to the healthcare market analytical platforms that perform therapeutic drug monitoring (TDM) without relying on mAb specific reagents will be needed. In this study we demonstrate that liquid-chromatography-mass spectrometry (LC-MS) can be used to perform TDM of mAbs in the same manner as smaller nonbiologic drugs. The assay uses commercially available reagents combined with heavy and light chain disulfide bond reduction followed by light chain analysis by microflow-LC-electrospray ionization-quadrupole-time-of-flight mass spectrometry (ESI-Q-TOF MS). Quantification is performed using the peak areas from multiply charged mAb light chain ions using an in-house developed software package developed for TDM of mAbs. The data presented here demonstrate the ability of an LC-MS assay to quantify a therapeutic mAb in a large cohort of patients in a clinical trial. The ability to quantify any mAb in serum via the reduced light chain without the need for reagents specific for each mAb demonstrates the unique capabilities of LC-MS. This fact, coupled with the ability to phenotype a patient's polyclonal repertoire in the same analysis further shows the potential of this approach to mAb analysis.
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http://dx.doi.org/10.1021/acs.analchem.6b00544DOI Listing
June 2016

Factors Determining the Clinical Utility of Serial Measurements of Antineutrophil Cytoplasmic Antibodies Targeting Proteinase 3.

Arthritis Rheumatol 2016 07;68(7):1700-10

Mayo Clinic and Mayo Foundation for Medical Education and Research, Rochester, Minnesota.

Objective: Relapse following remission is common in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), particularly with ANCAs directed at proteinase 3 (PR3). This study was undertaken to evaluate the association of an increase in PR3-ANCA level with subsequent relapse.

Methods: Data from the Rituximab versus Cyclophosphamide for ANCA-Associated Vasculitis (RAVE) trial were used. Starting from the time of achieving complete remission, serial measurements by direct and capture enzyme-linked immunosorbent assays (ELISAs) were analyzed in 93 patients with PR3-ANCA, using Cox proportional hazards regression.

Results: An increase in PR3-ANCA level was identified in 58 of 93 subjects (62.4%) by direct ELISA and in 59 of 93 (63.4%) by capture ELISA. Relapses occurred in 55 of 93 subjects (59.1%), with 25 and 21 occurring within 1 year after an increase by direct ELISA and capture ELISA, respectively. An increase by direct ELISA was associated with subsequent severe relapses (hazard ratio [HR] 4.57; P < 0.001), particularly in patients presenting with renal involvement (HR 7.94; P < 0.001) and alveolar hemorrhage (HR 24.19; P < 0.001). Both assays identified increased risk for severe relapse in the rituximab group (HR 5.80; P = 0.002 for direct ELISA and HR 4.54; P = 0.007 for capture ELISA) but not the cyclophosphamide/azathioprine group (P = 0.103 and P = 0.197, respectively).

Conclusion: The association of an increase in PR3-ANCA level with the risk of subsequent relapse is partially affected by the PR3-ANCA detection methodology, disease phenotype, and remission induction treatment. An increase in PR3-ANCA level during complete remission conveys an increased risk of relapse, particularly severe relapse, among patients with renal involvement or alveolar hemorrhage and those treated with rituximab. Serial measurements of PR3-ANCA may be informative in this subset of patients, but the risk of relapse must be weighed carefully against the risks associated with therapy.
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http://dx.doi.org/10.1002/art.39637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7137903PMC
July 2016

Allosteric modulation of proteinase 3 activity by anti-neutrophil cytoplasmic antibodies in granulomatosis with polyangiitis.

J Autoimmun 2015 May 3;59:43-52. Epub 2015 Mar 3.

Comprehensive Pneumology Center, Institute of Lung Biology and Disease (iLBD), University Hospital, Ludwig Maximilians University and Helmholtz Zentrum München, Member of the German Center of Lung Research, Max-Lebsche-Platz 31, 81377 Munich, Germany; Max Planck Institute of Neurobiology, Planegg-Martinsried, Germany. Electronic address:

Anti-neutrophil cytoplasmic antibodies (ANCA) with proteinase 3 (PR3) specificity are a useful laboratory biomarker for the diagnosis of Granulomatosis with Polyangiitis (GPA) and are believed to be implicated in the pathogenesis. It has been repeatedly suggested that disease activity of GPA is more closely related to the appearance and rise of PR3-inhibiting ANCA than to an increase of total ANCA. Previous studies on a limited number of patient samples, however, have yielded inconclusive results. To overcome the previous methodological limitations, we established a new ultrasensitive method to quantify the inhibitory capacity of PR3-ANCA using small volumes of plasma from patients with GPA. A large collection of longitudinally-collected samples from the Wegener Granulomatosis Etanercept Trial (WGET) became available to us to determine the functional effects of ANCA on PR3 in comparison to clinical disease manifestations. In these patient samples we not only detected PR3-ANCA with inhibitory capacity, but also PR3-ANCA with enhancing effects on PR3 activity. However no correlation of these activity-modulating PR3-ANCA with disease activity at either the time of enrollment or over the course of disease was found. Only patients with pulmonary involvement, especially patients with nodule formation in the respiratory tract, showed a slight, but not significant, decrease of inhibitory capacity. Epitope mapping of the activity-modulating PR3-ANCA revealed a binding on the active site surface of PR3. Yet these ANCA were able to bind to PR3 with an occupied active site cleft, indicating an allosteric mechanism of inhibition. The recently described signal ratio between the MCPR3-3 and MCPR3-2 capture ELISA was consistent with the binding of activity-modulating ANCA to the active site surface. Evidence for a shared epitope between activity-modulating PR3-ANCA and MCPR3-7, however, was very limited, suggesting that a majority of PR3-ANCA species do not inhibit PR3 by the same mechanism as previously reported for MCPR3-7.
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http://dx.doi.org/10.1016/j.jaut.2015.02.002DOI Listing
May 2015

A monoclonal antibody (MCPR3-7) interfering with the activity of proteinase 3 by an allosteric mechanism.

J Biol Chem 2013 Sep 31;288(37):26635-48. Epub 2013 Jul 31.

From the Comprehensive Pneumology Center, Institute of Lung Biology and Disease (iLBD), University Hospital, Ludwig Maximilians University and Helmholtz Zentrum München, Member of the German Center for Lung Research (DZL), Max-Lebsche-Platz 31, 81377 Munich, Germany.

Proteinase 3 (PR3) is an abundant serine protease of neutrophil granules and a major target of autoantibodies (PR3 anti-neutrophil cytoplasmic antibodies) in granulomatosis with polyangiitis. Some of the PR3 synthesized by promyelocytes in the bone marrow escapes the targeting to granules and occurs on the plasma membrane of naive and primed neutrophils. This membrane-associated PR3 antigen may represent pro-PR3, mature PR3, or both forms. To discriminate between mature PR3 and its inactive zymogen, which have different conformations, we generated and identified a monoclonal antibody called MCPR3-7. It bound much better to pro-PR3 than to mature PR3. This monoclonal antibody greatly reduced the catalytic activity of mature PR3 toward extended peptide substrates. Using diverse techniques and multiple recombinant PR3 variants, we characterized its binding properties and found that MCPR3-7 preferentially bound to the so-called activation domain of the zymogen and changed the conformation of mature PR3, resulting in impaired catalysis and inactivation by α1-proteinase inhibitor (α1-antitrypsin). Noncovalent as well as covalent complexation between PR3 and α1-proteinase inhibitor was delayed in the presence of MCPR3-7, but cleavage of certain thioester and paranitroanilide substrates with small residues in the P1 position was not inhibited. We conclude that MCPR3-7 reduces PR3 activity by an allosteric mechanism affecting the S1' pocket and further prime side interactions with substrates. In addition, MCPR3-7 prevents binding of PR3 to cellular membranes. Inhibitory antibodies targeting the activation domain of PR3 could be exploited as highly selective inhibitors of PR3, scavengers, and clearers of the PR3 autoantigen in granulomatosis with polyangiitis.
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http://dx.doi.org/10.1074/jbc.M113.495770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772210PMC
September 2013

Discrimination and variable impact of ANCA binding to different surface epitopes on proteinase 3, the Wegener's autoantigen.

J Autoimmun 2010 Dec;35(4):299-308

Thoracic Diseases Research Unit, Stabile Bldg. 8-56, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic antibodies (ANCA) are highly specific for the autoimmune small vessel vasculitis, Wegener's granulomatosis (WG). PR3-ANCA have proven diagnostic value but their pathogenic potential and utility as a biomarker for disease activity remain unclear. PR3-ANCA recognize conformational epitopes, and epitope-specific PR3-ANCA subsets with variable impact on biological functions of PR3 have been postulated. The aims of this study were to identify specific PR3 surface epitopes recognized by monoclonal antibodies (moAbs) and to determine whether the findings can be used to measure the functional impact of epitope-specific PR3-ANCA and their potential relationship to disease activity. We used a novel flow cytometry assay based on TALON-beads coated with recombinant human (H) and murine (M) PR3 and 10 custom-designed chimeric human/mouse rPR3-variants (Hm1-5/Mh1-5) identifying 5 separate non-conserved PR3 surface epitopes. Anti-PR3 moAbs recognize 4 major surface epitopes, and we identified the specific surface location of 3 of these with the chimeric rPR3-variants. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3 was measured indirectly using a capture-ELISA system based on the different epitopes recognized by capturing moAbs. Epitope-specific PR3-ANCA capture-ELISA results obtained from patient plasma (n=27) correlated with the inhibition of enzymatic activity of PR3 by paired IgG preparations (r=0.7, P<0.01). The capture-ELISA results also seem to reflect disease activity. In conclusion, insights about epitopes recognized by anti-PR3 moAbs can be applied to separate PR3-ANCA subsets with predictable functional qualities. The ability of PR3-ANCA to inhibit the enzymatic activity of PR3, a property linked to disease activity, can now be gauged using a simple epitope-based capture-ELISA system.
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http://dx.doi.org/10.1016/j.jaut.2010.06.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2963671PMC
December 2010

Functional characterization of antineutrophil cytoplasmic antibodies in patients with cocaine-induced midline destructive lesions.

Arthritis Rheum 2008 May;58(5):1546-51

Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA.

Objective: Antineutrophil cytoplasmic antibodies (ANCA) binding to neutrophil elastase (NE) and proteinase 3 (PR3) are detectable in most patients with cocaine-induced midline destructive lesions (CIMDL), but the pathogenic role and antigen specificity of these antibodies are unknown. This study was undertaken to assess the effects of NE ANCA on the enzymatic activity of NE, to determine whether these antibodies interfere with the physiologic effect of secretory leukoprotease inhibitor (SLPI), and to investigate the antigen specificity of both NE and PR3 ANCA in patients with CIMDL. We also compared the binding of PR3 ANCA in patients with CIMDL with that in patients with Wegener's granulomatosis (WG).

Methods: PR3 ANCA and NE ANCA were detected by capture enzyme-linked immunosorbent assays (ELISAs) and by indirect immunofluorescence. IgG was purified from the patients' sera, and the influence of NE ANCA on the enzymatic activity of NE and on the inhibitory activity of SLPI was investigated by determining the hydrolysis of N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide by NE.

Results: IgG from NE ANCA-positive sera of patients with CIMDL inhibited the enzymatic activity of NE and did not interfere with the activity of SLPI. In contrast to the findings in WG sera, measurement of PR3 ANCA in CIMDL sera showed only fair to moderate concordance between the 2 different capture ELISAs. Cross-inhibition experiments demonstrated that NE ANCA and PR3 ANCA represent distinct autoantibodies in patients with CIMDL.

Conclusion: The functional effects of NE ANCA on the enzymatic activity of NE or on the activity of SLPI cannot be implicated in the pathogenesis of CIMDL. The autoimmune reaction that targets neutrophil serine proteases in patients with CIMDL is frequently directed against more than one antigen. The ANCA response, including the reactivity of PR3 ANCA, in patients with CIMDL differs from what has been described in patients with WG.
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http://dx.doi.org/10.1002/art.23469DOI Listing
May 2008

Antiproteinase 3 antineutrophil cytoplasmic antibodies and disease activity in Wegener granulomatosis.

Ann Intern Med 2007 Nov;147(9):611-9

Mayo Clinic, Rochester, Minnesota 55905, USA.

Background: The utility of antineutrophil cytoplasmic antibody (ANCA) levels to guide the management of patients with Wegener granulomatosis remains controversial.

Objective: To determine whether pro-proteinase 3 (PR3)-ANCA levels are a better measure of disease activity than mature-PR3-ANCA levels, whether decreases in either level are associated with shorter time to remission, and whether increases are followed by relapse.

Design: Prospective, observational cohort study.

Setting: 8 United States medical centers that participated in a treatment trial for Wegener granulomatosis.

Patients: 156 patients with Wegener granulomatosis enrolled during periods of active disease.

Measurements: PR3-ANCA levels (by capture enzyme-linked immunosorbent assay) and disease activity (by the Birmingham Vasculitis Activity Score for Wegener granulomatosis).

Results: The ANCA levels were only weakly associated with disease activity across patients. The longitudinal association within patients was stronger, but changes in ANCA levels explained less than 10% of the variation in disease activity. Decreases in mature- and pro-PR3-ANCA levels were not statistically significantly associated with shorter time to remission, and increases in mature-PR3-ANCA levels (adjusted hazard ratio, 0.8 [95% CI, 0.4 to 1.9]; P = 0.67) and pro-PR3-ANCA levels (adjusted hazard ratio, 1.0 [CI, 0.5 to 2.1]; P = 0.99) were not associated with relapse. The proportion of patients who had relapse within 1 year of an increase in PR3-ANCA levels was 40% for mature-PR3 (CI, 18% to 56%) and 43% for pro-PR3 (CI, 22% to 58%).

Limitations: Samples were collected approximately every 3 months. Sensitivity and specificity of ANCA levels for detecting remission and relapse could not be calculated because each patient had different follow-up times.

Conclusion: Pro-PR3-ANCA is no better than mature-PR3-ANCA as a measure of Wegener granulomatosis activity. Decreases in PR3-ANCA levels are not associated with shorter time to remission, and increases are not associated with relapse. These findings suggest that ANCA levels cannot be used to guide immunosuppressive therapy.
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http://dx.doi.org/10.7326/0003-4819-147-9-200711060-00005DOI Listing
November 2007

ANCA are detectable in nearly all patients with active severe Wegener's granulomatosis.

Am J Med 2007 Jul;120(7):643.e9-14

Thoracic Disease Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, Rochester, Minn 55905, USA.

Background: The pathogenic significance of antineutrophilic cytoplasmic antibodies (ANCA) in Wegener's granulomatosis is controversial. Their presence is influenced by the extent, severity, and activity of the disease at the time of sampling. The objective of this study was to determine the frequency of ANCA in patients with active Wegener's granulomatosis and to assess the influence of disease severity on test results.

Methods: Baseline serum samples from the 180 participants in a multicentric prospective trial were tested for ANCA by indirect immunofluorescence, direct enzyme-linked immunosorbent assay (ELISA), and capture ELISA. Disease activity was measured using the Birmingham Vasculitis Activity Score for Wegener's granulomatosis. All patients had active disease at enrollment. Patients were categorized as having severe (n=128) or limited (n=52) Wegener's granulomatosis.

Results: When all ANCA detection methods were combined, 166 patients (92%) were ANCA positive, including 96% with severe disease and 83% with limited disease.

Conclusion: ANCA are detectable in nearly all patients with active severe Wegener's granulomatosis, but approximately 1 of 5 patients with active limited disease are ANCA negative. Immunofluorescence and both direct and capture ELISAs are required for optimal detection, suggesting that ANCA are not recognized equally well by all testing methods.
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http://dx.doi.org/10.1016/j.amjmed.2006.08.016DOI Listing
July 2007

Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies.

J Biochem 2007 Jan 11;141(1):101-12. Epub 2006 Dec 11.

Thoracic Disease Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905, USA.

Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.
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http://dx.doi.org/10.1093/jb/mvm008DOI Listing
January 2007

A novel capture-ELISA for detection of anti-neutrophil cytoplasmic antibodies (ANCA) based on c-myc peptide recognition in carboxy-terminally tagged recombinant neutrophil serine proteases.

J Immunol Methods 2005 Dec 3;307(1-2):62-72. Epub 2005 Oct 3.

Thoracic Disease Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic and Foundation, Rochester, MN 55905, USA.

Testing for antineutrophil cytoplasmic antibodies (ANCA) reacting with proteinase 3 (PR3) is part of the routine diagnostic evaluation of patients with small vessel vasculitis. For PR3-ANCA detection, capture ELISAs are reported to be superior to direct ELISAs. Standard capture ELISAs, in which PR3 is anchored by anti-PR3 monoclonal antibodies (moAB), have two potential disadvantages. First, the capturing moAB may compete for epitopes recognized by some PR3-ANCA, causing occasional false-negative results. Second, the capture of recombinant PR3 mutant molecules becomes unpredictable as modifications of specific conformational epitopes may not only affect the binding of PR3-ANCA, but also the affinity of the capturing anti-PR3 moAB. Here, we describe a new capture ELISA, and its application for PR3-ANCA detection. This new assay is based on the standardized capture of a variety of different carboxy-terminally c-myc tagged recombinant ANCA target antigens using anti-c-myc coated ELISA plates. Antigen used include c-myc tagged human rPR3 variants (mature and pro-form conformations), mouse mature rPR3 and human recombinant neutrophil elastase. This new anti-c-myc-capture ELISA for PR3-ANCA detection has an intra- and inter-assay coefficient of variation of 3.6% to 7.7%, and 15.8% to 18.4%, respectively. The analytical sensitivity and specificity for PR3-ANCA positive serum samples were 93% and 100%, respectively when rPR3 with mature conformation was used as target antigen, and 83% and 100% when the pro-enzyme conformation was employed. In conclusion, this new anti-c-myc capture ELISA compares favorably to our standard capture ELISA for PR3-ANCA detection, enables the unified capture of different ANCA target antigens through binding to a c-myc tag, and allows capture of rPR3 mutants necessary for PR3-ANCA epitope mapping studies.
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http://dx.doi.org/10.1016/j.jim.2005.09.004DOI Listing
December 2005

Differences between human proteinase 3 and neutrophil elastase and their murine homologues are relevant for murine model experiments.

FEBS Lett 2005 Oct;579(24):5305-12

Thoracic Diseases Research Unit, Division of Pulmonary and Critical Care Medicine, Mayo Clinic Foundation, Rochester, MN 55905, USA.

Direct comparisons of human (h) and murine (m) neutrophil elastase (NE) and proteinase 3 (PR3) are important for the understanding and interpretation of inflammatory and PR3-related autoimmune processes investigated in wild-type-, mNE- and mPR3/mNE knockout mice. To this end, we purified recombinant mPR3 and mNE expressed in HMC1 and 293 cells and compared their biophysical properties, proteolytic activities and susceptibility to inhibitors with those of their human homologues, hPR3 and hNE. Significant species differences in physico-chemical properties, substrate specificities and enzyme kinetics towards synthetic peptide substrates, oxidized insulin B chain, and fibrinogen were detected. MeOSuc-AAPV-pNA and Suc-AAPV-pNA were hydrolyzed more efficiently by mPR3 than hPR3, but enzymatic activities of mNE and hNE were very similar. Fibrinogen was cleaved much more efficiently by mPR3 than by hPR3. All four proteases were inhibited by alpha(1)-antitrypsin and elafin. Eglin C inihibited mNE, hNE, mPR3, but not hPR3. SLPI inhibited both NEs, but neither PR3. The custom-designed hNE inhibitor, Val(15)-aprotinin, is a poor inhibitor for mNE. In conclusion, appropriate interpretation of experiments in murine models requires individual species-specific assessment of neutrophil protease function and inhibition.
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http://dx.doi.org/10.1016/j.febslet.2005.08.056DOI Listing
October 2005

Effects of carboxy-terminal modifications of proteinase 3 (PR3) on the recognition by PR3-ANCA.

Kidney Int 2003 Feb;63(2):756-60

Thoracic Diseases Research Unit and Hematology Research Unit, Mayo Graduate School of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.

Background: Autoantibodies directed against neutrophil proteinase 3 (PR3-ANCA) from patients with Wegener's granulomatosis and microscopic polyangiitis recognize conformational epitopes of PR3. During maturation of neutrophils, PR3 undergoes amino-terminal and carboxy-terminal processing. In contrast to amino-terminal processing, the effects of carboxy-terminal processing on recognition of PR3 by PR3-ANCA remain unknown. Carboxy-terminally modified or tagged recombinant PR3 (rPR3) molecules may be useful for the refinement of diagnostic assays and for the study of biological processes.

Methods: This study was designed to determine whether 293 cells can be used to express specifically designed carboxy-terminal variants of rPR3, and to evaluate the effects of different carboxy-terminal modifications on the recognition by PR3-ANCA in the capture ELISA.

Results: The rPR3-variants secreted into the media supernatants of transfected 293 cells escaped proteolytic processing. Furthermore, in contrast to the effects of amino-terminal pro-peptide deletion on PR3-ANCA binding, carboxy-terminal modifications (deletion and additions) did not significantly affect recognition by PR3-ANCA.

Conclusions: This expression system is ideally suited for the expression of custom-designed carboxy-terminal rPR3 variants, and major conformational effects of carboxy-terminal modifications seem unlikely.
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http://dx.doi.org/10.1046/j.1523-1755.2003.00765.xDOI Listing
February 2003
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