Cloning and functional expression of the murine homologue of proteinase 3: implications for the design of murine models of vasculitis

AbstractAnti-neutrophil cytoplasmic autoantibodies recognizing conformational epitopes (c-ANCA) of proteinase 3 (PR3) from azurophil granules are a diagnostic hallmark in Wegener's granulomatosis (WG). Because a functional PR3 homologue has not been identified in rodents, it is difficult to assess immunopathological responses in rats or mice immunized with patients' derived c-ANCA or human PR3. Here we report the full length cDNA cloning and functional expression of murine PR3 in HMC-1 cells. Recombinant murine PR3 shows highly similar substrate specificities towards synthetic peptides and is inhibited by human α1-proteinase inhibitor like human PR3. However, neither human c-ANCA, rabbit sera nor mouse monoclonal antibodies to human PR3 recognize the murine homologue. Consequently, it is unlikely that disease observed in mice after immunization with c-ANCA or human PR3 is caused by pathogenic antibodies directed against mouse PR3. Recombinant human-mouse chimaeric variants will be a valuable new tool to localize the disease-specific immunodominant epitopes in human PR3

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

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

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

BACKGROUND: Little 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. METHODS: Multicolor 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). RESULTS: The 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). CONCLUSION: This 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 REGISTRATION: ClinicalTrials.gov NCT00104299. FUNDING: The Vasculitis Foundation, the National Institute of Allergy and Infectious Diseases of the NIH, and the Mayo Foundation for Education and Research

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Auto-reactive B cells escape peripheral tolerance checkpoints in patients with PR3-ANCA associated vasculitis

International audienc

Identification and phenotyping of circulating autoreactive proteinase 3-specific B cells in patients with PR3-ANCA associated vasculitis and healthy controls

International audienc

Auto-reactive B cells escape peripheral tolerance checkpoints in patients with PR3-ANCA associated vasculitis

International audienc

Tailoring work participation support for cancer survivors using the stages of change: perspectives of (health care) professionals and survivors

Purpose: Interventions to support work participation in cancer survivors (CSs) have shown limited effectiveness. Applying a behavioral change framework (e.g., stages of change) could make work participation interventions for CSs more timely and tailored. We aimed to explore the application of the stages of change framework to work participation support for CSs and to generate stage-specific intervention content. Methods: Eighteen experts (e.g., occupational physicians, reintegration consultants) were individually interviewed, and three focus groups with CSs (n = 6, n = 5, n = 4) were conducted. Data were analyzed along the six work-related behavioral change stages purported by the readiness for return to work framework, which is based on the stages of change. Results: The following themes were identified: (1) pre-contemplation: emotional support and staying connected—encourage contact with the employer/colleagues; (2) contemplation: considering return to work is stressful—facilitate the deliberation process; (3) preparation self-evaluative: assess current capabilities—seek guidance from, e.g., occupational physicians; (4) preparation behavioral: planning return to work—allow for personalized solutions and encourage supervised return to work; (5) uncertain maintenance: guard against overload—train self-efficacy strategies and communication tactics; and (6) proactive maintenance: accept and prepare for the long term. Conclusions: Our results support the potential utility of tailoring CSs’ work participation support along the stages of change. Implications for Cancer Survivors: We provided recommendations for intervention content and developed a stage-specific work participation intervention for CSs, the effectiveness of which will be evaluated in an upcoming randomized controlled trial

Exostosin 1/Exostosin 2–Associated Membranous Nephropathy

F.C.F. and P.R. contributed equally to this work.International audienceBACKGROUND: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