Memories of Bob Sim-Genius Complementologist and Cheerful Travel Companion.

Shortly before his 70th birthday, Robert B [...].

Anti-plasminogen antibodies in ANCA-associated vasculitis: An optimized anti-plasminogen assay.

Anti-plasminogen antibodies (α-PLG) were previously detected in a subpopulation of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients, showing a relation to renal lesions and outcome. Several studies showed different proportions of α-PLG positive AAV patients, possibly due to differences in the assays used. We here present a new, optimized α-PLG Enzyme-Linked Immuno Sorbent Assay (ELISA) and validate the presence of α-PLG in AAV. Different ELISA set-ups were tested regarding plasminogen (PLG) antigen, concentrations, coating buffers, blocking agents, and environmental conditions. Purified lysine-PLG (lys-PLG) showed better differentiation between positive samples and negative samples than glutamic acid-PLG (glu-PLG). Therefore, lys-PLG was used as coating antigen. With the optimized α-PLG ELISA we found α-PLG in 14.3% of the myeloperoxidase (MPO)-ANCA patients, whereas all our proteinase-3 (PR3)-ANCA patients tested in our new assay were negative. Concluding, in this study we have combined important technical findings and methods from previous studies to optimize the α-PLG assay, which can be used for future research purposes and will aid in uniform reporting of α-PLG status of patients.

Stabilized Cyclic Peptides as Scavengers of Autoantibodies: Neutralization of Anticitrullinated Protein/Peptide Antibodies in Rheumatoid Arthritis.

The occurrence of autoantibodies is a hallmark of rheumatoid arthritis, specifically those autoantibodies targeting proteins containing the arginine-derived amino acid citrulline. There is strong evidence showing that the occurrence of anticitrullinated protein/peptide antibodies (ACPA) are involved in disease progression, and ACPA was recently shown to induce pain in animals. Here, we explore a novel concept useful for research, diagnostics, and possibly therapy of autoimmune diseases, namely, to directly target and neutralize autoantibodies using peptide binders. A high-affinity peptide-based scavenger of ACPA was developed by grafting a citrullinated epitope derived from human fibrinogen into a naturally occurring stable peptide scaffold. The best scavenger comprises the truncated epitope α-fibrinogen, [Cit573]fib(566-580), grafted into the scaffold sunflower trypsin inhibitor-1, SFTI-1. The final peptide demonstrates low nanomolar apparent affinity and superior stability.

Circulating secretory IgA antibodies against cyclic citrullinated peptides in early rheumatoid arthritis associate with inflammatory activity and smoking.

BACKGROUND: A possible association between mucosal immunization and inflammation, as well as the initiation and propagation of rheumatoid arthritis (RA), is attracting renewed interest. The aim of this study was to evaluate the possible occurrence and clinical correlations of circulating secretory immunoglobulin A (SIgA) antibodies against the second-generation cyclic citrullinated peptides (CCP) among patients with recent-onset RA followed prospectively over 3 years. METHODS: Baseline serum samples from 636 patients with recent-onset RA were analyzed for SIgA anti-CCP antibodies by using an enzyme-linked immunosorbent assay with a secondary antibody directed against secretory component. SIgA anti-CCP status at baseline was analyzed in relation to smoking, HLA-DRB1/shared epitope (SE), and the disease course over 3 years. Significant findings were evaluated in regression analysis that included age, sex, smoking, and SE. RESULTS: Seventeen percent of the patients tested positive for circulating SIgA anti-CCP, and the occurrence was confirmed by detection of secretory component in an affinity-purified IgA anti-CCP fraction. SIgA anti-CCP positivity at baseline was associated with slightly higher baseline erythrocyte sedimentation rate (ESR) (mean 38 vs. 31 mm/first hour, p = 0.004) and C-reactive protein (CRP) (mean 30 vs. 23 mg/L, p = 0.047). During follow-up, SIgA anti-CCP-positive patients had a higher mean AUC regarding ESR (adjusted p = 0.003), although there were no significant differences regarding CRP, tender and swollen joint counts, or radiological joint damage (median Larsen progression 1.0 vs. 1.0, p = 0.22). SIgA anti-CCP was associated significantly with smoking (79 % ever smokers among SIgA anti-CCP-positive patients vs. 59 % in SIgA anti-CCP-negative patients, adjusted OR 2.19, 95 % CI 1.01-4.37, p = 0.027) but not with carriage of the SE (80 % vs. 73 %, p = 0.62). CONCLUSIONS: Circulating SIgA anti-CCP, which is present in a subgroup of patients with early RA, is not related to SE, but it is environmentally linked to cigarette smoking. This finding strengthens the hypothesis that immunization against citrullinated peptides and/or proteins may occur at mucosal surfaces of the airways. Analysis of SIgA antibodies in serum may be a convenient and more versatile means to investigate the "mucosal connection" in RA compared with analyses in mucosal fluid samples.

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens.

INTRODUCTION: We have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA). METHODS: The autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit. RESULTS: Two peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen ß chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65%, 15%, 35%, and 53% of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5% (Cit573), 6% (Cit591), 8% (Cit72), and 4% (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84% and 63% respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency. CONCLUSIONS: Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.

European Union funded project on the development of a whole complement deficiency screening ELISA-A story of success and an exceptional manager: Mohamed R. Daha.

A whole complement ELISA-based assay kit, primarily designed to screen for deficiencies in components of the complement system was developed during a European Union grant involving more than a dozen European scientists and a small-medium enterprise company (Wieslab, which later merged into Eurodiagnostica). The consortium was led by Prof. Mohamed R. Daha who had already guided a preceding European grant which prepared the ground for this endeavor to create a novel and sophisticated complement measurement tool. The final result of the grant was a scientific publication (Seelen et al., 2005, J. Immunol. Methods 296, 187-198) and a commercially available complement deficiency screening kit, WIESLAB(®) Complement system Screen. Thereafter, the group decided to carry on with a grant, located at Innsbruck Medical University, and supported by royalties and unrestricted educational grants from Eurodiagnostica, Malmö, entitled "Search for Applications for WIESLAB(®) Complement system Screen (SAW)" with the aim to look for further applications of this assay. During the latter project the group organized several scientific meetings aimed at evaluating the use of the assay as well as developing further branches of its platform. A look back over almost two decades reveals a great story of excellent research which was also commercially successful, fulfilling the aims of European Union grants. It is also a story of ageless friendship, only possible due to the vision and guidance of an exceptional manager: Moh Daha.

Affinity purified anti-citrullinated protein/peptide antibodies target antigens expressed in the rheumatoid joint.

INTRODUCTION: A major subset of patients with rheumatoid arthritis (RA) is characterized by the presence of circulating autoantibodies directed to citrullinated proteins/peptides (ACPAs). These autoantibodies, which are commonly detected by using an enzyme-linked immunosorbent assay (ELISA) based on synthetic cyclic citrullinated peptides (CCPs), predict clinical onset and a destructive disease course. In the present study, we have used plasma and synovial fluids from patients with RA, for the affinity purification and characterization of anti-CCP2 reactive antibodies, with an aim to generate molecular tools that can be used in vitro and in vivo for future investigations into the pathobiology of the ACPA response. Specifically, this study aims to demonstrate that the surrogate marker CCP2 can capture ACPAs that bind to autoantigens expressed in vivo in the major inflammatory lesions of RA (that is, in the rheumatoid joint). METHODS: Plasma (n = 16) and synovial fluid (n = 26) samples were collected from RA patients with anti-CCP2 IgG levels of above 300 AU/mL. Total IgG was isolated on Protein G columns and subsequently applied to CCP2 affinity columns. Purified anti-CCP2 IgG was analyzed for reactivity and specificity by using the CCPlus® ELISA, in-house peptide ELISAs, Western blot, and immunohisto-/immunocytochemistry. RESULTS: Approximately 2% of the total IgG pool in both plasma and synovial fluid was CCP2-reactive. Purified anti-CCP2 reactive antibodies from different patients showed differences in binding to CCP2 and differences in binding to citrullinated peptides from α-enolase, vimentin, fibrinogen, and collagen type II, illustrating different ACPA fine-specificity profiles. Furthermore, the purified ACPA bound not only in vitro citrullinated proteins but, more importantly, in vivo-generated epitopes on synovial fluid cells and synovial tissues from patients with RA. CONCLUSIONS: We have isolated ACPAs from plasma and synovial fluid and demonstrated that the CCP2 peptides, frequently used in diagnostic ELISAs, de facto act as surrogate antigens for at least four different, well-characterized, largely non-cross-reactive, ACPA fine specificities. Moreover, we have determined the concentration and proportion of CCP2-reactive IgG molecules in rheumatoid plasma and synovial fluid, and we have shown that the purified ACPAs can be used to detect both in vitro- and in vivo-generated citrullinated epitopes by various techniques. We anticipate that these antibodies will provide us with new opportunities to investigate the potential pathogenic effects of human ACPAs.

Salivary IgA antibodies to cyclic citrullinated peptides (CCP) in rheumatoid arthritis.

Circulating IgG anti-cyclic citrullinated peptide antibodies (CCP) are highly specific for rheumatoid arthritis (RA) and prognostic of poor outcome. Serum IgA anti-CCP occurs in a subset of IgG-positive cases and relates to still more aggressive disease. Mucosal IgA-class antibodies, however, are generally associated with anti-inflammatory actions and systemic tolerance induction. In the present study, unstimulated salivary samples from 63 patients with established RA and 20 healthy persons were analysed by enzyme-linked immunoassay for the presence of IgA anti-CCP antibodies. To ensure antigen specificity, IgA-reactivity with the corresponding uncitrullinated antigen, cyclic arginine peptide (CAP), was analysed and anti-CCP/anti-CAP ratios calculated. Retrospective data regarding disease activity and radiological outcome were achieved via medical records. Salivary IgA anti-CCP was found in 14/63 (22%) patients and one (5%) control (positive test=anti-CCP/anti-CAP ratio>1.5). Salivary IgA reactivity was dose-dependently inhibited by pre-incubation with soluble CCP to a degree strongly correlating with anti-CCP/anti-CAP ratio. In salivary IgA anti-CCP positive patients, joint erosions within 6 years of diagnosis was significantly lower (p=0.043), and at the time for diagnosis there was a trend towards lower erythrocyte sedimentation rate (p=0.071) and C-reactive protein (p=0.085). Contrasting to circulating IgG and IgA anti-CCP, our results imply that salivary IgA antibodies may be associated with a less severe outcome of RA. Hypothetically, this relates to an anti-inflammatory and protective immunomodulating role of secretory IgA-class autoantibodies against citrullinated antigens presented at mucosal surfaces.