Bead arrays for antibody and complement profiling reveal joint contribution of antibody isotypes to C3 deposition

The development of antigen arrays has provided researchers with great tools to identify reactivities against self or foreign antigens from body fluids. Yet, these approaches mostly do not address antibody isotypes and their effector functions even though these are key points for a more detailed understanding of disease processes. Here, we present a bead array-based assay for a multiplexed determination of antigen-specific antibody levels in parallel with their properties for complement activation. We measured the deposition of C3 fragments from serum samples to reflect the degree of complement activation via all three complement activation pathways. We utilized the assay on a bead array containing native and citrullinated peptide antigens to investigate the levels of IgG, IgM and IgA autoantibodies along with their complement activating properties in serum samples of 41 rheumatoid arthritis patients and 40 controls. Our analysis revealed significantly higher IgG reactivity against the citrullinated fibrinogen β and filaggrin peptides as well as an IgA reactivity that was exclusive for citrullinated fibrinogen β peptide and C3 deposition in rheumatoid arthritis patients. In addition, we characterized the humoral immune response against the viral EBNA-1 antigen to demonstrate the applicability of this assay beyond autoimmune conditions. We observed that particular buffer compositions were demanded for separate measurement of antibody reactivity and complement activation, as detection of antigen-antibody complexes appeared to be masked due to C3 deposition. We also found that rheumatoid factors of IgM isotype altered C3 deposition and introduced false-positive reactivities against EBNA-1 antigen. In conclusion, the presented bead-based assay setup can be utilized to profile antibody reactivities and immune-complex induced complement activation in a high-throughput manner and could facilitate the understanding and diagnosis of several diseases where complement activation plays role in the pathomechanism

Citizens AND HYdrology (CANDHY): conceptualizing a transdisciplinary framework for citizen science addressing hydrological challenges

Widely available digital technologies are empowering citizens who are increasingly well informed and involved in numerous water, climate, and environmental challenges. Citizen science can serve many different purposes, from the "pleasure of doing science" to complementing observations, increasing scientific literacy, and supporting collaborative behaviour to solve specific water management problems. Still, procedures on how to incorporate citizens' knowledge effectively to inform policy and decision-making are lagging behind. Moreover, general conceptual frameworks are unavailable, preventing the widespread uptake of citizen science approaches for more participatory cross-sectorial water governance. In this work, we identify the shared constituents, interfaces, and interlinkages between hydrological sciences and other academic and non-academic disciplines in addressing water issues. Our goal is to conceptualize a transdisciplinary framework for valuing citizen science and advancing the hydrological sciences. Joint efforts between hydrological, computer, and social sciences are envisaged for integrating human sensing and behavioural mechanisms into the framework. Expanding opportunities of online communities complement the fundamental value of on-site surveying and indigenous knowledge. This work is promoted by the Citizens AND HYdrology (CANDHY) Working Group established by the International Association of Hydrological Sciences (IAHS)

Prospective randomized study comparing the Teleflex Medical SaphLITE Retractor to the Ethicon CardioVations Clearglide Endoscopic System

BACKGROUND: Several minimally invasive saphenous vein harvesting techniques have been developed to reduce morbidities associated with coronary artery bypass grafting. This prospective, randomized study was designed to compare two commonly used minimally invasive saphenous vein harvesting techniques, the SaphLITE Retractor System (Teleflex Medical) and the Clearglide Endoscopic Vessel Harvesting System (Ethicon CardioVations, Inc.). METHODS: Between January 2003 and March 2004, a total of 200 patients scheduled for primary, nonemergent coronary artery bypass grafting, with or without concomitant procedures were randomized into two groups: SaphLITE (n = 100) and Clearglide (n = 100). Pre-, intra- and postoperative data was collected and subjected to statistical analysis. Randomization provided homogenous groups with respect to preoperative risk factors. RESULTS: Harvest location for the SaphLITE group was thigh (n = 40), lower leg (n = 5) and both lower leg and thigh (n = 55). The location of harvest for the Clearglide group was thigh (n = 3), lower leg (n = 16) and both lower leg and thigh (n = 81). The mean incision length was 3.6 cm (range, 2–6) in the SaphLITE group versus 2.1 cm (range, 1–4) in the Clearglide group (p < 0.05). The total incision length was 12.9 cm versus 8.9 (p < 0.05) in the SaphLITE and Clearglide groups. Conversion to the open technique occurred in 5 SaphLITE patients and 7 Clearglide patients. Intraoperative leg exploration for bleeding occurred in two of the Clearglide patients and none of the SaphLITE patients. Post-operative complications specifically related to minimally invasive harvesting technique, including a two-week post-discharge visit, were not statistically different between the groups. CONCLUSION: The saphenous vein can be safely harvested utilizing the SaphLITE and Clearglide systems. While the Clearglide system allows for fewer incisions (number and length) and less harvest time, these benefits may be outweighed by the increased cost of the Clearglide system compared to the SaphLITE retractor

Clinical features of patients with homozygous complement C4A or C4B deficiency

Introduction Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. Material and methods Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. Results Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%Cl = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%Cl = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%Cl = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%Cl = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%Cl = 1.22-4.88, p = 0.010). Conclusion This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.Peer reviewe

Genotypic Diversity of Complement Component C4 Does Not Predict Kidney Transplant Outcome

Gene copy number of complement component C4, which varies among individuals, may determine the intrinsic strength of the classical complement pathway. Presuming a major role of complement as an effector in transplant rejection, we hypothesized that C4 genetic diversity may partially explain the variation in allograft outcomes. This retrospective study included 1969 deceased-donor kidney transplants randomly selected from the Collaborative Transplant Study DNA bank. We determined recipient and donor gene copy number of total C4, C4 isotypes (C4A and C4B), and C4 gene length variants (C4L and C4S) by quantitative real-time PCR analysis. Groups defined according to recipient C4 gene copy number (low, intermediate, and high) had similar 10-year allograft survival. Genotypic groups showed comparable rates of graft dysfunction, treatment for rejection, immunological graft loss, hospitalization for infection, malignant disease, and death. Similarly, separate analyses of C4A, C4B, C4L, and C4S; combined evaluation of donor and recipient C4 genotype; or analysis of recipients with higher risk for rejection did not reveal considerable outcome effects. In conclusion, we did not demonstrate that C4 gene copy number associates with transplant outcome, and we found no evidence that the resulting variation in the strength of classical complement activation influences susceptibility to rejection

Frontiers in Immunology / Neuropilin-1 Acts as a Receptor for Complement Split Products

Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a KD value of 0.71 M. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.(VLID)490490