A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.

Alterations in Saliva and Plasma Cytokine Concentrations During Long-Duration Spaceflight

Long-duration spaceflight is known to cause immune dysregulation in astronauts. Biomarkers of immune system function are needed to determine both the need for and effectiveness of potential immune countermeasures for astronauts. Whereas plasma cytokine concentrations are a well-established biomarker of immune status, salivary cytokine concentrations are emerging as a sensitive indicator of stress and inflammation. For this study, to aid in characterizing immune dysregulation during spaceflight, plasma and saliva cytokines were monitored in astronauts before, during and after long-duration spaceflight onboard the International Space Station. Blood was collected from 13 astronauts at 3 timepoints before, 5 timepoints during and 3 timepoints after spaceflight. Saliva was collected from 6 astronauts at 2 timepoints before spaceflight, 2 timepoints during and 3 timepoints following spaceflight. Samples were analyzed using multiplex array technology. Significant increases in the plasma concentration of IL-3, IL-15, IL-12p40, IFN-α2, and IL-7 were observed during spaceflight compared to before flight baseline. Significant decreases in saliva GM-CSF, IL-12p70, IL-10 and IL-13 were also observed during spaceflight as compared to compared to before flight baseline concentrations. Additionally, plasma TGFβ1 and TGFβ2 concentrations tended to be consistently higher during spaceflight, although these did not reach statistical significance. Overall, the findings confirm an in-vivo hormonal dysregulation of immunity, appearing pro-inflammatory and Th1 in nature, persists during long-duration orbital spaceflight. These biomarkers may therefore have utility for monitoring the effectiveness of biomedical countermeasures for astronauts, with potential application in terrestrial research and medicine.

Anticitrullinated protein/peptide antibody multiplexing defines an extended group of ACPA-positive rheumatoid arthritis patients with distinct genetic and environmental determinants

IntroductionThe second generation anticycliccitrullinated peptide (anti-CCP2) assay detects the majority but not all anticitrullinated protein/peptide antibodies (ACPA). Anti-CCP2-positive rheumatoid arthritis (RA) is associated with HLA-DRB1* shared epitope (SE) alleles and smoking. Using a multiplex assay to detect multiple specific ACPA, we have investigated the fine specificity of individual ACPA responses and the biological impact of additional ACPA reactivity among anti-CCP2-negative patients.MethodsWe investigated 2825 patients with RA and 551 healthy controls with full data on anti-CCP2, HLA-DRB1* alleles and smoking history concerning reactivity against 16 citrullinated peptides and arginine control peptides with a multiplex array.ResultsThe prevalence of the 16 ACPA specificities ranged from 9% to 58%. When reactivity to arginine peptides was subtracted, the mean diagnostic sensitivity increased by 3.2% with maintained 98% specificity. Of the anti-CCP2-negative patients, 16% were found to be ACPA positive. All ACPA specificities associated with SE, and all but one with smoking. Correction for arginine reactivity also conveyed a stronger association with SE for 13/16 peptides. Importantly, when all ACPA specificities were analysed together, SE and smoking associated with RA in synergy among ACPA positive, but not among ACPA-negative subjects also in the anti-CCP2-negative subset.ConclusionsMultiplexing detects an enlarged group of ACPA-positive but anti-CCP2-negative patients with genetic and environmental attributes previously assigned to anti-CCP2-positive patients. The individual correction for arginine peptide reactivity confers both higher diagnostic sensitivity and stronger association to SE than gross ACPA measurement.