Heightened Proinflammatory Glycosylation of Borrelia burgdorferi IgG Antibodies in Synovial Fluid in Patients With Antibiotic-Refractory Lyme Arthritis.

OBJECTIVE: Terminal glycans on the Fc portion of IgG antibodies are critical for antibody-triggered, proinflammatory or antiinflammatory responses. We undertook this study to compare glycan profiles of total IgG1 and Borrelia burgdorferi (Bb)-specific IgG1 antibodies in patients with oral antibiotic-responsive or antibiotic-refractory Lyme arthritis (LA). METHODS: Following affinity-column processing, glycan profiles of IgG antibodies were determined in serum and synovial fluid (SF) samples of 21 LA patients using glycoblotting with hydrazide glycan enrichment and determination of glycan structure by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Correlations between glycan profiles and treatment outcomes were analyzed. RESULTS: Compared with patients with antibiotic-refractory LA, those with antibiotic-responsive LA had total and Bb-specific IgG1 antibody glycans with less intense inflammatory profiles, containing lower percentages of N-acetylglucosamine (GlcNAc) and bisecting GlcNAc and higher percentages of galactose and fucose. In contrast, patients with antibiotic-refractory LA prior to receiving IV antibiotic therapy had total IgG1 and Bb IgG1 antibodies with maximal, minimally opposed, proinflammatory glycan profiles, containing high percentages of GlcNAc and bisecting GlcNAc, intermediate percentages with galactose and fucose, and low percentages with N-acetylneuraminic acid (sialic acid). Patients with refractory LA who were first seen with synovitis after receiving IV antibiotic therapy still had Bb IgG1 antibodies with strongly inflammatory glycan profiles, but their inflammatory potential appeared to be waning. CONCLUSION: Patients with oral antibiotic-responsive LA had Bb IgG1 antibodies with more balanced proinflammatory/antiinflammatory glycan profiles, whereas patients with antibiotic-refractory LA had Bb IgG1 antibodies with maximal, minimally opposed, proinflammatory glycan profiles. Among patients with antibiotic-refractory LA, antibodies with this unbalanced inflammatory glycan profile may have a role in sustaining maladaptive joint inflammation.

Glycoblotting of Egg White Reveals Diverse N-Glycan Expression in Quail Species.

The glycan part of glycoproteins is known to be involved in the structure and modulatory functions of glycoproteins, serving as ligands for cell-to-cell interactions, and as specific ligands for cell-to-microbe interactions. It is believed that intraspecies and interspecies variations in glycosylation exist. As an approach to better understand glycan diversity, egg whites (EW) from four different quail species are studied by the well-established glycoblotting procedure, a glycan enrichment and analysis method. N-Glycans were classified and the profiles were established for quail egg white samples which showed 21 relevant glycan peaks; 18 peaks were expressed significantly, and 10 glycan peaks are found to be abundant in certain species. The result establishes glycan profiles for Blue Scaled, Bobwhite, Japanese, and Mountain Quail egg whites and shows a unique difference among glycan expressions, particularly, high mannose in Japanese Quail and tetra-antennary glycan structure for other quail species.

Method comparison for analyzing wound healing rates.

Wound healing scratch assay is a frequently used method to characterize cell migration, which is an important biological process in the course of development, tissue repair, and immune response for example. The measurement of wound healing rate, however, varies among different studies. Here we summarized these measurements into three types: (I) direct rate average; (II) regression rate average; and (III) average distance regression rate. Using Chinese hamster ovary (CHO) cells as a model, we compared the three types of analyses on quantifying the wound closing rate, and discovered that type I & III measurements are more resistant to outliers, and type II analysis is more sensitive to outliers. We hope this study can help researchers to better use this simple yet effective assay.

Gypsy moth pheromone-binding protein-ligand interactions: pH profiles and simulations as tools for detecting polar interactions.

Pheromone-binding proteins (PBPs) are believed to control diffusion of pheromones in sensory hairs of insects. The interactions of gypsy moth (Lymantria dispar) PBPs with the sex attractant pheromone, (+)-Disparlure ((7R,8S)-epoxy-2-methyloctadecane), and the enantioselectivity of recognition are not completely understood. Enantioselectivity is important for L. dispar, because (-)-disparlure cancels the attraction of (+)-disparlure, so these moths use enantiopure (+)-disparlure for communication. We performed docking simulations of the protonated homology PBP models with the enantiomers of disparlure, 5-oxadisparlure, 10-oxadisparlure, 5-thiadisparlure and 10-thiadisparlure, together with a binding assay experiment, in which the pH profiles for the PBP-ligand combinations were surveyed. The molecular simulations revealed different amino acid residues in the binding sites, movement of specific amino acid residues at certain pH values, distinct amino acid-ligand interactions (side chain donors/acceptors, H-arene bonding, backbone donors/acceptors) and differences in the conformations of each protein-ligand complex. The pKa values obtained from the binding experiment and the results from the molecular simulations served as tools for detecting polar interactions between the PBPs and ligands. The differences found between structures docked with ligand enantiomers reveal the enantioselectivity of the gypsy moth PBPs towards the pheromone and its antipode, as well as towards enantiomers of pheromone analogs with heteroatom substitutions.