Separation of the principal HDL subclasses by iodixanol ultracentrifugation

HDL subclasses detection, in cardiovascular risk, has been limited due to the time-consuming nature of current techniques. We have developed a time-saving and reliable separation of the principal HDL subclasses employing iodixanol density gradient ultracentrifugation (IxDGUC) combined with digital photography. HDL subclasses were separated in 2.5 h from prestained plasma on a three-step iodixanol gradient. HDL subclass profiles were generated by digital photography and gel scan software. Plasma samples (n = 46) were used to optimize the gradient for the resolution of HDL heterogeneity and to compare profiles generated by IxDGUC with gradient gel electrophoresis (GGE); further characterization from participants (n = 548) with a range of lipid profiles was also performed. HDL subclass profiles generated by IxDGUC were comparable to those separated by GGE as indicated by a significant association between areas under the curve for both HDL₂ and HDL₃ (HDL₂, r = 0.896, P < 0.01; HDL₃, r = 0.894, P < 0.01). The method was highly reproducible, with intra- and interassay coefficient of variation percentage < 5 for percentage area under the curve HDL₂ and HDL₃, and < 1% for peak Rf and peak density. The method provides time-saving and cost-effective detection and preparation of the principal HDL subclasses.     

Solution structures of human myeloma IgG3 antibody reveal extended Fab and Fc regions relative to the other IgG subclasses

Human immunoglobulin G subclass 3 (IgG3) possesses a uniquely long hinge region that separates its Fab antigen-binding and Fc receptor-binding regions. Owing to this hinge length, the molecular structure of full-length IgG3 remains elusive, and the role of the two conserved Fc glycosylation sites are unknown. To address these issues, we subjected glycosylated and deglycosylated human myeloma IgG3 to multidisciplinary solution structure studies. Using analytical ultracentrifugation, the elongated structure of IgG3 was determined from the reduced sedimentation coefficients s⁰_20,w of 5.82 to 6.29 S for both glycosylated and deglycosylated IgG3. X-ray and neutron scattering showed that the Guinier R_G values were 6.95 nm for glycosylated IgG3 and were unchanged after deglycosylation, again indicating an elongated structure. The distance distribution function P(r) showed a maximum length of 25 to 28 nm and three distinct maxima. The molecular structure of IgG3 was determined using atomistic modeling based on molecular dynamics simulations of the IgG3 hinge and Monte Carlo simulations to identify physically realistic arrangements of the Fab and Fc regions. This resulted in libraries containing 135,135 and 73,905 glycosylated and deglycosylated IgG3 structures, respectively. Comparisons with the X-ray and neutron scattering curves gave 100 best-fit models for each form of IgG3 that accounted for the experimental scattering curves. These models revealed the first molecular structures for full-length IgG3. The structures exhibited relatively restricted Fab and Fc conformations joined by an extended semirigid hinge, which explains the potent effector functions of IgG3 relative to the other subclasses IgG1, IgG2, and IgG4.     

Early decreases in plasma lipid transfer proteins during weight reduction

Objective: To determine the effect of short‐term weight loss in obese women on concentrations of plasma cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP), two new risk factors for cardiovascular disease. Research Methods and Procedures: Plasma CETP and PLTP mass concentrations were measured in 38 obese, non‐diabetic women before and after a moderate, 4% weight loss that was obtained by a 1250 kcal/d diet for 4 weeks. Anthropometric and biological parameters were measured before and after weight loss. Results: Plasma CETP concentration decreased substantially after weight loss (2.76 ± 0.79 before and 2.31 ± 0.69 mg/L after; p = 0.000), and the same was true for plasma PLTP concentration (9.01 ± 2.44 mg/L before vs. 8.34 ± 2.57 after; p = 0.043). The HDL profile shifted toward the small‐sized range, with significant decreases in the relative abundance of HDL_2b and HDL_2a at the expense of HDL_3b after weight loss. A significant, positive correlation between CETP and PLTP mass concentrations is reported for the first time in obese patients (r = 0.43, p = 0.004), and weight reduction was accompanied by early, concomitant, and parallel decreases in plasma CETP and PLTP levels (r = 0.47, p = 0.003). The significant relationship between CETP and PLTP levels was lost after the dietary intervention (r = 0.27; p = 0.11). Discussion: CETP and PLTP correlate positively and significantly in obese patients. The hypocaloric dietary manipulation constitutes a relevant intervention to reduce rapidly and simultaneously plasma levels of CETP and PLTP. The impact of reduced PLTP activity on HDL size appeared to be more prominent than the impact of concomitant reduction in CETP activity.     

Effect of Liposomal Formulations and Immunostimulating Peptidoglycan Monomer (PGM) on the Immune Reaction to Ovalbumin in Mice

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDpm(εNH₂)-d-Ala-d-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).     

Isolation of High Density Lipoprotein Subclasses by Electrofiltration and Their Chemical Components

Abstract

The exact role of high density lipoprotein in atheroprotection is not well understood yet due to its complex nature; it comprises more than ten subclasses that vary in size, composition, and function. Isolation and characterization of these subclasses is an important step for further studies addressing their functions in health and disease. In this work, we present a novel method that is relatively simple and efficient for isolation of high density lipoprotein subclasses. The method depends on fractional filtration of the subclasses through a preformed gel membrane system under the effect of an electric field, where the stepwise isolation of the subclasses depends on differences in their rates of migration in polyacrylamide gel. Using this design, we were able to isolate seven high density lipoprotein subclasses with relative molecular masses of 42,000–50,000; 71,000; 103,000; 124,000; 150,000; 182,000; and 219,000. All the subclasses contained apolipoprotein A-I, phosphatidylcholine, sphingomyelin, free cholesterol, esterified cholesterol, and triacylglycerols. Some fractions of some samples contained the apolipoproteins A-II, C-I, C-II, C-III, and E. A subclass of molecular mass of 106,000 was identified and isolated from a healthy young subject that contained albumin and apoA-I with some free and esterified cholesterol, but with no triacylglycerols. This electrofiltration technique offers a novel tool for isolating pure native high density lipoprotein subclasses in a concentrated form that can be used directly for detailed studies of their physicochemical and physiological properties.     

The lipoprotein subfraction profile: heritability and identification of quantitative trait loci

The HDL and LDL subclass profile is an emerging cardiovascular risk factor. Yet, the biological and genetic mechanisms controlling the lipoprotein subclass distribution are unclear. Therefore, we aimed 1) to determine the heritability of the entire spectrum of LDL and HDL subclass features and 2) to identify gene loci influencing the lipoprotein subfraction pattern. Using NMR spectroscopy, we analyzed the lipoprotein subclass distribution in 1,275 coronary artery disease patients derived from the Regensburg Myocardial Infarction Family Study. We calculated heritabilities, performed a microsatellite genome scan, and calculated linkage. HDL and LDL subclass profiles showed heritabilities ranging from 23% to 67% (all P < 10(-3)) of traits using univariate calculation. After multivariate adjustment, we found heritabilities of 27-48% (all P < 0.05) for HDL and 21-44% for LDL traits. The linkage analysis revealed a significant logarithm of the odds (LOD) score (3.3) for HDL particle concentration on chromosome 18 and a highly suggestive signal for HDL particle size on chromosome 12 (2.9). After multivariate adjustment, we found a significant maximum LOD score of 3.7 for HDL size. Our study is the first to analyze heritability and linkage for the entire spectrum of LDL and HDL subclass features. Our findings may lead to the identification of genes controlling the lipoprotein subclass distribution.     

From Rhesus macaque to human: structural evolutionary pathways for immunoglobulin G subclasses

The Old World monkey, Rhesus macaque (Macaca mulatta, Mm), is frequently used as a primate model organism in the study of human disease and to test new vaccines/antibody treatments despite diverging before chimpanzees and orangutans. Mm and humans share 93% genome identity with substantial differences in the genes of the adaptive immune system that lead to different functional IgG subclass characteristics, Fcγ receptors expressed on innate immune cells, and biological interactions. These differences put limitations on Mm use as a primary animal model in the study of human disease and to test new vaccines/antibody treatments. Here, we comprehensively analyzed molecular properties of the Fc domain of the four IgG subclasses of Rhesus macaque to describe potential mechanisms for their interactions with effector cell Fc receptors. Our studies revealed less diversity in the overall structure among the Mm IgG Fc, with MmIgG1 Fc being the most structurally like human IgG3, although its C_H2 loops and N²⁹⁷ glycan mobility are comparable to human IgG1. Furthermore, the Fcs of Mm IgG3 and 4 lack the structural properties typical for their human orthologues that determine IgG3’s reduced interaction with the neonatal receptor and IgG4’s ability for Fab-arm exchange and its weaker Fcγ receptor interactions. Taken together, our data indicate that MmIgG1-4 are less structurally divergent than the human IgGs, with only MmIgG1 matching the molecular properties of human IgG1 and 3, the most active IgGs in terms of Fcγ receptor binding and Fc-mediated functions. PDB accession numbers for deposited structures are 6D4E, 6D4I, 6D4M, and 6D4N for MmIgG1 Fc, MmIgG2 Fc, MmIgG3 Fc, and MmIgG4 Fc, respectively.     

Characterization of cellular and humoral immune responses to alkaline phosphatase from fertile hydatid cysts in the human peripheral blood

Hydatid cyst, the larval stage of the tapeworm Echinococcus granulosus and a causative agent of cystic echinococcosis, possesses a vast number of antigenic peptides that are constantly presented in the host immune system during infection. Here, we sought to provide more information about the cellular/humoral components engaged in the peripheral immune reactions to the fertile-cyst-derived Echinococcus alkaline phosphatase (E.ALP) in human hosts. Lymphoproliferative and cytokine responses after recall of E.ALP suggested the presence of specific immune reactions against the antigen. Interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin (IL)-10 had the highest fold increase over the spontaneous levels in response to hydatid crude antigen (HCA). Recall of E.ALP, as well as its encounter, boosted IFN-γ, TNF-α, IL-2, and IL-6 responses in peripheral blood mononuclear cells cultures (PBMCs). The HCA-driven levels of all the cytokines in the culture supernatants of normal PBMCs were higher than those measured after E.ALP encounter. Immunoglobulin G (IgG)-profile in response to HCA showed the dominance of specific IgG1, IgG2, and IgG4 antibodies, but relatively lower affinity of IgG3 to this antigen. IgG1 and IgG3 were the only isotypes detected in serum responses to E.ALP. Our findings suggested that E.ALP contributes to the early phase of immune responses to the parasite, likely by induction of proinflammatory profiles and clonal expansion of high-affinity IgG1- and IgG3-secreting plasma cells, suggesting the value of E.ALP as a candidate to develop novel therapeutic and immunization strategies.     

IgG binding kinetics to oligo B protein A domains on lipid layers immobilized on a 27 MHz quartz-crystal microbalance

Although molecular recognitions between membrane receptors and their soluble ligands have been analyzed using their soluble proteins in bulk solutions, molecular recognitions of membrane receptors should be studied on lipid membranes considering their orientation and dynamics on membrane surfaces. We employed Staphylococcal Protein A (SpA) oligo B domains with long trialkyl-tags from E. coli (LppBx, x = 1, 2, and 5) and immobilized LppBx on lipid layers using hydrophobic interactions from the trialkyl-tag, while maintaining the orientation of B domain-chains on a 27 MHz quartz-crystal microbalance (QCM; AT-cut shear mode). The binding of IgG Fc regions to LppBx on lipid layers was detected by frequency decreases (mass increases) on the QCM. The maximum amount bound (Delta m(max)), association constants (K(a)), association and dissociation rate constants (k(1) and k(-1), respectively) were obtained. Binding kinetics of IgG to LppB2 and LppB5 were quite similar, showing a simple 1:1 binding of the IgG Fc region to the B domain, when the surface coverage of LppB2 and LppB5 on the lipid surface is low (1.4%). When LppB5 was immobilized at the high surface coverage of 3.5%, the complex bindings of IgG such as one IgG bound to one or two LppB5 on the membrane could be observed. IgG-LppB1 binding was largely restricted because of steric hindrance on lipid surfaces. This gives a suggestion why Protein A has five IgG binding domains.     

Kinetic analysis of thermal stability of human low density lipoproteins: a model for LDL fusion in atherogenesis

Fusion of modified LDL in the arterial wall promotes atherogenesis. Earlier we showed that thermal denaturation mimics LDL remodeling and fusion, and revealed kinetic origin of LDL stability. Here we report the first quantitative analysis of LDL thermal stability. Turbidity data show sigmoidal kinetics of LDL heat denaturation, which is unique among lipoproteins, suggesting that fusion is preceded by other structural changes. High activation energy of denaturation, E(a) = 100 ± 8 kcal/mol, indicates disruption of extensive packing interactions in LDL. Size-exclusion chromatography, nondenaturing gel electrophoresis, and negative-stain electron microscopy suggest that LDL dimerization is an early step in thermally induced fusion. Monoclonal antibody binding suggests possible involvement of apoB N-terminal domain in early stages of LDL fusion. LDL fusion accelerates at pH < 7, which may contribute to LDL retention in acidic atherosclerotic lesions. Fusion also accelerates upon increasing LDL concentration in near-physiologic range, which likely contributes to atherogenesis. Thermal stability of LDL decreases with increasing particle size, indicating that the pro-atherogenic properties of small dense LDL do not result from their enhanced fusion. Our work provides the first kinetic approach to measuring LDL stability and suggests that lipid-lowering therapies that reduce LDL concentration but increase the particle size may have opposite effects on LDL fusion.