Fc gamma receptor IIIb binding of individual antibody proteoforms resolved by affinity chromatography–mass spectrometry

The crystallizable fragment (Fc) of immunoglobulin G (IgG) activates key immunological responses by interacting with Fc gamma receptors (FcɣR). FcɣRIIIb contributes to neutrophil activation and is involved in antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). These processes present important mechanisms-of-actions of therapeutic antibodies. The very low affinity of IgG toward FcɣRIIIb (KD ~ 10 µM) is a technical challenge for interaction studies. Additionally, the interaction is strongly dependent on IgG glycosylation, a major contributor to proteoform heterogeneity. We developed an affinity chromatography–mass spectrometry (AC-MS) assay for analyzing IgG-FcɣRIIIb interactions in a proteoform-resolved manner. This proved to be well suited to study low-affinity interactions. The applicability and selectivity of the method were demonstrated on a panel of nine different IgG monoclonal antibodies (mAbs), including no-affinity, low-affinity and high-affinity Fc-engineered or glycoengineered mAbs. Thereby, we could reproduce reported affinity rankings of different IgG glycosylation features and IgG subclasses. Additional post-translational modifications (IgG1 Met252 oxidation, IgG3 hinge-region O-glycosylation) showed no effect on FcɣRIIIb binding. Interestingly, we observed indications of an effect of the variable domain sequence on the Fc-binding that deserves further attention. Our new AC-MS method is a powerful tool for expanding knowledge on structure–function relationships of the IgG-FcɣRIIIb interaction. Hence, this assay may substantially improve the efficiency of assessing critical quality attributes of therapeutic mAbs with respect to an important aspect of neutrophil activation

Increased Hemoglobin Mass and VO2max With 10 h Nightly Simulated Altitude at 3000 m

Purpose: To quantify the changes of hemoglobin mass (Hbmass) and maximum oxygen consumption (VO2max) after 22 days training at 1300-1800 m combined with nightly exposure to 3000-m simulated altitude. We hypothesized that with simulated 3000-m altitude, an adequate beneficial dose could be as little as 10 h/24 h. Methods: Fourteen male collegiate runners were equally divided into 2 groups: altitude (ALT) and control (CON). Both groups spent 22 days at 1300-1800 m. ALT spent 10 h/night for 21 nights in simulated altitude (3000 m), and CON stayed at 1300 m. VO2max and Hb mass were measured twice before and once after the intervention. Blood was collected for assessment of percent reticulocytes (%retics), serum erythropoietin (EPO), ferritin, and soluble transferrin receptor (sTfR) concentrations. Results: Compared with CON there was an almost certain increase in absolute VO2max (8.6%, 90% confidence interval 4.8-12.6%) and a likely increase in absolute Hbmass (3.5%; 0.9-6.2%) at postintervention. The %retics were at least very likely higher in ALT than in CON throughout the 21 nights, and sTfR was also very likely higher in the ALT group until day 17. EPO of ALT was likely higher than that of CON on days 1 and 5 at altitude, whereas serum ferritin was likely lower in ALT than CON for most of the intervention. Conclusions: Together the combination of the natural and simulated altitude was a sufficient total dose of hypoxia to increase both Hbmass and VO2max.</p

3D-cardiomics: A spatial transcriptional atlas of the mammalian heart.

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/