Lynch, G. W. | Search bioRxiv

bioRxiv2021

Translational Proteomics for Transfusion Medicine: Resolution of the IVIG Proteomes of Different Geographically Sourced and Prepared IVIG Immunotherapies.

Intravenous Immunoglobulins (IVIGs) are prepared from thousands of donor plasmas and as a result comprise an extreme broad mix and depth of Antibody (Ab) specificities. IVIG formulations available in Australia are from both local and overseas donor sources and extracted using a variety of purification methods and immunoglobulin purities, with the Australia-derived and prepared IVIG listed at >98% and the overseas-derived preparations at [≥] 95%. Because of these differences it was predicted that the formulations might individually vary in composition and that together with obvious genetic and geographic antigenic (Ag) environment differences may result in notable variability between formulations. Hence a focussed comparative proteomic profiling of IVIG formulations was undertaken to identify notable similarities and differences across products. Comparisons between formulations did reveal marked qualitative differences in 2D-gel Antibody profiling that included parameters of isoelectric charge (pI), as well as immunoglobulin (Ig) monomer to dimer ratio variability between products, including high molecular weight (MW) immunoglobulin multimers for some. These notable differences were in part quite likely a product of the respective purification methods used, and capacity to select (or de-select) for antibodies of such different properties. Furthermore, for identification of non-Ig proteins carried over from plasma through purifications Mass spectrometry was performed. This identified a few such ancillary proteins, and their identities, in general, differed between formulations. Proteins detected included the most abundant protein of plasma, albumin, as well as other mostly large and abundant proteins; RAG1 - V(D)J recombination activating protein1, gelsolin, complement Factor-B, serotransferrin, tetranectin, NADH ubiquinone oxidoreductase, caspase 3 and VEGFR1. An alternate strategy used commercial Multiplex xMAP assay to detect cytokines, which are small and present in plasma at trace but highly active quantities. This revealed various different cytokine profiles across the formulations studied. The identification of additional proteins, and especially cytokines in IVIGs, is particularly notable, and the positive, negative or null biological relevance for clinical use, needs resolution. Collectively these findings reveal marked differences between Australian and overseas-derived (non-Australian) IVIGs in immunoglobulin composition and biochemical characteristics, and presence of additional carry-over proteins from plasma. These findings prompt the need for further evaluation of the micro-compositions of individual formulations. Perhaps detailed mining and improved comparative understanding of each IVIG formulation, may enable highly tailored and strategic clinical use of certain formulations that are personalised best-fit treatments for particular conditions. Such as in treatment of a neuropathy, as compared to another formulation, more suited for treating a particular infectious disease. The most salient and overarching study conclusion is need for caution in attributing equivalence across IVIGs.

immunology↗

bioRxiv2019

Cell-projection pumping: A hydrodynamic cell-stiffness dependent mechanism for cytoplasmic transfer between mammalian cells

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fib) and malignant cells (MC). Others report similar transfer via either tunneling nanotubes (TNT) or shed membrane vesicles and this changes the phenotype of recipient cells. Our current time-lapse microscopy showed most exchange was from Fib into MC, with less in the reverse direction. Although TNT were seen, we were surprised transfer was not via TNT, but was instead via fine and often branching cell projections that defied direct visual resolution because of their size and rapid movement. Their structure was revealed nonetheless, by their organellar cargo and the grooves they formed indenting MC, while this was consistent with holotomography. Discrete, rapid and highly localized transfer events, evidenced against a role for shed vesicles. Transfer coincided with rapid retraction of the cell-projections, suggesting a hydrodynamic mechanism. Increased hydrodynamic pressure in retracting cell-projections normally returns cytoplasm to the cell body. We hypothesize cell-projection pumping (CPP), where cytoplasm in retracting cell-projections partially equilibrates into adjacent recipient cells via micro-fusions that form temporary inter-cellular cytoplasmic continuities. We tested plausibility for CPP by combined mathematical modelling, comparison of predictions from the model with experimental results, and then computer simulations based on experimental data. The mathematical model predicted preferential CPP into cells with lower cell stiffness, expected from equilibration of pressure towards least resistance. Predictions from the model were satisfied when Fib were co-cultured with MC, and fluorescence exchange related with cell stiffness by atomic force microscopy. When transfer into 5000 simulated recipient MC or Fib was studied in computer simulations, inputting experimental cell stiffness and donor cell fluorescence values generated transfers to simulated recipient cells similar to those seen by experiment. We propose CPP as a potentially novel mechanism in mammalian inter-cellular cytoplasmic transfer and communication. SIGNIFICANCETime-lapse observations of co-cultured cells led us to hypothesize what we believe to be a novel hydrodynamic mechanism transferring cytoplasm between cells. Similar transfer by other mechanisms markedly affects cell behavior. Combined mathematical modelling, satisfaction of predictions from the mathematical model in cell culture experiments, and separate computer simulations that generate outcomes similar to experimental observations, support our hypothesized mechanism.

cell biology↗