Gas propulsion of microprojectiles for the transformation of biological cells

Bombardment of intact cells and tissue with DNA-coated microprojectiles represents a novel approach to the genetic transformation of biological material. In this thesis, a gas propulsion particle gun is developed for such a purpose. The particle gun utilises gas dynamics to control particle velocity and spread, thereby enabling optimisation of transformation efficiencies for varying types of target material. The dynamics associated with particle acceleration are shown to relate to transient, shock tube flow. Measurements from schlieren high-speed video photographs of the shock structure of an underexpanded jet demonstrate good agreement with empirical correlations of previous researchers; pitot tube measurements of the nozzle exit Mach number are made and shown to be in good agreement with theoretical contact surface velocities. A novel optical particle velocimeter is used to measure particle time-offlight between axial locations in the system's target chamber, providing a consistent, distance-averaged measurement of particle velocities. Particle velocities are measured for a range of system pressure ratios and driver gases. Variation in particle velocities is seen to be similar to theoretical variation in contact surface velocities. Analytical theory is used to predict small gas-particle velocity lag. Particle velocities with helium as a driver gas are shown to be considerably higher than those with air. High speed video recording of chalk particles exiting the nozzle is used to visualise the spatial and temporal variation in particle spread as a function of nozzle pressure ratio. This technique demonstrates that particle spread increases with increasing nozzle pressure ratio, as gas dynamics theory indicates. Conditions for bombardment of maize suspension cells are experimentally optimised. Significant rates of transient genetic expression are achieved with both air and helium as driver gases. High levels of transient genetic expression are also found with bombardment of maize coleoptiles and the leaves of various dicot species. Transformation efficiencies for bombardment of HL60 human leukaemia cells show dramatic increases over efficiencies seen with conventional techniques not involving cell bombardment. For other cell types the gas propulsion device described here appears to give rates of transient genetic expression similar to those reported for commercial systems using microprojectiles. Other data for the performance of such systems are too limited at present to allow comparisons of controllability and reproducibility of bombardment efficiency.</p

High generation of reactive oxygen species from neutrophils in patients with severe COVID-19.

Neutrophilia and an elevated neutrophil:lymphocyte ratio are both characteristic features of severe COVID-19 infection. However, functional neutrophil responses have been poorly investigated in this setting. We utilised a novel PMA-based stimulation assay to determine neutrophil-derived reactive oxygen species (ROS) generation in patients with severe COVID-19 infection, non-COVID related sepsis and healthy study participants. ROS production was markedly elevated in COVID-19 patients with median values ninefold higher than in healthy controls and was particularly high in patients on mechanical ventilation. ROS generation correlated strongly with neutrophil count and elevated levels were also seen in patients with non-COVID related sepsis. Relative values, adjusted for neutrophil count, were high in both groups but extreme low or high values were seen in two patients who died shortly after testing, potentially indicating a predictive value for neutrophil function. Our results show that the high levels of neutrophils observed in patients with COVID-19 and sepsis exhibit functional capacity for ROS generation. This may contribute to the clinical features of acute disease and represents a potential novel target for therapeutic intervention