In vitro characterization of PrismaLung+: a novel ECCO2R device.

BACKGROUND: Invasive mechanical ventilation is lifesaving in the setting of severe acute respiratory failure but can cause ventilation-induced lung injury. Advances in extracorporeal CO2 removal (ECCO2R) technologies may facilitate more protective lung ventilation in acute respiratory distress syndrome, and enable earlier weaning and/or avoid invasive mechanical ventilation entirely in chronic obstructive pulmonary disease exacerbations. We evaluated the in vitro CO2 removal capacity of the novel PrismaLung+ ECCO2R device compared with two existing gas exchangers. METHODS: The in vitro CO2 removal capacity of the PrismaLung+ (surface area 0.8 m2, Baxter) was compared with the PrismaLung (surface area 0.35 m2, Baxter) and A.L.ONE (surface area 1.35 m2, Eurosets) devices, using a closed-loop bovine blood-perfused extracorporeal circuit. The efficacy of each device was measured at varying pCO2 inlet (pinCO2) levels (45, 60, and 80 mmHg) and blood flow rates (QB) of 200-450 mL/min; the PrismaLung+ and A.L.ONE devices were also tested at a QB of 600 mL/min. The amount of CO2 removed by each device was assessed by measurement of the CO2 infused to maintain circuit equilibrium (CO2 infusion method) and compared with measured CO2 concentrations in the inlet and outlet of the CO2 removal device (blood gas analysis method). RESULTS: The PrismaLung+ device performed similarly to the A.L.ONE device, with both devices demonstrating CO2 removal rates ~ 50% greater than the PrismaLung device. CO2 removal rates were 73 ± 4.0, 44 ± 2.5, and 72 ± 1.9 mL/min, for PrismaLung+, PrismaLung, and A.L.ONE, respectively, at QB 300 mL/min and pinCO2 45 mmHg. A Bland-Altman plot demonstrated that the CO2 infusion method was comparable to the blood gas analysis method for calculating CO2 removal. The resistance to blood flow across the test device, as measured by pressure drop, varied as a function of blood flow rate, and was greatest for PrismaLung and lowest for the A.L.ONE device. CONCLUSIONS: The newly developed PrismaLung+ performed more effectively than PrismaLung, with performance of CO2 removal comparable to A.L.ONE at the flow rates tested, despite the smaller membrane surface area of PrismaLung+ versus A.L.ONE. Clinical testing of PrismaLung+ is warranted to further characterize its performance.

Challenges and Opportunities in the Development of Aptamers for TNFα.

RNA aptamers for tumor necrosis factor-alpha (TNFα), for which functionality was demonstrated in L929 cells, show only little affinity for the protein in vitro. Detailed investigation of the aptamer-protein interaction by surface plasmon resonance and quartz crystal microbalance analysis revealed that affinity is not the only crucial parameter for efficacy and functionality of those aptamers. Instead, the sensitive equilibrium of the monomeric and homotrimeric form of soluble TNFα decides on aptamer binding. Our results show that the field of application and the source of TNFα have to be carefully defined before selection of aptamer sequences.

Fabrication of Homogeneous High-Density Antibody Microarrays for Cytokine Detection.

Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids can contribute to the diagnosis of diseases. Here we report on the development of an array comprised of different anti-cytokine antibodies on an activated solid support coupled with a fluorescence readout mechanism. Optimization of the array preparation was done in regard of spot homogeneity and spot size. The proinflammatory cytokines Tumor Necrosis Factor alpha (TNFα) and Interleukin 6 (IL-6) were chosen as the first targets of interest. First, the solid support for covalent antibody immobilization and an adequate fluorescent label were selected. Three differently functionalized glass substrates for spotting were compared: amine and epoxy, both having a two-dimensional structure, and the NHS functionalized hydrogel (NHS-3D). The NHS-hydrogel functionalization of the substrate was best suited to antibody immobilization. Then, the optimization of plotting parameters and geometry as well as buffer media were investigated, considering the ambient analyte theory of Roger Ekins. As a first step towards real sample studies, a proof of principle of cytokine detection has been established.

Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs).

Although the identification of the multigene family encoding mammalian olfactory receptors were identified more than 20 years ago, we are far from understanding olfactory perception because of the difficulties in functional expression of these receptors in heterologous cell systems. Cell-free (CF) or in vitro expression systems offer an elegant alternative route to cell based protein expression, as the functional expression of membrane proteins can be directly achieved from the genetic template without the need of cell cultivation and protein isolation. Here we investigated in detail the cell-free expression and membrane insertion of the olfactory receptor OR5 in dependence of different experimental conditions like probing different origins of the cell-free expression system (from bacteria, via plants and insects toward mammalian system) and lipid composition of the respective extracts. We provided substantial biochemical indications by radioactive labeling based on [(35)S]-methionine, followed by proteolytic digestion, and we found that the insertion of the olfactory receptor OR5 into liposomes resulted in an unidirectional orientation with the binding side exposed into the aqueous space, resembling the native orientation in the cilia of the olfactory neurons. We report the different results in synthesis capacity for the different in vitro systems employed as we like to demonstrate the first in vitro kit toward and ex situ and ex vivo odorant receptor array.

Cytochrome c biosensor--a model for gas sensing.

This work is about gas biosensing with a cytochrome c biosensor. Emphasis is put on the analysis of the sensing process and a mathematical model to make predictions about the biosensor response. Reliable predictions about biosensor responses can provide valuable information and facilitate biosensor development, particularly at an early development stage. The sensing process comprises several individual steps, such as phase partition equilibrium, intermediate reactions, mass-transport, and reaction kinetics, which take place in and between the gas and liquid phases. A quantitative description of each step was worked out and finally combined into a mathematical model. The applicability of the model was demonstrated for a particular example of methanethiol gas detection by a cytochrome c biosensor. The model allowed us to predict the optical readout response of the biosensor from tabulated data and data obtained in simple liquid phase experiments. The prediction was experimentally verified with a planar three-electrode electro-optical cytochrome c biosensor in contact with methanethiol gas in a gas tight spectroelectrochemical measurement cell.