An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

Water relations of Picea abies. I. Comparison of water relations parameters of spruce shoots examined at the end of the vegetation period and in winter

Seasonal changes of some water relations parameters of Norway spruce shoots ( Picea abies [L.] Karst.) were studied during two experiments using the pressure-volume analysis. For each experiment only shoots of a single tree were used.
During the first study, the course of the turgor loss point (as bulk osmotic pressure when turgor first reaches zero, πp) of shoots developed in late 1986 vegetation period, were measured in 1987. The turgor loss point decreased temporarily from –2.5 MPa at the beginning of the year to –3.3 MPa at the end of March, but then increased to the original level for the rest of the year.
During the second study, water relations parameters were measured in late summer 1987 and in late winter 1988. Winter shoots at full water saturation contained up to 20% less water than in late summer. Accordingly, the bulk osmotic pressure at full water saturation (π_p) decreased from –1.7 MPa in late summer to –1.9 MPa in winter, π_p decreased also from –2.2 MPa to –2.8 MPa. However, the amount of osmotically active substances (mOsmol, N) remained unchanged. The relative amount of apoplastic water in the total shoot water content appeared to drop insignificantly from 17% to 15%.
The results show that the decrease in π_o and π_p in late winter is not due to an accumulation of osmotically active substances in the vacuoles but is due to a decrease in tissue water content. The temporary reduction of the symplastic volume by deposition of osmotically inert substances seems to be the most probable cause of this phenomenon.     

The radiation response of hypoxic cells in EMT6 spheroids in suspension culture does model data from EMT6 tumors

Radiation survival curves of EMT6/Ed spheroids have been obtained under conditions which eliminate changes in oxygen concentration between growth and irradiation. These curves show a high-dose, resistant component which is nearly parallel to the curves obtained when spheroids were irradiated under nitrogen. Thus EMT6 spheroids appear to model accurately the radiation responses of EMT6 tumors. In contrast, when spheroids were grown to relatively high density (300-400 spheroids per 250-ml spinner flask), then separated into several flasks for irradiation, an increase in oxygen concentration in the medium occurred which fully oxygenated the previously hypoxic cells. The two causes for the oxygen depletion in sealed growth flasks were quantitated. Depletion of total oxygen in the flask occurred, and, more importantly, oxygen consumption kept the growth medium well below equilibrium with the oxygen in the gas phase. Smaller but similar effects on oxygen concentration were found in flasks containing V79 spheroids.