Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.     

The Relative Abundance of Mountain Pine Beetle Fungal Associates Through the Beetle Life Cycle in Pine Trees

The mountain pine beetle (MPB) is a native bark beetle of western North America that attacks pine tree species, particularly lodgepole pine. It is closely associated with the ophiostomatoid ascomycetes Grosmannia clavigera, Leptographium longiclavatum, Ophiostoma montium, and Ceratocystiopsis sp.1, with which it is symbiotically associated. To develop a better understanding of interactions between beetles, fungi, and host trees, we used target-specific DNA primers with qPCR to assess the changes in fungal associate abundance over the stages of the MPB life cycle that occur in galleries under the bark of pine trees. Multivariate analysis of covariance identified statistically significant changes in the relative abundance of the fungi over the life cycle of the MPB. Univariate analysis of covariance identified a statistically significant increase in the abundance of Ceratocystiopsis sp.1 through the beetle life cycle, and pair-wise analysis showed that this increase occurs after the larval stage. In contrast, the abundance of O. montium and Leptographium species (G. clavigera, L. longiclavatum) did not change significantly through the MPB life cycle. From these results, the only fungus showing a significant increase in relative abundance has not been formally described and has been largely ignored by other MPB studies. Although our results were from only one site, in previous studies we have shown that the fungi described were all present in at least ten sites in British Columbia. We suggest that the role of Ceratocystiopsis sp.1 in the MPB system should be explored, particularly its potential as a source of nutrients for teneral adults.     

Passive irrigation and functional morphology of crustacean burrows in a tropical mangrove swamp

Flushing measurements and a resin cast of a burrow inhabited by Sesarma messa and Alpheus cf macklay were taken from a Rhizophoraspp. forest. The burrow had 9 openings and occupied a swamp surface area of 0.64 m². Passive irrigation of the burrow was investigated by recording change in conductivity of burrow water in a chamber 45 cm below the swamp surface during tidal inundation of the swamp. The chamber was completely flushed within approximately one hour, i.e. by a single tidal event. Burrow morphology was determined by means of resin casting. The investigated burrow was of discrete structure, with an overall depth of 1.2 m and a total volume of 68 l, i.e. ca. 9% of the volume of swamp soil. The below ground surface area of chambers and tunnels was 3.8 m². The mean and maximum chamber/tunnel diameter was 7 cm and 11 cm respectively. The soil in the close vicinity of the burrow was extensively penetrated by roots, and any two parts of the burrow were located no further than 20 cm away from each other. By reducing diffusion distances within the soil and by being well flushed, the burrows provide an efficient mechanism for removal of excess salt accumulated in the soil around mangrove roots due to exclusion.     

Modulating effect of cerulein on benzodiazepine receptors

Subcutaneous administration of caerulein (100-500 micrograms/kg) significantly reduced the development of picrotoxin (8 mg/kg) seizures in male mice. The same doses of caerulein inhibited 3H-flunitrazepam binding in in vivo experiments. Proglumide, an antagonist of cholecystokinin receptors, in low dose (5 mg/kg) potentiated the effects of caerulein (100 micrograms/kg), whereas the administration of proglumide in high dose (25 mg/kg) reduced the action of caerulein on 3H-flunitrazepam binding and picrotoxin seizures. Caerulein (5-1000 nM) decreased 3H-flunitrazepam binding in in vitro experiments only after supplementation of the binding medium with 120 mM NaCl and 5mM KCl. The results suggest the possible interaction of caerulein with chloride ionophor. It seems probable that the direct interaction of caerulein with chloride ionophor in involved in the inhibitory effect of caerulein on picrotoxin seizures and 3H-flunitrazepam binding.     

Purification and properties of thyrotrophin releasing hormone (TRH)-containing particles from the rat hypothalamus

The subcellular distribution of the TRH-like immunoreactivity in the rat hypothalamus and brain was studied. In differential centrifugation, the 900 g for 10 min supernatant (S1) of the hypothalamus or brain contained 61--79% of the total TRH. At 11,000 g for 20 min, 51--73% of the TRH in S1 was sedimented. When the hypothalamic S1 was fractioned under non-equilibrium conditions at 25 degrees C, two populations of TRH-containing particles were observed in several types of continuous linear density gradients. Metrizamide and sucrose gradients affected TRH-assay. TRH-particles were very light in Percol-gradients. Isotonic dextran 40,000-sucrose gradients gave the most reproducible results. In these gradients, the large TRH-particles (35%) equilibrated at 1.055--1.060 kg/l and the small ones (23%) at 1.041--1.047 kg/l. Working at 4 degrees C decreased the amount of large TRH-particles. The apparently larger particles contained cytoplasmic and mitochondrial enzymes and were sensitive to hypoosmotic shock like synaptosomes. Electron micrographs confirmed that these particles were synaptosomes. The true nature of the small particles remained unclear but morphologically a part of them were also synaptosomes. Treatment of the animals with reserpine (10 mg/kg i.p., 24 h), with 6-hydroxydopamine (100 microgram/rat i.c.v.) or with 5,7-dihydroxytryptamine (200 microgram/rat i.c.v.) did not affect significantly TRH-recovery or distribution in the hypothalamus.     

Regulation of cardiac cyclic GMP-dependent protein kinase

An assay method based on the ability of high concentrations of Mg²⁺ to stimulate phosphorylation of histone in the presence of low concentrations of ATP was developed for the measurement of cyclic GMP-dependent protein kinase activity ratios (activity -cyclic GMP/activity + cyclic GMP). In tissues which contain only trace amounts of cyclic GMP-dependent protein kinase, the basal activity ratios were high due to interference from a cyclic nucleotide-independent protein kinase. In order to study the regulation of the cardica cyclic GMP-dependent protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal or elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated witth the acetylcholine-induced protein kinase, factors affecting the equilibrium between the active and inactive forms of the enzyme were determined. Since the rate of dissociation of cyclic GMP from its binding site(s) was relatively slow at 0–4°C at pH 7.0, the amount of time required to process tissue samples was the major limiting factor for preserving the equilibrium between active and inactive forms of the enzyme. Dilution of heart tissue extracts at 0–4°C did not significantly alter the activity ratio of the enzyme under conditions of basal elevated cyclic GMP levels. Experiments using charcoal or exogenous cyclic GMP-dependent protein kinase in the homogenizing medium demonstrated that the release of sequestered cyclic GMP was not responsible for the elevation of the cyclic GMP-dependent protein kinase activity ratios by agents like acetylcholine. Therefore, the assay reflected in part, at least, the retention of kinase-bound cyclic GMP in the tissue extracts. The effects of acetylcholine and sodium nitroprusside on cyclic GMP levels, the cyclic GMP-dependent protein kinase activity ratios, and force of contraction were studied in the perfused rat heart. Both agents produced rapid, dose-dependent increases in cardiac cyclic GMP. Optimal concentrations of acetylcholine produced a 2–3-fold increase in the levels of cyclic GMP and an increase in the cyclic GMP-dependent protein kinase activity ratio. No significant effect of acetylcholine on cyclic nucleotide-independent protein kinase activity was observed. Associated with the acetylcholine-induced increase in cyclic GMP and the cyclic GMP-dependent protein kinase activity ratio was a reduction in the force of contraction. In contrast, nitroprusside produced little or no increase in the cyclic GMP-dependent protein kinase activity ratio despite increasing the level of cyclic GMP 8–10-fold. Nitroprusside also had no effect on contractile force. In combination, nitroprusside and acetylcholine produced additive effects on cyclic GMP levels, but protein kinase activation and force of contraction were similar to those seen with acetylcholine alone. The results suggest that the cyclic GMP produced by acetylcholine in the rat heart is coupled to activation of the cyclic GMP-dependent protein kinase, while that produced by nitroprusside is not.     

Functional outline of the epidemic process

The present study has shown that on the level of the parasitic system the epidemic process is a biological system, wherein the host population serves as the internal regulator, the mechanism of transmission serves as the external regulator and the parasite population, as the regulated object. The biological regulating mechanisms of the epidemic process have fundamental differences in the groups of infectious with various mechanisms of transmission, and the specific nature of the mechanism of transmission determines the peculiar features of the biological mechanism which governs the self-regulation of the epidemic process. In contrast, on a higher level of the organization of the epidemic process, i. e. on the level of the socio-ecological system, the epidemic process is a biosocial system, wherein the human society serves as the regulator, the parasitic system serves as the regulated object and the mechanism of transmission plays the role of the filter which determines the scope of social factors, most important in the regulation of the epidemic process in a given infection. The spontaneous regulation of the epidemic process is the freed forward channel from the regulator to the regulated object, and the controlled regulation is the feedback channel.