Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo.

Human natural killer (NK) cells play an important role in anti-viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon-inducible molecules CD69 and tetherin peaked 24-48 h post-infection, coincident with a peak of interferon-induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post-infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus-based therapy. Furthermore, our results suggest the existence of a post-infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.

Isolation of ribosomes from cysts of entamoeba invadens.

A reproducible method for the preparation of 75S ribosomes from cysts of Entamoeba invadens is presented. The method depends on the inactivation of the cyst's ribonuclease by high levels of Bentonite. The ribosomes are found to be extremely sensitive to ribonuclease, and to be stabilized by the addition of manganous and calcium ions to the magnesium customarily employed. Reasons are given for equating these ribosomes with the particles of which the crystalline chromatoid bodies are made.

Structure of ribosomes of chromatoid bodies: three-dimensional Fourier synthesis at low resolution.

A three-dimensional Fourier synthesis at an axial resolution of 75 angstroms of optical diffraction data from electron micrographs of stained sections of chromatoid bodies reveals the position of the large and small ribosomal subunits within these crystals of ribosomes.

Zinc in Entamoeba invadens.

Atomic absorption spectroscopy, electron microprobe analysis, and dithizone staining of trophozoites and cysts of Entamoeba invadens demonstrate that these cells have a high concentration of zinc (approximately 10(-6) microgram per cell or 1 percent of their dry weight). In the cysts of this organism, the zinc is confined to the chromatoid bodies, which previous work has shown to contain crystals of ribosomes. The chemical state and function of this zinc are unknown.

Nature of the packing of ribosomes within chromatoid bodies.

Optical Fourier transformns, made of electron mnicrographs of the crystalline array of ribonucleoprotein known as chromatoid bodies of Entamoeba invadens, have been interpreted as the transforms of a helix of 12 nodes in five turns. A model shows that this helix may be built of spheres 180 angstroms in diameter, placed at a radius of 150 angstroms. The optical transform of the model is very similar to the transform of the original electron micrograph.

A new method for computing the macromolecular electric potential.

A general methodology is developed for the rigorous computation of the electrostatic potential for a protein of arbitrary shape, assuming the presence of linear dielectric media. The theory proceeds by considering the distribution of induced polarization charge at the dielectric interface, rather than by attempting a direct solution of Poisson's equation (as in the finite-difference approach of Warwicker & Watson). The method is applied to a study of two-dimensional model proteins, where it is shown that the presence of a cleft is associated with a region of relatively high potential in the solvent medium. The results of a preliminary calculation in three dimensions for the protein lysozyme are also discussed; again, a region of enhanced potential is observed near the cleft at the active site. Our computational evidence supports the suggestion of Warwicker & Watson that clefts are associated with important electrostatic effects.

Regression of atheroma in the rabbit.

Ten studies in the literature concerning regression of rabbit atheroma were re-examined. In studies where cholesterol content was referred to weight, a degree of regression was noted in 3/4 studies. Such regression might at least partly have resulted from the dilution effect of the atheroma contents when results were expressed on a weight basis. By contrast, when results were referred to length or protein, partial regression was seen in only 1/4 studies. Mild atheroma induced by short-term cholesterol feeding did seem to regress in 2/2 studies.

Exchange of plasma radioactive cholesterol with atheroma lipids in situ and implanted subcutaneously.

Physicochemical exchange of radioactive cholesterol was investigated in mildly hypercholesterolaemic rabbits. Such exchange was examined in erythrocytes and atheroma lipids in situ in the aortic wall and in such lipids when implanted subcutaneously. The implanted atheroma lipids gained radioactivity from intravenously injected [3H]cholesterol, even though cholesterol was being resorbed from the implants. Autoradiography showed exchange between plasma cholesterol and both intracellular and dispersed extracellular lipid in these implants. Cholesterol crystals in both implants and atheromatous lesions labelled only sluggishly. The results are consistent with the view that physicochemical exchange is a major factor in cholesterol movement in vivo.

The resorption rate of atheroma lipids in situ and implanted subcutaneously.

Resorption rates have been investigated in the rabbit for atheroma-lipids and cholesterol injected subcutaneously, atheroma lipids in situ in the mildly atheromatous aorta and lipid in the degenerating peripheral nerve. Atheroma lipids injected subcutaneously resorb relatively rapidly, subcutaneous cholesterol and lipid in Wallerian degeneration are intermediate in this respect, whereas only the group with an initially low blood cholesterol (small than approximately equal to 100 mg/100 ml) showed even a suggestion of lipid resorption from their mild or trivial atheromatous lesions. Ti is not clear whether or not atheroma resorbed at all in the groups with initial blood cholesteroles above 100 mg//00 ml. The relative inertia of the arthromatous lesion is mainly attributed to the relative absence of reticuloendothelial phagocytes in it.

Permeability in atherosclerosis: fluorescence test in green light with trypan blue.

A new microscopic fluorescence method for trypan blue at 570 nm has been used to follow the entry of albumin into the atheromatous rabbit aorta. Permeability into the inner aortic wall increases before the onset of gross lesions and seems just to precede intraendothelial deposition of lipid. Thereafter, permeability of the inner wall progressively increases until streaks or small plaques develop. These raised lesions stain and fluoresce variably, some intensely so while others are almost unreactive. This variability might reflect the difference between progressive and quiescent lesions. However, a zone of increased permeability surrounds many raised lesions, suggesting that the edge is a major site of growth and progression.

Lipoprotein levels and tissue lipids in fatty-fibrous atherosclerosis induced in rabbits by two years' cholesterol feeding at a low level.

A group of 12 young NZW rabbits of the same breeding strain were fed a diet enriched with 0.1% cholesterol by weight. The resulting modest hypercholesterolaemia resolved after 4-5 months. Two animals that died during this period showed no gross or microscopic atherosclerosis. After 6 months, the dietary cholesterol was increased to 0.2%. In some animals this resulted in moderate hypercholesterolaemia. One animal that died at this time showed no atherosclerosis with a mean serum cholesterol level of 224 mg/dl. Just after one year, dietary cholesterol was increased to 0.3%. This resulted in definite hypercholesterolaemia in some animals, but a few resisted the treatment with mean serum cholesterol levels around 40-60 mg/dl. In general, animals with established hypercholesterolaemia showed severe atherosclerosis, but often of a more fibrous and less cellular nature than is usual in the rabbit. Aortic wall cholesterol content (on a weight basis) correlated positively with serum cholesterol concentration (r = + 0.69, P approximately 0.05) and negatively with the ratio of HDL cholesterol to (LDL plus VLDL) cholesterol (double log plot: r = -0.79, P less than 0.025).