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.

Evidence for a strong sulfur-aromatic interaction derived from crystallographic data.

We have uncovered new evidence for a significant interaction between divalent sulfur atoms and aromatic rings. Our study involves a statistical analysis of interatomic distances and other geometric descriptors derived from entries in the Cambridge Crystallographic Database (F. H. Allen and O. Kennard, Chem. Design Auto. News, 1993, Vol. 8, pp. 1 and 31-37). A set of descriptors was defined sufficient in number and type so as to elucidate completely the preferred geometry of interaction between six-membered aromatic carbon rings and divalent sulfurs for all crystal structures of nonmetal-bearing organic compounds present in the database. In order to test statistical significance, analogous probability distributions for the interaction of the moiety X-CH(2)-X with aromatic rings were computed, and taken a priori to correspond to the null hypothesis of no significant interaction. Tests of significance were carried our pairwise between probability distributions of sulfur-aromatic interaction descriptors and their CH(2)-aromatic analogues using the Smirnov-Kolmogorov nonparametric test (W. W. Daniel, Applied Nonparametric Statistics, Houghton-Mifflin: Boston, New York, 1978, pp. 276-286), and in all cases significance at the 99% confidence level or better was observed. Local maxima of the probability distributions were used to define a preferred geometry of interaction between the divalent sulfur moiety and the aromatic ring. Molecular mechanics studies were performed in an effort to better understand the physical basis of the interaction. This study confirms observations based on statistics of interaction of amino acids in protein crystal structures (R. S. Morgan, C. E. Tatsch, R. H. Gushard, J. M. McAdon, and P. K. Warme, International Journal of Peptide Protein Research, 1978, Vol. 11, pp. 209-217; R. S. Morgan and J. M. McAdon, International Journal of Peptide Protein Research, 1980, Vol. 15, pp. 177-180; K. S. C. Reid, P. F. Lindley, and J. M. Thornton, FEBS Letters, 1985, Vol. 190, pp. 209-213), as well as studies involving molecular mechanics (G. Nemethy and H. A. Scheraga, Biochemistry and Biophysics Research Communications, 1981, Vol. 98, pp. 482-487) and quantum chemical calculations (B. V. Cheney, M. W. Schulz, and J. Cheney, Biochimica Biophysica Acta, 1989, Vol. 996, pp.116-124; J. Pranata, Bioorganic Chemistry, 1997, Vol. 25, pp. 213-219)-all of which point to the possible importance of the sulfur-aromatic interaction. However, the preferred geometry of the interaction, as determined from our analysis of the small-molecule crystal data, differs significantly from that found by other approaches.

A new classification of the amino acid side chains based on doublet acceptor energy levels.

We describe a new classification of the amino acid side chains based on the potential energy level at which each will accept an extra (doublet) electron. The doublet acceptor energy level, and the doublet acceptor orbital were calculated using semiempirical INDO/2-UHF molecular orbital theory. The results of these calculations show that the side chains fall into four groups. We have termed these groups repulsive, insulating, semiconducting, and attractive in accordance with where each lies on the relative energy scale. We use this classification to examine the role of residues between the donor and acceptor in modulating the rate and mechanism of electron transfer in proteins. With the calculated acceptor levels, we construct a potential barrier for those residues between the donor and acceptor. It is the area beneath this barrier that determines the decay of electronic coupling between donor and acceptor, and thus the transfer rate. We have used this schematic approach to characterize the four electron transfer pathways in myoglobin recently studied by Mayo et al. (Mayo, S.L., W.R. Ellis, R.J. Crutchley, and H.B. Gray. 1986. Science [Wash. DC]. 233:948-952).

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.

Interactions between soluble IgG, complement and cells in lymphocyte and monocyte ADCC.

The interactions of K lymphocyte- and monocyte-mediated ADCC (antibody-dependent cell-mediated cytotoxicity) systems with monomeric and aggregated IgG in solution are compared. K-lymphocyte ADCC is inhibited by aggregate, but not by monomer, while monocyte ADCC is inhibited by both. The difference may depend on monocytes having Fc receptors of high affinity. The inhibition of K-lymphocyte ADCC by aggregates is reduced by the binding of complement to the aggregates. Adherent cells can inhibit K-lymphocyte ADCC if IgG is absent from the culture medium, but have no effect in its presence. This effect may also depend on the presence of high affinity Fc receptors on monocytes. The properties of K-lymphocyte ADCC are compatible with a significant role in vivo; by contrast monocyte ADCC is strongly inhibited by physiological concentrations of IgG.

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).

Predictor for sulfur-aromatic interactions in globular proteins.

The predictor, Y = 2.54 (formula: see text), where Y is the number of sulfur-aromatic interactions in a globular protein and N is the total number of amino acids in the protein, accounts for 75% of the variation in this type of interaction that occurs in 22 globular proteins whose three-dimensional structure has been determined. We find that S-pi interactions are not random events but rather are the outcome of a competition between the dipolar groups, amides and sulfur-containing, for ring neighbors. This outcome is strongly weighted in favor of S-pi interactions by the presence in the protein of positively charged side-chains; it is not affected by the number of strictly non-polar side-chains.

Chains of alternating sulfur and pi-bonded atoms in eight small proteins.

This paper demonstrates the existence of regions in eight small globular proteins in which the side chains of sulfur-containing amino acids (cysteine and methionine) alternate in space with side chains of aromatic amino acids (histidine, phenylalanine, tryptophan and tyrosine). The proteins are: rubredoxin, high potential iron protein, cytochrome c, flavodoxin, deoxyhemoglobin, trypsin inhibitor, ribonuclease-S, and lysozyme. The sulfur-pi-bonded 'chains' involve a minimum of five and a maximum of 10 amino acids, and contain the most polarizable atoms within proteins. S-pi-chains give extra stability to the folding of proteins; they may also afford paths for the step-wise movement of electrons.

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.

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.

Dietary restriction and regression of atherosclerosis.

Rabbits were fed a cholesterol-enriched diet to render them atheromatous. After 3 months on this diet they were switched to a low-lipid stock diet. Some animals were killed at this point, while the rest were divided into (a) a group allowed to eat ad-libitum and (b) a resticted group allowed to eat half by weight of what the ad-libitum group consumed. Most animals were killed at 9 months (i.e. after 6 months' regression). The group allowed the restricted diet showed a 27% weight loss, but their serum cholesterol fell slightly more slowly than that of the ad-libitum animals. Likewise, atherosclerosis was slightly worse in the restricted than in the ad-libitum group. The results do not support the view that severe dietary restriction causes atherosclerosis to regress.

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.

Frequency-dependent diffraction from enzymatic breathing modes.

The intensity of the time-dependent Fourier transform of any macro-molecule which contains a cleft of the sort that exists in the protein lysozyme is derived. It is shown that there is a region of this transform in which this intensity varies nearly sinusoidally with time with a frequency equal to that of the macromolecule's breathing mode of vibration, and where, therefore, the diffracted power will be primarily at this single frequency. Observation of this power will allow the experimental determination of the frequency of this mode, which is expected to be much lower than those observable by Raman spectroscopy.