George E. Gifford

Comparison of in vitro cell cytotoxic assays for tumor necrosis factor

Four published in vitro assays which measure cell cytotoxicity were compared utilizing murine tumor necrosis factor. These included determination of residual cell number by crystal violet staining in the presence and absence of actinomycin D, lack of viability as determined by neutral red uptake, and [3H]thymidine release in cytotoxin treated L929 cells. Treatment of cells with actinomycin D followed by crystal violet staining was the most sensitive method measured. However, addition of actinomycin D to the neutral red uptake assay could be shown to be even more sensitive. Additionally, it was shown how actinomycin D dosage, cell seeding density and time of incubation affect TNF titer.

Gamma Interferon Priming of Mouse and Human Macrophages for Induction of Tumor Necrosis Factor Production by Bacterial Lipopolysaccharide

Priming of macrophages from both murine and human sources by recombinant immune interferons from Escherichia coli (r-IFN-gamma s) and activation by lipopolysaccharide (LPS) resulted in the production of tumor necrosis factor (TNF). r-IFN-gamma alone did not induce TNF production by macrophages; for this to occur, the second signal provided by small amounts (nanograms) of LPS was required. The small amounts of LPS alone were insufficient to activate the macrophages for TNF production. Priming by r-IFN-gamma was not necessary when larger amounts of LPS were employed, although an enhancement of yield resulted. Priming could also be demonstrated in vivo. Inoculation of r-IFN-gamma into mice resulted in increased yields of TNF following LPS challenge 12 hours later.

Stimulation of RNA Synthesis in L-929 Cells by Rabbit Tumor Necrosis Factor

Summary BCG or Corynebacterium par-vum primed rabbits injected with endotoxin contained a serum substance called tumor necrosis factor (TNF) that killed transformed mouse L-929 cells but not secondary normal mouse embryo fibroblasts. L-929 cells treated with TNF showed a six-fold stimulation of RNA synthesis that reached a maximum at approximately the same time as they started to die. Non-sensitive normal mouse embryo fibroblasts as well as TNF resistant L-929 cells did not show this large stimulation of RNA synthesis. When actinomycin D was employed to inhibit RNA synthesis, in the presence of TNF, a synergistic effect on killing of the transformed cells occurred.

A photometric and plaque assay for macrophage mediated tumor cell cytotoxicity

Pretreatment of L-929 cells for 2-3 h with 2 micrograms/ml actinomycin D followed by washing rendered them exceedingly sensitive to the direct cytotoxic effects of murine and rabbit macrophages. We exploited the tremendous increase in sensitivity of L-929 cells to effector cell mediated cytotoxicity by demonstrating simple and convenient photometric and plaque assays capable of being completed in 18 h for the quantitation of macrophage mediated tumor cell killing. The plaques demonstrated were generally visible to the unaided eye and were linearly related to the number of effector cells plated indicating that a plaque represented multitarget cytolysis attributed to a single effector cell. Unlike previously described assays, the effector:target ratios demonstrated were extremely low. Using the described techniques, it was estimated that a single macrophage could kill from approximately 10 to 1000 actinomycin D pretreated and washed target cells. In the presence of LPS-activated effector cells, the majority of these targets that stained with eosin Y did so in a cluster pattern, indicating cytotoxicity rather than mere detachment from the monolayer surface.

Factors affecting the adsorption of polio virus to magnetite in water and wastewater

Abstract The process of magnetic filtration involves the use of magnetite for the removal of various pollutants from water and wastewater. A study was undertaken to investigate the interaction between magnetite and polio virus. It was found that the adsorption process was enhanced by the presence of cations, conformed to the Freundlich isotherm, and did not vary significantly when the pH was varied from 5 to 9. The influence of two wastewater effluents on the adsorption process was also studied and their interference with the adsorption of polio virus to magnetite is discussed.

Studies on Tissue Specificity of Interferon

SUMMARY: The antiviral effects of chick and mouse interferons on chick and mouse cells were studied. In both systems a small amount of antiviral activity was found in the heterologous assays which varied from approximately 1 to 10 % of the homologous antiviral effect. A difference in the uptake of interferon by homologous and heterologous cells was observed which only partly explained the relative species specificity of the antiviral action of the two interferons. Destruction of interferon by supernatants from homologous or heterologous cultures could not be shown. The slopes of the dose response curves of mouse or chick interferon seemed to be significantly different and were independent of the two cell strains employed.

Retinoic Acid Induces a G1 Cell Cycle Block in HeLa Cells

Summary Evidence is presented that retinoic acid-induced anchorage dependent growth of HeLa cells is cell cycle dependent. Spinner cultures of retinoic acid-inhibited cells were blocked in the G1 phase and this block was reversed by simultaneous removal of retinoic acid and attachment of the cells to a plastic tissue culture surface. Studies with a synchronized cell population demonstrated that the retinoic acid had to be added during or before late S phase if the cells were to be blocked in the subsequent G1 phase. These findings are consistent with the hypothesis that vitamin A establishes normal growth controls to some transformed cells.

Enhancement of vaccinia virus plaque formation by trypsin.

Summary The size and numbers of vaccinia plaques in chick embryo cell cultures are enhanced by trypsin.

Effect of solar radiation on poliovirus: Preliminary experiments

Abstract Solar radiation was found to have some inactivating effect on poliovirus type 1 in the absence of any natural or synthetic photosensitizing agent. Nontronite, a three-layer clay, displayed a protective effect against light or thermal inactivation. Photoinactivation of poliovirus in lake water was retarded by the presence of blue-green algae. The light inactivation was less important at a depth of 6 in.

Effect of proteolytic enzymes on vaccinia virus replication

Chymotrypsin and trypsin enhance vaccinia virus replication in chick embryo cell cultures. Two phenomena are described: an enhancement of plaque numbers and an increase in plaque size. Low doses of chymotrypsin (1.3μg/4 million cells) acting on the virus particles are responsible for the increase in vaccinia virus plaque numbers. The effect is not due to disaggregation of virus aggregates by the enzyme. Doses of 3 to 10 (μg of chymotrypsin per culture have an effect on chick embryo cell monolayers. A linear dose response is found when plaque size is measured as a function of the dose of chymotrypsin. Preliminary studies indicate that some of the enzyme becomes cell-associated. When one replication cycle was followed the final yield of plaque forming virus from both enzyme treated and control cells was the same. However, the process of replication in enzyme treated monolayers was accelerated by about 5 hours. The enzyme appears to have an effect on some early event in virus replication.