Biomedical subjects
Publications and source records attributed to D A Flick.
Tumour necrosis factor was originally thought to kill tumour cells but not normal cells and to be a principal mediator in macrophage-mediated killing of tumour cells. It is now known to have a broad spectrum of additional activity ranging from regulatory effects on normal cells to inhibitory effects on viruses and parasites.
Tumour necrosis factor (TNF) was first described as an oncolytic factor found in sera of animals injected (primed) with reticuloendothelial stimulators and subsequently (days later) given lipopolysaccharide (LPS). TNF is not found in the serum of 'primed' animals but can be found in animals given LPS alone when sensitive assays are employed. TNF appears almost immediately upon LPS injection, reaches a maximum from about 1.5-2 hours and disappears rapidly thereafter, and is almost undetectable by 4-6 hours. When such mice are injected again with LPS, they are unresponsive (tolerized) and do not produce TNF again, at least for seven days. Other unrelated substances, such as muramyl dipeptide, viruses and mitogens, also induce TNF production. A high percentage of patients with some parasitic infections (but not cancers) demonstrate low levels of TNF in their sera; thus, they do not seem to be tolerized but produce it continuously. TNF can also be produced in macrophage cultures by treatment with LPS, muramyl dipeptide and other substances. Again, it appears almost immediately and synthesis is maintained for about 8-12 hours. Synthesis is dependent upon the continuous presence of LPS. After synthesis stops it cannot be reinitiated by adding more LPS; thus, the macrophages also appear to be tolerized. Macrophage cell lines eventually become sensitive again after cultivation in LPS-free conditions. Synthesis of TNF is inhibited by actinomycin D or cycloheximide, indicating that it is an inducible protein. Its production is also inhibited by glucocorticoids and prostaglandin E2, indicating that these substances play important roles in the regulation of TNF synthesis.
Tumor necrosis factor (TNF) was detected in the sera of normal mice, unprimed by reticuloendothelial system (RES) stimulators, when such mice were injected with lipopolysaccharide (LPS). Amounts of TNF were approximately 200-fold less than those found in Corynebacterium parvum-primed mice. No TNF activity was detected in the sera of mice not administered LPS. TNF induction in unprimed mice was refractory to repeated administration of endotoxin, thus exhibiting a tolerance phenomenon. TNF produced in unprimed mice eluted similarly to Mycobacterium bovis, strain BCG-primed TNF on Sephacryl S-200 and DEAE Sephacel columns and was neutralized by rabbit antisera raised to partially purified BCG-primed TNF. When BALB/c mice having 7-day old subcutaneous Meth A tumor implants were administered TNF antiserum, endotoxin-induced hemorrhagic necrosis was largely prevented. These findings strongly suggest that endotoxin-induced hemorrhagic necrosis of tumors is mediated through TNF production and action.
The in vivo administration of tumor necrosis factor (TNF) provides a new approach to the immunotherapeutic treatment of tumors. We evaluated the pharmacokinetics of murine tumor necrosis factor in mice as a model for application in the human system. TNF had a clearance of 0.013 ml/min/g and a serum half life of 10.5 minutes. Its volume of distribution was consistent with the extracellular space. This information can provide parameters by which to select optimal modes of treatment for eradication of in vivo neoplasms.
A series of copolymers related in structure to the 1:2 alternating cyclocopolymer of divinyl ether and maleic anhydride (DIVEMA) have been shown to possess antitumor properties. The synthesis and structures of these copolymers are discussed, and their effectiveness as antitumor agents is presented. Certain of the copolymers have been prepared in controlled molecular weight ranges using chain transfer agents, and the resultant copolymers finally fractionated via use of solvent-nonsolvent systems. These samples of narrow molecular weight distribution have been evaluated for their antitumor properties and have been found to be quite effective.
Macrophages in the presence of bacteria-derived lipopolysaccharide (LPS) stimuli produce a soluble cytotoxin which is toxic to tumor cells. In this study, we examined various parameters of cytotoxin production from pulmonary lavage cells obtained from Fisher 344 cesarean-derived rats. Cultures of macrophages were derived from pulmonary lavage cells and stimulated in vitro with LPS. Cytotoxin production was assayed in vitro using an L-929 cell target assay. Pulmonary lavage preparations contained a relatively pure population of macrophages, and adherence studies revealed that nonadherent lavage cells contributed negligible amounts of cytotoxin, indicating that macrophages were responsible for cytotoxin production. After LPS stimulation, cytotoxin production became maximal within 10 h and thereafter plateaued. Doses of LPS above 0.1 microgram/ml were optimal for production, and in the absence of LPS, no cytotoxin was detected. Because cigarette smoke is the major etiological factor in the development of lung cancers and because smoking is known to profoundly alter the function of alveolar macrophages in humans and experimental animals, subsequent experiments examined the role of chronic cigarette smoke exposure on tumoricidal activity of lung macrophages. Rats were exposed in vivo for 8 wk to either cigarette smoke or air (sham-treated controls). When lavage cells were cultured and stimulated with LPS (1 microgram/ml), 5- to 10-fold less cytotoxin was produced by lavage cells from rats exposed to cigarette smoke. Similarly, using a direct cytotoxicity assay, lung macrophages of smoke-exposed animals also revealed marked impairment in cytotoxicity against L-929 cell targets, and this was noted over a wide range of macrophage:tumor target cell ratios. Another product of macrophages, interferon, was also decreased in rats exposed in vivo to cigarette smoke when compared to sham-treated controls. These results suggest that cigarette smoke exposure may impair pulmonary macrophage-mediated tumor defense mechanisms.
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.
Human promyelocytic leukemia cells, when differentiated into macrophages by treatment with phorbol myristate acetate, secrete a cytolytic factor. Enhanced production was achieved when the cultures were treated with bacterial lipopolysaccharide (LPS). Production of the factor was inhibited when cultures were treated with dactinomycin immediately after LPS treatment. Tritirachium alkaline proteinase treatment inactivated the factor, indicating that it has an essential protein moiety. The molecular weight was found to be approximately 40,000 by Sephacryl S-200 gel filtration. The factor was stable at 56 degrees C for 30 minutes, but 80% of the activity was inactivated at 70 degrees C in 30 minutes. The factor was destroyed (96%) by dialysis against 0.01 M HCl (pH 2) for 14 hours. The cytolytic factor had little activity on normal fibroblasts, but it was able to significantly kill transformed cells in vitro.
Natural cell-mediated cytotoxic activity in the mouse has been associated with two types of effector cells, the natural killer (NK) cell and the natural cytotoxic (NC) cell, which seem to differ with regard to their patterns of target selectivity, cell surface characteristics and susceptibility to regulatory factors. During studies on the mechanism of action of cytotoxic molecules, it became evident that WEHI-164, the prototype NC target cell, was highly susceptible to direct lysis by both human and mouse recombinant tumour necrosis factor (TNF). Here we show that NC, but not NK activity mediated by normal splenocytes, is abrogated by rabbit antibodies to recombinant and natural TNF, respectively. Thus, the cell-mediated activity defined as NC is due to release of TNF by normal spleen cells and does not represent a unique natural effector mechanism.