Following single intravenous injections of a foreign protein antigen, bovine γ-globulin, into mice and rabbits, antigenic material persisting in the liver could be detected for several weeks. Ground liver tissue—taken from the mice and rabbits, just mentioned, either 4 or 6 weeks after injecting the antigen—when transferred repeatedly, at 2 or 3 day intervals, to the peritoneal cavities of normal, or unilaterally adrenalectomized, recipient mice, rendered the recipients sensitive to active anaphylaxis when they were challenged after a suitable interval by intravenous injections of the original antigen. The work throws some light on the state of the antigenic material that persists for 4 to 6 weeks in the livers of the donor animals. Obviously it is sufficiently unchanged, at least in its reactive groups, to engender in the recipient mice antibodies capable of reacting with the original antigen.

A sensitive biological test has been used to detect the persistence of minute traces of a foreign protein, bovine γ-globulin, in the blood and livers of rabbits intravenously injected with it, as an antigen. At various intervals after injecting these rabbits (donors) serum or liver tissue was transferred from them to the peritoneal cavities of normal or unilaterally adrenalectomized mice (recipients) with the aim of rendering the latter hypersensitive to the antigen that might be persisting in the transferred materials; a state of affairs detectable, 2 days later, by the appearance of signs of reversed passive anaphylaxis when the recipient mice were intravenously challenged with a strong anti-bovine γ-globulin rabbit serum. The protein persisted in the blood of the donor rabbits, in readily demonstrable amounts for 1 month, and in the blood of one animal, in minute traces, or as long as 6 weeks. It was detectable in the livers for 8 weeks. The persistence of bovine γ-globulin in rabbits, which form circulating antibodies to it well, is not as long as that in mice, which form antibodies to it poorly, since in previous work with the mouse the antigen was found (1) in the blood after 8 weeks and in the liver for 14 weeks. Nevertheless the antigen persists in the rabbit much longer than is generally supposed. Indeed it can be found in the liver all through the period in which circulating antibody is demonstrable in the blood. Explanations for the phenomenon have been suggested. Its significance in relation to the mechanisms of antibody formation is obvious.

1. Mammary tissue of pregnant rabbits is found to contain an agent which inhibits the growth of Bashford Adenocarcinoma 63 in mice but this material is without effect on the Crocker Sarcoma 180. 2. Of the tissues so far studied the mammary gland of rabbits has yielded the most active product. 3. From the results it is probable that pregnancy enhances the production of the agent but this cannot be considered as established beyond doubt. 4. The factor is found, with diminished activity, only in the protein fraction obtained from the aqueous extract by full saturation with ammonium sulfate.

Extracts of desiccated homologous embryo skin and placenta decrease markedly the rate of postoperative local recurrence after the surgical removal of spontaneous cancer of mice. Autografts after a short period of contact with these extracts either failed to grow, or, in the majority of instances, their subsequent growth was definitely retarded. Intraperitoneal injection of the extracts was followed by cessation of growth of established tumors in more than two-thirds of the animals treated, and among these many of the tumors regressed and over 20 per cent were completely absorbed. Tumor mice treated with either extract rarely developed new malignant foci, though this happened frequently in untreated mice.

Extracts of desiccated embryo skin and placenta have been found to exert a definite retarding action on the growth of two transplantable carcinomas of mice, but they were without effect on sarcomas. In tests involving some 828 inoculations of the tumor cells and the extracts, complete suppression of growth occurred in from 55 to 71 per cent of instances, as compared with 21 to 27 per cent in the controls, and where growth was not completely suppressed some retardation was found in practically every instance. To judge from findings in rabbits the inhibitor is not demonstrable in the placenta until the beginning of the second third of pregnancy, reaches its maximum by the last third, but disappears about 2 or 3 days before term. Extracts of fresh placenta are without effect, and no very definite inhibition was noted in extracts of a variety of other desiccated or fresh tissues. The conclusions here reported are based on the results of over 3800 inoculations.

The inhibiting factor present in certain relatively slow-growing strains of Chicken Tumor I and in Chicken Tumor X is adsorbed from extracts of the desiccated tumors by aluminum hydroxide (Willstätter Type C) and can be released in effective quantities from this combination by treatment with basic sodium phosphate.

Extracts prepared from the testicle tissue of the rat, rabbit, fowl or bull, injected together with extracts of Chicken Tumor I or with cells of this tumor, showed no definite effect of either enhancement or inhibition as concerned the resulting tumors.

Water extracts of desiccates of certain relatively slow-growing strains of Chicken Tumors I and X, or the exudates from such tumors, definitely inhibited the growth of a mouse sarcoma (Crocker 180), and were without effect on a mouse carcinoma (Bashford 63) or Mouse Tumor S/37, a rapidly growing sarcoma derived from the stroma of a carcinoma. Extensive control tests with extracts from rapidly growing chicken tumors, and from tissues of normal and immune chickens showed no inhibiting action. There was no demonstrable action on the mouse tumors of sera from immunized rabbits, which neutralize the chicken tumor agent, nor of the sera from chickens highly immune to the chicken tumors.

The presence of an inhibiting substance in the chicken tumor is shown by the fact that a desiccate of the tumor is more active after it has been washed two or three times with water, and that an extract of the tumor is more potent after some factor is removed by adsorption on aluminum hydroxide. When the tumor-producing factor in an extract of a slow-growing tumor has been destroyed by heating at 55°C. it is found to have the property of neutralizing a highly active tumor extract. This inhibiting property is destroyed by heating over 65°C.

The injection of tumor extracts and their active protein fractions into rabbits induced the formation of precipitins and neutralizing antibodies. When the major portion of proteins in the tumor extract had been eliminated it induced the formation of neutralizing antibodies, but not of precipitins. The tumor agent, more highly purified by removal of the viscous fraction, did not induce precipitins, and only 2 out of the 15 sera gave any evidence of neutralizing bodies. After the removal of the major portion of protein, the extracts showed insufficient interaction with the sera to fix complement.

By two methods a protein fraction can be separated out from a Chicken Tumor I extract, which carries all the tumor-producing agent. The precipitate can be dissolved and reprecipitated a number of times without loss of activity. The agent can be largely dissociated from the protein as shown by the fact that aluminum hydroxide will adsorb the protein from an extract and leave the agent behind. This purified material has a very low protein content, if any, as shown by both chemical and biological tests.

Even though part of the energy of the incident light is probably absorbed by chemical entities which play no part in the specific reaction of inactivation, nevertheless the wave lengths most active in destroying biological cells or agents will presumably be found to be among those absorbed in the highest proportion. This would indicate that the curves here presented are approximately reciprocal to the coefficients of absorption of particular substances, the destruction of which caused the inactivation of the agents or the death of the cells. The similarity between the curves for bacteria, virus, and phage, both in shape and in total involved energies, suggests the presence of a common factor, or of closely related chemical entities, sensitive to ultra-violet light, whereas the data for the tumor agent suggest that its inactivation is due to the destruction of a substance having an essentially different spectral absorption, and therefore of a different chemical character. While the amount of ultra-violet energy required to affect the tumor agent is great, it is still less than that involved in the inactivation of some of the enzymes (7). A study is under way to compare the deduced spectral analysis with the actual coefficients of absorption of the highly purified tumor agent.

Numerous attempts have been made by us to separate from the cells of a tar sarcoma of the chicken (Chicken Tumor 9) a causative agent for the growth. Experiments with filtrates and desiccates injected as such or in combinations with embryonic tissues have all failed to give positive results. So too have injections of filtrates and desiccates into developing chick embryos failed to yield a response. The results confirm those of previous work with the tumor in this laboratory. The growth would appear to differ in a fundamental respect from all tumors of the fowl previously studied.

A localization out of the blood stream of the agent causing a chicken tumor, with the subsequent development of the growth can be brought about in the breast muscle, by inducing in this tissue a reaction by such substances as Scharlach R, tar, embryonic tissue and kieselguhr. Localization in the reaction tissue elicited by kieselguhr takes place relatively infrequently. The earlier stages of the reaction induced by these substances localize the tumor agent more regularly than the later stages of the reaction.

The external application of tar to a number of separated areas on the surface of mice, in such fashion that no single area is irritated sufficiently long to cause lesions of the skin, has resulted in a very high incidence of lung tumors. This incidence ranged from 60.0 per cent in one experiment to 78.3 per cent in another. Control mice from the same stock but from 3 to 6 months older, and for that reason the more liable to spontaneous lung tumors, failed to show a single instance of such growths. Even in a stock in which spontaneous lung tumors had been frequent, the incidence for corresponding age periods has never been above 5.5 per cent while the average has been between 1 and 2 per cent over a period of years. The tumors in the tar-painted animals occur as small white nodules, either single or multiple. They are typical epithelial neoplasms, identical histologically with those described by previous authors as occurring spontaneously in mice. Some possible factors in the causation of the tumors are briefly discussed.

1. The minimum dose of x-ray, of each of three degrees of hardness, required to produce persistent alopecia of the abdominal skin of normal white rabbits has been studied. 40 kilovolt rays filtered by 0.5 mm., 50 kilovolt rays filtered by 1 mm., and 70 kilovolt rays filtered by 1.5 mm. of aluminium were employed. The 70 kilovolt work is unfinished. 2. Under constant conditions of treatment, the animals were found to react with a high degree of consistency. 3. The values of the critical minimum doses expressed in terms of air ionization (pair of ions per gm. of air divided by 10 15 ), for rays produced at 30, 40, 50, and 70 kilovolt peak, are 2.04, 3.7, 4.5, and over 5.1 respectively. The 30 kilovolt value is taken from a previous paper. 4. It is shown that the alopecia reaction does not run parallel to the erythema and pigmentation reactions.

Male and female mice castrated during the first 7 weeks of life and implanted with cancer at later periods show a resistance definitely higher than do intact animals of the same age. This increased refractiveness is evident at 3 months after the operation but is more pronounced at 8 months to a year. Even castration in early adult life seems to increase the refractory state to later cancer inoculation. On the other hand, adult mice inoculated within a week after castration show slight if any evidence of increased resistance.

Rabbits x-rayed in doses sufficient to reduce the amount of their lymphoid tissue without damage to the bone marrow showed a definite deficiency in the production of precipitins, bacterial agglutinins, and protective antibodies. On the other hand, rabbits subjected to exposures of dry heat sufficient to increase the activity of the lymphoid organs, on immunization develop antibodies in larger quantity than do untreated animals immunized by the same process.

Seven areas on the abdomen of each of fourteen rabbits were exposed to soft Roentgen radiation of constant quality in doses varying, in the region of the value critical for the production of persistent alopecia, by regular steps of about 4 per cent. Without exception the critical dose was found to lie between two such values, the upper limit being represented by exposures on eight of the animals and the lower on ten of them. With certain reservations, the critical dose corresponds to the production of 2.04 x 10 15 ions of either sign per gm. of tissue.

Autografts from spontaneous cancers of mice when replanted into areas previously exposed to an erythema dose of x-rays, failed to grow in the majority of instances (71.4 per cent), while similar grafts inoculated into untreated areas grew in a large proportion of the animals (83.6 per cent). Autografts of spontaneous cancer, established and growing in the skin, disappeared in 76 per cent of animals after the tumor and surrounding tissues had been exposed to an erythema dose of x-rays, whereas other autografts of similar derivation that had been given a like dose of x-rays outside of the body and had been implanted in the same animals grew progressively in 96 per cent of instances. That this result was not due to a greater susceptibility of the cancer cells x-rayed in situ was shown by the fact that tumors treated in situ with x-rays and then replanted in an unrayed location on the same animal grew actively. Evidently the ray had done no direct damage to the cancer cells.