PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : V. ANTIGENIC PROPERTIES OF THE CHICKEN TUMOR I

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.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : IX. THE EFFECT OF AQUEOUS EXTRACTS OF CHICKEN TUMOR I ON YEAST NUCLEIC ACID

1. Aqueous extracts of Chicken Tumor I cause a disintegration of yeast nucleic acid. 2. Two enzyme activities, a polynucleotidase and a phosphatase, are demonstrated. 3. These activities are distinguished by different reactions to an environment of hydrogen, different optima of hydrogen ion concentration, and different rates of activity. 4. Adsorption with aluminum hydroxide Type C removes substances inhibiting the polynucleotidase activity. 5. There is a rough proportionality between the polynucleotidase activity and the tumor-inducing power of different fractions of extracts of Chicken Tumor I. 6. There is a rough proportionality between the inhibition of polynucleotidase activity and the inhibition of the tumor agent. 7. The polynucleotidase activity of Chicken Tumor I is as great as that of spleen, slightly less than that of kidney and liver, but much greater than that of muscle. There are also qualitative differences.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : I. THE SPECIFIC FIXATION BY TISSUES OF SUSCEPTIBLE ANIMALS

Ground muscle from susceptible chickens fixes in vitro in a proportion of instances the agent of the filterable Chicken Tumor I, and in a lesser degree inactivates it, whereas the muscle from resistant animals such as rabbit and pigeon, is without effect. It is shown that the power of fixation of the chicken muscle is far greater than its inactivating properties. Brain and liver from chicken, rabbit and pigeon seem devoid of any action on the agent. The desiccated chicken muscle tissue shares the properties of the fresh organ; and the process of desiccation does not release the agent from the inactive or slightly active mixture of fresh muscle and filtrate.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : XI. CHEMICAL COMPOSITION OF PURIFIED CHICKEN TUMOR EXTRACTS CONTAINING THE ACTIVE PRINCIPLE

The fractionation of active chicken tumor extracts has been continued. 1. By a rapid method of dialysis the diffusible fraction of the treated extract, representing about 75 per cent of the solids, has been eliminated without reducing the tumor-producing activity. 2. Combined methods of fractionation have resulted in the elimination of as much as 95 per cent of the total solids of the extract as inactive constituents. Since there was some concomitant enhancement of the activity of the agent the results were equivalent to a 25-fold concentration of it in terms of dry weight. 3. The chemical content of the undiffusible fraction has been determined in terms of total nitrogen, reducing substances, sulfur, phosphorus, and lipoids. 4. The evidence points to a protein and a phospholipoid as the principal constituents of the active residue. Further fractionation is being attempted with a view to connecting the tumor-producing activity with one of its remaining constituents.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : VII. SEPARATION OF THE ASSOCIATED INHIBITOR FROM TUMOR EXTRACTS

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.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : VI. ACTION OF THE ASSOCIATED INHIBITOR ON MOUSE 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.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : XIV. RELATION BETWEEN A TUMOR NUCLEOPROTEIN AND THE ACTIVE PRINCIPLE

1. The tumor-producing fraction, isolated from Chicken Tumor I by means of differential centrifugation at high speed, has been investigated as regards its power to absorb ultraviolet light. A characteristic absorption spectrum was found, with a maximum at λ2575. The absorbing power of the material in that region was largely due to the presence of nucleic acid, or of a closely related compound. 2. Inactivation of the purified tumor fraction with ultraviolet light depressed the absorbing power of the material, especially in the region of 2600–2500Å. These changes were those which nucleic acid would present under the same conditions. 3. Inactivation of the tumor agent with acid or alkali was accompanied by decomposition of the tumor nucleoprotein and passage of free nucleic acid into solution. 4. Partial or complete inactivation of the tumor agent by heat, at 50° or 65deg;C., was attended by liberation of nucleic acid of low molecular weight. 5. The parallelism between tumor-producing activity and the integrity of the tumor ribonucleoprotein suggests that the nucleoprotein may be an essential part of the active principle.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : II. THE INACTIVATION OF THE TUMOR-PRODUCING AGENT BY MONOCHROMATIC ULTRA-VIOLET LIGHT

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.     

PROPERTIES OF THE CAUSATIVE AGENT OF A CHICKEN TUMOR : IV. ASSOCIATION OF AN INHIBITOR WITH THE ACTIVE PRINCIPLE

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 EFFECT OF TESTICLE EXTRACT ON THE GROWTH OF TRANSPLANTABLE MOUSE TUMORS

Grafts of a transplantable mouse sarcoma designated as No. 180, and those of an attenuated strain of a more malignant Sarcoma S/37, treated with testicle extract, either fail to grow on inoculation or result in tumors of a lower growth rate than the controls. Autografts of spontaneous mouse tumors so treated show little if any effect, while the Bashford adenocarcinoma and the unattenuated S/37 are unaffected. The factor in testicle extract responsible for the retarding activity passes readily through a Berkefeld filter and is thermostable.     

THE EFFECT OF ANTITESTICULAR SERUM ON THE ENHANCEMENT VALUE OF TESTICLE EXTRACT

The experiments reported here show that the infection-enhancing factor of testicle extract is neutralized in vitro by an antiserum against homologous testicle extract. An antiserum developed against a testicle extract of one species does not influence the enhancing and spreading factor of the extract from another species. Rabbits immunized against testicle extract do not exhibit any alteration in the spreading or enhancing effect of extracts employed later, even when the testicle extract used is from the same species as that employed for the immunization. Whether the in vitro inactivation of the active factor is a specific neutralization, or is the result of adsorption of the factor on the flocculate formed, has not been definitely determined. The fact that there is no neutralization in vivo, and that the antiserum acts only on extracts from the same species, when definite flocculation takes place, tends to emphasize the probability that the neutralization is not a direct one, but is incidental to the flocculation mentioned.     

THE EFFECT OF TESTICLE EXTRACT ON RED BLOOD CELLS IN VITRO

Testicle extract possesses a notable power to increase red cell fragility. Testicle extracts of the rat, rabbit and guinea pig all exhibit this property, which is most pronounced with the extract from the rat and least so with that from the guinea pig. Splenic extract does not possess the property, or possesses it only to a very slight degree. These results support the hypothesis that the factor in testicle extract which enhances infections (the Reynals factor) does so by altering the permeability of the host tissue.     

SOME FACTORS DETERMINING THE LOCALIZATION OF A CHICKEN TUMOR AGENT

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 ENHANCING EFFECT OF AZOPROTEINS ON THE LESIONS PRODUCED BY VACCINE VIRUS,THE SHOPE FIBROMA VIRUS,AND THE AGENT TRANSMITTING CHICKEN TUMOR I

It is known that azoprotein solutions, like testicular extracts, possess the property of causing particles to spread through the dermis. The present work shows that azoproteins exhibit, like testicular extract, the power to increase the size of virus lesions in the skin of rabbits, and the size of tumors in chickens. The results indicate that the extent of the lesion is roughly proportional to the spreading power of the solution. This suggests that the spread of the infective material, over a larger area of skin, is directly responsible for the enhancing effect. The production of extensive lesions by means of spreading agents may have a practical value when large amounts of working material are needed.     

STUDIES ON LYMPHOGRANULOMA VENEREUM : I. DEVELOPMENT OF THE AGENT IN THE YOLK SAC OF THE CHICKEN EMBRYO

Making use of the fact that the cells of the yolk sac of the developing embryo are readily infected with the agent of lymphogranuloma venereum and that the virus bodies can be readily observed in these cells because of the structure of the latter, the development of this agent has been followed at short intervals. It has been found to go through a regular cycle of development similar to that described for psittacosis in the spleen and less fully for lymphogranuloma venereum in the brain of infected mice. The development as observed microscopically can be shown to run parallel to changes in the infective titre of the yolk sac as tested in other eggs.     

THE EFFECT OF A GROWTH-PROMOTING EXTRACT OF THE ANTERIOR PITUITARY ON THE EARLY GROWTH OF THE ALBINO RAT

Castrate male albino rats were injected with a growth-promoting extract of bovine anterior pituitary from the 21st day of life (day of castration) to the 56th day (average for group) at which time the difference in weight between the pituitary-injected animals and controls was first clearly discernible. In comparison to controls the pituitary-injected animals at this stage showed: (1) a decrease in the weight of the pituitary gland; (2) an increase in nose-anus length; (3) an increase in the weight of heart, lungs, liver, and kidneys which is shown to be of the nature of a splanchnomegaly; (4) an increase in the weight of the blood removed; (5) an increase in the water content of the skin and the kidneys; (6) a tendency toward an increase in weight of the carcass. The significance of these findings is discussed.     

PHYSIOLOGICAL ONTOGENY : A. CHICKEN EMBRYOS. V. ON THE RATE OF THE HEART BEAT DURING THE DEVELOPMENT OF CHICKEN EMBRYOS.

These experiments show, then, that there are changes in rate which take place during the incubation period and that the direction of the curve expressing them changes in an orderly fashion. No attempt has yet been made to ascertain the influences operative in bringing about these alterations. It is our intention, however, to attempt to correlate them with such processes as changes in the rate of the general metabolism of the body, changes in structure of the heart muscle, and if possible with changes in the inherent dynamics of the muscle. It will be seen that the number of experiments at a given age is relatively speaking small. The temperature in spite of the precaution mentioned has likewise not been uniform, the range in the greatest number (38) of experiments having been 38°C. ± 1°. A few (four) counts have been made when the temperature was as low as 33.6°C. and a few (two) also at a temperature as high as 40°C. Although a curve as smooth as the one which has been drawn in all probability represents the general course of heart rate change, an explanation of the deviations which have been found is desirable. At least two influences which may be operative suggest themselves. First may be considered the effect of the unavoidable injury incident to cutting a window in the shell and removing so much of the shell membrane as to make the object visible. It has been a uniform practise to discard those eggs in which hemorrhage took place, so that if opening the egg has a share in the difficulty, this operation may be thought of as exerting a mechanical stimulus on a nervous or other controlling mechanism. But whether, especially at the early ages, a mechanism of such a nature exists within the embryo, we do not know. The search for one lies for the present beyond the range of our interest. A second influence responsible for the relatively large deviation in rate from the average may be that of fluctuations in temperature. The air of the room in which the observations were made was, in a given situation, fairly uniform at 38°C. ± 1.0°. as the temperature records show. In the earlier experiments the temperature was read at a distance about 1 foot away from the egg. Between this point and the egg a difference unknown to us may well have existed. It is improbable, however, that it can have been more than 2°C., that is to say, 38° ± 2°. Roughly if the change in rate is approximately 15 beats per degree a maximum deviation of 30 beats may be expected. In point of fact a deviation as great as this did not take place. In later experiments the temperature was read in the immediate vicinity of the eggs, so that a correction can be made more accurately. There is in all probability a high degree of uniformity in the rate of the heart of the chicken embryo at each stage of development, although variation is also found. What the extent of the deviation is that may properly be encountered we do not yet know. It is desirable to pursue this subject further. Observations will accordingly be made and will be reported later.     

THE INFLUENCE OF TESTICLE EXTRACT ON THE INTRADERMAL SPREAD OF INJECTED FLUIDS AND PARTICLES

The experiments in this paper show that testicle extract causes India ink particles and those of Prussian blue to spread much more extensively through the intercellular spaces than similar suspensions made with Ringer''s solution. Methylene blue inoculated intravenously localizes more extensively in areas previously injected with testicle extracts than in control areas receiving injections of tissue extracts without enhancing power. Kidney extracts have this property to a less degree, whereas spleen extracts and blood serum are devoid of it. The spreading power of extracts is destroyed by heating at 60°C. for 30 minutes, as is also the power to enhance infections. The precise mode of action of the Reynals factor is not known, but the results of the experiments here presented suggest that it may depend at least in part on the property whereby testicle extract increases the spread of injected material and alters the permeability of tissue cells. It is not inconceivable that changes in permeability facilitate the passage of vaccine virus through the endothelial cells of the blood and lymph vessels, and lead to the generalized vaccinia which is of frequent occurrence in the reported results (20). It has been shown that fluids and suspensions of inert particles are spread by the extract.B. tetanus and B. coli exotoxins and trypsin were not enhanced at all in their action despite the fact that they were spread through a more extensive area in the tissues. Viruses, on the other hand, are markedly influenced and in this respect resemble bacteria, not toxins and enzymes. It appears probable that a definite capacity for multiplication on the part of an injected substance is required if its pathogenic effects are to be enhanced. It may be concluded tentatively that the enhancing power of the testicle extract may depend on that property which not only spreads the injected material through a larger area but renders the tissue cells more easily penetrable by the agents.     

STUDIES ON THE ETIOLOGY OF RABBIT POX : I. ISOLATION OF A FILTERABLE AGENT: ITS PATHOGENIC PROPERTIES

A filterable agent was isolated from 7 cases of spontaneous rabbit pox by the intratesticular injection in rabbits of a variety of tissues. The virus was transmitted for 15 consecutive testicle to testicle passages in rabbits by Berkefeld V filtrates of testicular tissue emulsions. Unfiltered emulsions were more potent than filtrates. The virulence of the virus was maintained by ice box storage of infected tissues for as long as 127 days. The pathogenic properties of the virus persisted under conditions of animal passage for the 9 months from January to October, 1933, covered by these studies. The reaction produced in rabbits by the inoculation of tissue-virus emulsions was of two principal types, the first of which has been described and analyzed in the present paper. In the case of intratesticular injections, particularly of testicular tissue inocula, an acute fulminating and rapidly fatal condition regularly developed. The outstanding features were a massive hemorrhagic orchitis with marked scrotal edema, fever, and death within a week. The second type of reaction which is taken up in the next paper of this series (1) was observed under conditions in which the animal survived a week or longer. Occasional examples occurred in rabbits of the so called intratesticular series. The reaction was characterized by the development of a disease syndrome with a diversity of clinical manifestations which, it may be stated here for the sake of continuity, was indistinguishable from spontaneous rabbit pox.