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have been elicited against a normal tissue capable of growth on transplantation (embryonic tissue).
Taken together the foregoing traits identify the chicken sarcoma as a typical malignant tumor. In them there is nothing to suggest the presence of a parasitic cause for the disease, but much that has been held to favor the view of an intrinsic cell-derangement.
For the first attempts to separate out the sarcoma's cause filtration was employed. The tissue of a rapidly growing tumor was ground with sand, taken up in Ringer's solution, shaken for some time, centrifugalized, and the supernatant fluid was passed through a Berkefeld filter which held back small bacteria. The injection of a few cubic centimeters of the limpid fluid thus obtained gave rise to the characteristic spindle-celled growth in fowls previously normal; and this growth was capable of further transplantation through an indefinite series of hosts. More recently the causative agent has been differentiated from the living tumor cells by drying, by glycerinization and by repeated freezing and thawing,-processes which the tumor cells fail to survive. It remains active for many months in dried sarcomatous tissue, and for at least one month in tissue placed in 50 per cent. glycerin. It is quickly rendered innocuous by temperatures above 53° C., by 50 per cent. alcohol, by 2 per cent. phenol, by saponin in high dilutions, by chloroform and toluol in the proportions which prevent bacterial growth during autolysis, and by autolysis itself. It will not pass through a dialyzing membrane, nor, in our experience, through a porcelain filter. These various features seem sufficient to identify it as a living organism in distinction from a ferment. The organism has never been directly observed in fresh or stained preparations; and the morphology of the individual tumor cells does not suggest its presence. Attempts to cultivate it in vitro have not as yet proven successful.
The neoplastic change brought about by the agent takes place slowly compared with the proliferation of the cells, once they have become sarcomatous. Growth of the tumor, dissemination, injury to the host, immune processes, all are referable to these cells suddenly endowed with new properties. The introduction into a susceptible fowl of a large amount of the filterable agent is not in itself sufficient to cause a tumor. The development of a growth under these circumstances is conditional upon the presence of a cellderangement, such, for example, as is produced by the injection of infusorial earth. Yet even when the element of cell-derangement has been supplied, and the agent injected in quantity, a considerable percentage of the fowls fail to develop a sarcoma. The nature of the factors responsible for this failure has not been determined. The importance of cell-derangement as a contributory cause of human sarcomata has long been recognized.
The chicken sarcoma is strikingly non-infective under ordinary conditions. During the last three years more than a thousand fowls, with or without the tumor, have been kept together in close quarters, yet no instance of natural transmission has been observed. An examination of numerous spontaneous chicken tumors from various sources has shown that the sarcoma is not epidemic. These facts find an explanation in the various factors by which the agent's action is conditioned.
In conclusion it should be stated that the experiments with the chicken sarcoma have not yielded a method whereby a causative agent can be separated from the tumors of rats and mice. But they clearly prove that the characteristics of malignant tumors in general are compatible with the presence of a living causative agent. Such a cause for them seems, indeed, far from improbable.
Note: Dr. James B. Murphy has shared, as joint author, in the work on the chicken sarcoma since the recognition of the latter's filterable cause; and more recently Dr. W. H. Tytler has aided in the study of some of the growth's problems. LABORATORY OF THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH,
PROC. AMER. PHIL. SOC., II. 205 K, PRINTED JULY 24, 1912.
THE PROTEIN POISON.
(Read April 19, 1912.)
For many years I have been studying the chemistry of the bacterial cell. In 1900 I devised the large tanks for growing massive cultures. These have proved quite satisfactory, and I have been able to get bacterial cellular substances quite free from all impurities, in large amount. After many years of unsuccessful efiort Wheeler and I, in 1903, succeeded in partially isolating the poisonous group from the cellular substance of certain pathogenic bacteria. This we did by heating the cellular substance with a two per cent. solution of sodium hydroxid in absolute alcohol. When this is done at the temperature of boiling alcohol the cell substance is split up into a poisonous and a non-poisonous part. The former is soluble in alcohol, while the latter is insoluble. This gives us not only a method of preparation, but also one of partial separation. I may say that the evidence that a distinct cleavage of the bacterial cell is secured is shown by the fact that all the carbohydrate and all the phosphorus in this cellular substance remains in the insoulble or non-poisonous part. The poisonous portion contains no phosphorus and no carbohydrate, but it does give the biuret and the Millon reaction, and must therefore be classed as a protein. This protein has never been obtained as yet in a state of chemical purity. The best preparation that we have been able to secure up to this time kills guinea pigs when injected intravenously, in doses of .5 of a mg. There are certain reasons for believing that its effect upon man is still more pronounced.
Having found this poison in pathogenic bacteria we next looked for it in non-pathogenic organisms, and we found it in these quite as abundantly as in the pathogenic forms. It therefore follows that the pathogenicity of the bacterial cell does not depend upon its capability of producing a poison, because all bacterial cells contain a poison. Whether a germ is pathogenic to a given species of animal or not depends upon its capability of growing and multiplying in that animal's body.
Next we looked for this poison in certain animal proteins, such as the white of egg, the proteins of blood serum, the casein of milk, etc. In all of these the same or a like poison was found by the same method. Later we tested vegetable proteins, such as the gluten of flour, the zein of corn meal, the edestin of hemp seed, etc. Up to the present time we have examined more than thirty proteins of bacterial, animal and vegetable origin, and in all of these the same poisonous group has been detected.
It has long been suspected, and indeed I may say, known, that the protein molecule contains a poisonous group. At first it was supposed that the diverse proteins which man takes in his food are but slightly altered in the alimentary canal, and before absorption. It is now known that this is not true, and that in the healthy man all proteins are broken down into amino acids by the ferments of the alimentary canal, and that these amino acids are, either during absorption or directly thereafter, resynthesized so as to form the proteins which are characteristic of man's body. The precipitin test has demonstrated that every species of animal has its own specific protein bodies. Every albuminous molecule contains a poisonous group. Peptones injected into the blood act as poisons; therefore the peptone group contains a poisonous molecule, and it is this poisonous molecule in the peptone group which we have succeeded in partially isolating. The symptoms induced by this protein poison are marked and characteristic. They divide themselves into three distinct groups. Soon after the injection of a minimum fatal dose in one of the lower animals there is evidence of peripheral irritation. This is shown by the fact that the animal becomes restless and attempts to scratch itself, not only the part adjacent to the point of injection, but every portion of its body which it can reach. This is known as the stage of peripheral irritation. In man it is characterized by itching and by an erythematous eruption which begins about the place of injection, and rapidly spreads over the
body. In the second stage the animal lies in a lethargic condition, with rapid, difficult respiration. It prefers not to move, and when urged to do so it shows that it has partially lost the power of coördinating its movements. It drags its posterior extremities, or it sways from side to side. This is known as the paralytic stage. The third stage manifests itself by clonic covulsions, which repeat themselves after intervals of rest, becoming more and more violent, until death results. After reaching the convulsive stage recovery is rare, although it does occasionally occur. The symptoms are produced by the injection of protein poison, whether obtained from bacterial, animal or vegetable proteins. It should be stated that in order to study these symptoms properly the dose should approach the minimum quantity. When the dose is excessive the first and even the second stage may not be observed. The animal is speedily thrown into a convulsion, and death results within a few minutes. When a non-fatal dose is given the first and second stages appear, and may last in guinea pigs for an hour, possibly two, but recovery is rapid and apparently complete. It is of importance to note this fact, that when recovery does take place it follows rapidly, and apparently the animal is as well as ever within two or three hours, and possibly earlier.
We had studied this protein poison and its effects upon animals when the phenomenon of protein sensitization, improperly called anaphylaxis, was discovered. All will understand that protein sensitization is demonstrated by injecting a protein, any protein, into an animal and waiting for a certain length of time, or until the animal becomes sensitized, when a second injection into the same animal causes the symptoms which I have described, in the same order as observed when the protein poison is administered, and that the final effects are the same. Comparing the phenomena of protein sensitization with those of protein poisoning Wheeler and I in 1907 offered the following explanation of protein sensitization: When a foreign protein is injected into an animal it must be disposed of in some way. Unless introduced in large amount it is not eliminated by the kidney. It soon disappears from the circulating blood and is deposited in various tissues, the exact place of deposition depending