Virus-induced Papillomas Research Articles

A DISTINCTIVE SUBSTANCE ASSOCIATED WITH THE BROWN-PEARCE RABBIT CARCINOMA

A substance is regularly present in saline extracts of the Brown-Pearce rabbit carcinoma growth which is capable of fixing complement specifically in mixture with the sera of certain rabbits bearing the tumor or in which this has recently retrogressed, as the foregoing experiments have shown. The substance was not demonstrable in extracts of the normal tissues, virus papillomas, or uterine cancers of rabbits, nor in extracts of rabbit tissues infected with certain viruses (vaccine virus, Virus III, fibroma virus). The sera of normal rabbits, of those immune to a variety of infectious diseases, including syphilis, vaccinia, fibromatosis, and Virus III, and of others with uterine cancers or virus-induced papillomas, failed to fix complement specifically in mixture with extracts containing the antigen of the Brown-Pearce tumor. The significance of the findings will be discussed in the succeeding paper, after consideration has been given to some of the properties of the serologically active material derived from the Brown-Pearce tumor.

A DISTINCTIVE SUBSTANCE ASSOCIATED WITH THE BROWN-PEARCE RABBIT CARCINOMA

The serologically active substance of the Brown-Pearce tumor, a complement-fixing antigen, differs notably from certain other tissue antigens (the Wassermann, Forssman, and organ- and species-specific tissue haptens, for example) in the fact that it is not effective after alcoholic extraction of the tissue containing it. Like many of the proteins and viruses it is in-activated upon heating to 65 degrees C. for 30 minutes; and, like them as well, its activity is lost upon treatment with acid (to pH 4.5 or lower) or alkali (to pH 11.5 or higher). Filtration and centrifugation experiments disclosed the fact that the antigen of the Brown-Pearce tumor passed readily through collodion membranes with average pore diameters of 383 mmicro and more, but was retained completely by those with average pore diameters of 348 mmicro and less. It was thrown down completely or almost completely upon centrifugation at 20,000 R.P.M. for one hour. The findings indicate that the Brown-Pearce tumor antigen has a large and nearly uniform particle size and weight,-as large as that of many of the viruses. They differentiate it sharply from the generality of "soluble antigens.rdquo; Upon direct comparison, the complement-fixing antigen of the Brown-Pearce tumor was found to be similar in a number of its general traits to the serologically active substance of the virus-induced papillomas of rabbits, which in turn is intimately associated or identical with the virus responsible for the papillomas. Extracts of the Brown-Pearce tumor containing the serologically active substance in quantity have given rise to no lesions, however, upon injection into normal or tarred rabbits. The significance of the findings is discussed.

Identity of "Inhibitor" and Antibody in Extracts of Virus-Induced Rabbit Papillomas

Saline extracts of the virus-induced papillomas of domestic rabbits often contain something that inhibits or neutralizes the virus in vitro, as Shope first noted.1 Serological studies have shown that when mixed with it the blood of rabbits carrying these papillomas also has in most instances the power to neutralize the virus in vitro.2 Experiments were undertaken to learn whether the “inhibitor” procured from the papillomas may not be specific antiviral antibody of the sort present in the blood.To test for “inhibitor”, 10% extracts of freshly procured papillomas were prepared by grinding in sand, suspending in saline, and centrifuging at about 4400 rpm for 20 minutes in an angle-head centrifuge. The clear supernatant fluids were then mixed in equal parts with a Berkefeld filtrate of the virus-induced growths of cottontail rabbits, containing virus of known titer, incubated 2 hours at 37°C, and rubbed into scarified areas on the skin of normal domestic rabbits according to a titration technic already descr...

IMMUNOLOGICAL REACTIONS WITH A VIRUS CAUSING PAPILLOMAS IN RABBITS

A study has been made of the yield of virus and of the complement-binding antigen from the virus-induced papillomas of cottontail and domestic rabbits. Extracts of the discrete, naturally occurring papillomas of cottontail rabbits usually contain virus in large amount; and, as a rule, they also contain the complement-binding antigen in high titer. The confluent growths produced experimentally with the virus in some cottontails, on the other hand, often fail to yield the virus, or furnish it in small amount; and extracts of them have little if any complement-binding capacity. The sera of cottontails with massive papillomas from which the virus cannot be recovered often have high antibody titers. Many extracts were tested of the virus-induced papillomas of domestic rabbits. None contained the virus in large amount, and the majority of them failed to manifest it on sensitive test. A few fixed complement in low titer when mixed with immune sera, but most failed to do so. Crude extracts of the "non-infectious," virus-induced papillomas of domestic rabbits stimulated the formation of virus-neutralizing and complement-binding antibodies in low titer when injected intraperitoneally into normal rabbits of the same breed, but Berkefeld filtrates of the same materials proved devoid of this immunizing effect. The significance of the findings described in the three papers is discussed. The evidence as a whole favors the view that the virus stimulates the formation of the virus-neutralizing and complement-binding antibodies in vivo, and many facts indicate that it is closely associated, and in all probability identical, with the antigen that reacts with immune serum to fix complement in vitro.

IMMUNOLOGICAL REACTIONS WITH A VIRUS CAUSING PAPILLOMAS IN RABBITS

The sera of rabbits bearing virus-induced papillomas have been found to bind complement when mixed with antigens consisting of extracts or filtrates of the growths containing the virus. The sera of normal rabbits, of those immune to other viruses (vaccinia, herpes, fibroma, myxoma), of rabbits with syphilis, or of those with papillomas consequent on tarring, did not fix complement upon admixture with the papilloma antigens. The complement-binding antibody was present in the serum specimens in the same relative proportions as the virus-neutralizing antibody, and both were present in greatest amount in the sera of rabbits that had borne large papillomas over considerable periods of time. A few sera were come upon that neutralized small amounts of the virus yet failed to bind complement to any noteworthy degree in the tests. The sera of cottontail rabbits fixed complement and neutralized the virus in much higher titer than the sera of domestic rabbits with comparable growths. The implications of the findings will be discussed in a subsequent paper, after the properties of the complement-binding antigen have been scrutinized.

THE COURSE OF VIRUS-INDUCED RABBIT PAPILLOMAS AS DETERMINED BY VIRUS, CELLS, AND HOST

An experimental analysis of the factors responsible for the observed differences in the course of virus-induced papillomas of the rabbit has shown that some are referable to the virus, others to the cells, and yet others to host influences. The interplay of these factors affords enlightening illustration of the nature of the cell-virus relationship in virus-induced tumors. Retrogression of the rabbit papillomas appears to be consequent on a generalized resistance of host origin, elicited by and directed against the proliferating, virus-infected cells.

A Complement-binding Antigen in Extracts of the Brown-Pearce Carcinoma of Rabbits

Summary and CommentA substance is present in extracts of the Brown-Pearce tumor which binds complement specifically when mixed with the sera of rabbits bearing the growth, or in which it has retrogressed. The substance is readily distinguishable from the complement-binding antigen which can be extracted from the virus-induced papillomas of rabbits, though it resembles the latter in its general traits. Further work is under way to determine the significance of the findings.

Ultracentrifugal Concentration of a Homogeneous Heavy Component from Tissues Diseased with Equine Encephalomyelitis

With the demonstration of the existence of a heavy protein in plant tissues diseased with tobacco mosaic virus1 it became inevitable that similar proteins should be sought in other kinds of virus-infected tissues. Most viruses are more or less unstable towards the chemicals which would naturally be used in efforts to concentrate and purify them. The air-ultracentrifuge2 has, however, furnished a new tool3 which has been successful in extracting and purifying several of the less stable virus proteins causing plant diseases4 and in preparing a similar substance5 from virus-induced rabbit papillomas (Shope). The same type of ultracentrifugal examination has now been made of tissues diseased with the virus of equine encephalomyelitis.∗As in the previous ultracentrifugal concentrations an angle-centrifuged tissue suspension was ultracentrifuged for one to one and a half hours in a maximum field of ca 50,000 times gravity. All but a trace of the virus activity was thus sedimented. The translucent pellet formed ...

PH Stability of Shope Papilloma Virus and of Purified Papilloma Virus Protein

We describedc the isolation of a high molecular weight protein from virus-induced papillomas of western cotton-tail rabbits. It was found that this protein could be extracted only from infectious suspensions of such papillomas and that virus activity was proportional to the amount of the protein present. This and other experiments, in which we have failed to dissociate virus activity from the protein, point to its being the purified agent responsible for the disease. We have now completed a detailed comparison of the influence of pH on (1) the infectiousness of the virus and (2) the stability of the molecules of the protein. Stability of the virus was determined by dissolving the virus protein in buffer solutions of accurately determined pH and titrating the solutions in domestic rabbits immediately, and after 6 hours, 24 hours, a week and a month. With the analytical air-driven ultracentrifuge,4 the molecular constitution of these solutions was found from their sedimentation diagrams. On the acid side of pH 7 the virus activity remains high until at a pH between 2.9 and 3.3 it suddenly is lost. The protein molecule splits at exactly this point. From pH 3.3 to pH 7, on both sides of its isoelectric point, the papilloma protein has a principal component with S20 ° = ca 260 × 10-13 cm. sec.-1 dynes-1; there ordinarily appears also a faint secondary boundary corresponding to S20° — ca 380 × 10-13. At pH 1.85 the completely inactive protein sediments with the sharp boundary corresponding to a single molecular species with S20° = ca 180 × 10-13. In contrast to conditions in acid solutions, full virus activity is not preserved for a long time at any pH greater than 7. Alkaline inactivation occurs in two ways. Above pH 10.1 virus solutions immediately become non-infectious. Below this point the titre of a virus solution gradually diminishes with time, at a rate that decreases as neutrality is approached. Study of the stability of the protein molecule with the analytical ultracentrifuge demonstrates that it fragments at the same pH (10.1-10.2) at which immediate inactivation is observed. This molecular disintegration is more complete than the one seen in strongly acid solutions; the largest piece of the original protein that can be photographed in strong alkali has a sedimentation constant of only S20° = ca 30 × 10-13. Below pH 10 the sedimentation constant of the papilloma protein molecule is not measurably changed nor is the molecular homogeneity visibly diminished even when solutions are kept until all virus activity has been lost. These solutions thus contain non-infectious protein material consisting of only slightly altered papilloma protein molecules. The biological properties of such non-infectious derivatives of the papilloma virus protein are being studied.

OBSERVATIONS ON THE RELATION OF THE VIRUS CAUSING RABBIT PAPILLOMAS TO THE CANCERS DERIVING THEREFROM

The papillomas caused by the Shope virus sometimes grow down spontaneously into the subcutaneous tissue and extend along the lymphatics in the same way as do many cancers of the human breast. They may even invade the voluntary muscle under such circumstances, taking on an aspect suggestive of squamous cell carcinomatosis, but ultimately they differentiate in the way characteristic of the papilloma. Slight operative interferences with papillomas may be followed by a development of secondary nodules in the lungs. These result from cell emboli, and the same local conditions determine their fate as are effective in the case of emboli composed of human cancer cells. The virus-induced papilloma is not only a neoplasm in its immediate aspect and habit but sometimes one that verges upon malignancy. The tumors, including the cancers, which eventually derive from it in favorable hosts, are representative of more than a mere enhancement of the activity of the growth. They develop within a relatively brief period of time but only after the papilloma has grown for a long while; and they are morphologically various whereas the parent tumor is remarkably constant in its form. Some of the new growths differ but little from the papilloma, however, even when possessed of the ability to metastasize, and many continue to be influenced by the virus. The Shope virus is heavily conditioned in its carcinogenic activity, yet it is the nearest cause for cancer now known.

SEROLOGICAL REACTIONS WITH A VIRUS CAUSING RABBIT PAPILLOMAS WHICH BECOME CANCEROUS

The serum of a rabbit with large cancers resulting from the transplantation of a squamous cell carcinoma that had arisen from a virusinduced papilloma, possessed the power to neutralize the virus, and so too in less degree did that of an animal of the same transplantation series in which a small nodule had developed. The sera of rabbits carrying tar papillomas or the Brown-Pearce carcinoma proved wholly devoid of effect on the virus. The implantation of Brown-Pearce tumor material mixed with virus did not lead to an enduring establishment of the latter in the resulting growths, nor to any immediate changes in their morphological character. The significance of the facts is discussed.

Carcinomatous Changes in Virus-induced Papillomas of the Skin of the Rabbit

The Shope rabbit papilloma, a skin growth caused by a virus, has been shown to possess the characters whereby tumors are recognized. When given opportunity, as on implantation within the host, the growth frequently looks and behaves like a malignant neoplasm. The present report is concerned with instances in which skin papillomas caused by the virus have spontaneously become carcinomatous. The change has been noted in 5 of 10 domestic rabbits with growths existing 4 to 8 months. During the early weeks of its development after virus inoculation the papilloma enlarges laterally, but later it is restricted by scar tissue and builds outwards only. At first it overlies the skin appendages, but these disappear after a time and it becomes bedded somewhat more deeply. The malignant change may first attract attention when a fissure exuding serosanguineous fluid opens in the midst of the papilloma; but more often there occurs a generalized, fleshy, discoid thickening of the base of the growth, which gradually raises the jagged, dry, papillomatous tissue some millimeters above the skin surface, and also bulges downwards. Soon the animal gnaws at this portion of the growth, opening in the one case a depressed ulceration with firm, gristly walls, and in the other laying bare a high, fungoid mass. On biopsy a squamous cell carcinoma is found, or an invasive, papillomatous, epithelial tumor, or most frequently, the 2 intermixed, with every gradation between them. The growth may for some weeks remain circumscribed and fungating, or it may rapidly extend under the skin, involving it and the muscle and becoming fixed upon the deep tissues. Some cancers less than 2 months old are already 5 cm. across and 2–3 cm. deep. As a rule they become infected with pus-producing organisms and the health of the host suffers, though most of our rabbits are still alive. Metastasis to a regional lymph-node (confirmed by section) has occurred once. Transplantation to the leg muscle of the host has resulted in a secondary, highly invasive, squamous cell carcinoma.