IMMUNOLOGICAL REACTIONS WITH A VIRUS CAUSING PAPILLOMAS IN RABBITS : I. DEMONSTRATION OF A COMPLEMENT FIXATION REACTION: RELATION OF VIRUS-NEUTRALIZING AND COMPLEMENT-BINDING ANTIBODIES

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 MASKING EFFECT OF EXTRAVASATED ANTIBODY ON THE RABBIT PAPILLOMA VIRUS (SHOPE)

The foregoing experiments have shown that the causative virus is usually "masked" in the large, disorderly, fissured and inflamed papillomas of cottontails when antiviral antibody is present in quantity in their blood, though virus can be recovered as a rule from the smaller, discrete, well ordered papillomas of these rabbits, almost irrespective of the amount of antibody in the blood of the individuals bearing them. Other findings are described which indicate that the masking of the virus in the large fissured growths is due to serum antibody present in them as result of exudation or hemorrhage, which neutralizes the virus when the growths are extracted or preserved in vitro. The local conditions that favor extravasation of serum (and the accumulation of antibody) prevail as a rule in the large, confluent growths arising after virus has been sown broadcast on scarified skin, but to lesser extent or not at all in the discrete papillomas that occur naturally or as result of tattoo inoculation. The state of affairs is notably different in the papillomas of domestic rabbits. The virus is regularly masked in these, and usually masked completely, even when there is little antibody in the blood and the local conditions do not favor its extravasation into the growths. The findings indicate that something other than antibody is primarily responsible for the masking in this species

A DISTINCTIVE SUBSTANCE ASSOCIATED WITH THE BROWN-PEARCE RABBIT CARCINOMA : II. PROPERTIES OF THE SUBSTANCE: DISCUSSION

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°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 mµ and more, but was retained completely by those with average pore diameters of 348 mµ 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

THE ENDURING PARTNERSHIP OF A NEOPLASTIC VIRUS AND CARCINOMA CELLS : CONTINUED INCREASE OF VIRUS IN THE V2 CARCINOMA DURING PROPAGATION IN VIRUS-IMMUNE HOSTS

The V2 carcinoma—a transplanted rabbit cancer derived originally from a virus-induced papilloma and carrying in masked or altered form the virus primarily responsible for it—was propagated in five successive groups of animals all previously hyperimmunized against the papilloma virus. The cancer grew as well in the hyperimmunized hosts as in normal animals implanted during the same months; and serological tests, made when the tumor was eventually returned to ordinary hosts, proved that the virus was still associated with the carcinoma cells: it had increased to the usual extent as the tumor grew in the hyperimmune animals. The continued increase of the neoplastic virus during propagation of the V2 carcinoma in hyperimmunized hosts contrasts sharply with the elimination of certain extraneous passenger viruses when the tumors they ride upon are grown in hosts previously immunized against them. The facts as a whole would seem to warrant a distinction between the enduring partnership of a neoplastic virus and carcinoma cells on the one hand and the casual association of passenger viruses with tumor cells on the other

SUPPRESSION OF GROWTH OF BROWN-PEARCE TUMOR CELLS BY A SPECIFIC ANTIBODY : WITH A CONSIDERATION OF THE NATURE OF THE REACTING CELL CONSTITUENT

Experiments are reported in detail which show that an antibody which appears in the blood of certain rabbits implanted with the Brown-Pearce tumor or injected with cell-free extracts of it is capable of suppressing the growth of the tumor cells under a variety of experimental conditions, the effects of the antibody being wholly distinct from those of unknown factors that frequently bring about regression of the growth. The implications of the findings are discussed with particular reference to facts indicating that the distinctive cell constituent with which the antibody reacts may play a significant part in the proliferative activities of the Brown-Pearce tumor cell

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

REGRESSION OF TRANSPLANTED LYMPHOMAS INDUCED IN VIVO BY MEANS OF NORMAL GUINEA PIG SERUM : I. COURSE OF TRANSPLANTED CANCERS OF VARIOUS KINDS IN MICE AND RATS GIVEN GUINEA PIG SERUM, HORSE SERUM, OR RABBIT SERUM

In the experiments here described transplanted lymphomas of two kinds regularly regressed following repeated injections of normal guinea pig serum intraperitoneally into mice carrying them, the animals meanwhile remaining lively and devoid of signs of illness or wasting. The lymphomas of untreated control mice, by contrast, usually grew progressively and killed their hosts within 20 to 30 days, and the same was true of the growth of other mice given repeated injections of horse serum or rabbit serum. In similar experiments, the cells of a transplanted lymphosarcoma of rats were temporarily kept from proliferating by repeated intraperitoneal injections of guinea pig serum, though the cells of two transplanted mammary carcinomas of mice, and those of fibrosarcoma, grew unimpeded in hosts likewise treated. Additional experiments related to the phenomenon here described, and a discussion of the findings as a whole, are given in an associated paper

REGRESSION OF TRANSPLANTED LYMPHOMAS INDUCED IN VIVO BY MEANS OF NORMAL GUINEA PIG SERUM : II. STUDIES ON THE NATURE OF THE ACTIVE SERUM CONSTITUENT: HISTOLOGICAL MECHANISM OF THE REGRESSION: TESTS FOR EFFECTS OF GUINEA PIG SERUM ON LYMPHOMA CELLS IN VITRO: DISCUSSION

In an extension of the experimental studies recorded in an associated paper; attempts were made to isolate and characterize the constituent of guinea pig serum responsible for inducing regression of transplanted lymphomas in vivo. The active material was precipitated readily from the whole serum, along with some of the globulins, by means of ammonium sulfate in concentrations of 2.0 molar or greater; it withstood heating at 56°C. for 20 or 30 minutes, but was inactivated upon heating at 66°C. for similar periods; it was completely inactivated by chymotrypsin in concentrations of 1 or 2 mg./cc. during 6 hours at 37°C. Furthermore, the inhibitory effects of small amounts of the guinea pig serum in vivo were enhanced upon admixture with immune sera prepared by injecting the lymphosarcoma cells into rabbits. The facts as a whole suggest that the active material is a protein, and that it may be one or another of the components of complement; yet they do not suffice to establish its identity. Microscopic studies showed that the cells of subcutaneous lymphomas rapidly died and were resorbed following injections of relatively large amounts of guinea pig serum intraperitoneally into mice carrying them, while similar changes followed more gradually after repeated injections of smaller amounts of guinea pig serum. No changes referable to the guinea pig serum were seen in the normal tissues or organs of mice receiving it. Mouse lymphoma cells, suspended artificially as individuals in a physiological saline solution, regularly remained viable following incubation in vitro in mixture with guinea pig serum during 6 hours at 37°C. The finding provides strong evidence that the regression of lymphomas that follows injection of guinea pig serum in vivo is brought about through some reaction in which the guinea pig serum and the host both participate. Some of the implications of the findings are discussed

A DISTINCTIVE SUBSTANCE ASSOCIATED WITH THE BROWN-PEARCE RABBIT CARCINOMA : I. PRESENCE AND SPECIFICITY OF THE SUBSTANCE AS DETERMINED BY SERUM REACTIONS

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

EFFECTS OF ARSENIC-AZOPROTEINS ON MOUSE LYMPHOMA CELLS IN VIVO : WITH OBSERVATIONS ON THE EFFECTS OF OTHER "ANTI-LYMPHOMA" AGENTS, AND ON THE SUSCEPTIBILITY TO THESE EFFECTS OF LYMPHOMA CELLS OF VARIOUS TYPES

Conjugates made by coupling diazotized arsanilic acid with one or another of a variety of proteins regularly brought about the complete regression of established 6C3HED lymphomas in living mice without perceptibly harming the latter, while untreated control animals regularly died with lymphomatosis. Histologic studies made plain that the lymphoma cells promptly die in mice treated with the arsenic-azoproteins, while those in untreated control animals continue to proliferate. Various inorganic and organic arsenicals (including arsanilic acid and 4-arsonophenyldiazotate) were essentially devoid of effect on the lymphoma cells in vivo, and this proved true as well of the proteins employed (serum albumins and globulins procured from several species, casein, and ovalbumin). Mixtures of arsanilic acid and the several proteins, various sulfur-azoproteins, and a number of other substances—viz., amethopterin, chlorambucil, 6-mercaptopurine, 8-azaguanine, azaserine, 6-azauracil, 5-fluorouracil, thioTEPA, and DON, each given in maximal tolerated amounts—also failed to influence notably the course of established 6C3HED lymphomas in vivo. Although readily overcoming Lymphoma E9514 cells growing in the subcutaneous tissues of susceptible mice, the arsenic-azoproteins had little or no effect once these cells had reached the livers and spleens of susceptible hosts. Furthermore the arsenic-azoproteins had little or no effect in vivo on the cells of Lymphoma AKRL1, L1210, and L4946. The findings were considered in relationship to the respective susceptibilities of several types of lymphoma cells to other anti-lymphoma agents—notably guinea pig serum, immune serums prepared in rabbits with mouse lymphoma cells as antigens, and a variety of chemical compounds. Taken together, the observations provide proof that lymphoma cells of various types, although resembling one another quite closely in growth characteristics following transplantation in susceptible hosts, and in morphology as disclosed by ordinary microscopy, differ notably in susceptibility to the effects of the several anti-lymphoma agents