Papillomas Of Domestic Rabbits Research Articles

Changes in RNA expression pattern during the malignant progression of cottontail rabbit papillomavirus-induced tumors in rabbits

Cottontail rabbit papillomavirus induces strictly epithelial tumors in both cottontail and domestic rabbits. A high proportion of the initial benign papillomas progress within 8 to 14 months to invasive carcinomas. With the help of mRNA-specific riboprobes for E6, E7, E1, E2, L1 and L2, we investigated by in situ hybridization the RNA expression pattern of cottontail rabbit papillomavirus in tissue sections of biopsies from different stages of tumor development. Common features of all lesions were high levels of E6 and E7 mRNAs and low levels of E1 and E2 mRNAs. In agreement with earlier reports, there was no evidence for a major mRNA class equivalent to the prominent E1-E4 RNA of human papillomavirus types 6/11 and 16. In cottontail rabbit papillomas, high levels of E6 and E7 mRNAs were present in the upper differentiated epithelial layers. These layers also contained most of the E1 and E2 mRNAs and the viral DNA. In contrast, papillomas of domestic rabbits revealed the opposite differentiation-dependent expression pattern for the E6 and E7 mRNAs; there were strong signals in the basal layers, and these declined with increased differentiation. Transcripts encoding the L1 mRNA were detected only in a few isolated cells of the granular layer. There was no difference between the amounts of E6, E7, E1, and E2 mRNAs present in highly dysplastic tissue and those present in adjacent normal papillomatous epithelium within a progressing papilloma. However, late transcripts and viral DNA detectable only in the upper layers of the papilloma were present throughout the thickness of the dysplastic tissue, indicating a newly acquired permissiveness of the dysplastic cells for viral DNA replication and late transcription. Carcinomas in general had the same expression patterns for E6, E7, and E1 but were dissimilar in the levels of expression of E2 and late transcripts.

Evidence for and localization of vegetative viral DNA replication by autoradiographic detection of RNA-DNA hybrids in sections of tumors induced by Shope papilloma virus.

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis.

Immunofluorescent studies of virus-induced rabbit papilloma (Shope) in vitro.

SummaryShope virus-induced papillomas of domestic rabbits were cultivated in vitro and were subjected to immunofluorescent investigation. Two morphologically different cells, round or polygonal cells and triangular or columnar cells, were observed to grow out of explanted papilloma tissues. Among them, the majority of round or polygonal cells exhibited intense fluorescence reaction in nucleus. A certain number of triangular or columnar cells also reacted with specific fluorescence in nucleus but the intensity of the staining was weaker and less conspicuous. The implication of these findings was discussed in connection with previous immunofluorescent studies on the tumorous tissues of the Shope system.

Studies of the mechanism of action of the Shope rabbit papilloma virus. I. Concerning the nature of the induction of arginase in the infected cells.

These studies make plain that the Shope papilloma virus induces the production of an arginase in rabbit squamous epithelium, and provide evidence that the information for the synthesis of the enzyme is derived from the virus rather than the rabbit. This form of induction is therefore different from that brought about by chemicals such as galactosides (36). Striking differences were shown between the physiochemical properties of Shope virus-induced arginase and other arginases of domestic and Kansas cottontail rabbits. The absence of a requirement for manganese suggests the mechanism of splitting off urea from arginine by papilloma arginase probably differs from that of previously described arginases. These findings (particularly the experiments in which it was demonstrated that papilloma growth could be greatly reduced by giving animals supplemental arginine in the presence of small amounts of the arginase inhibitor, canavanine) provide evidence that suggests the following mechanism of action for the virus: viral DNA introduces into rabbit epithelium the information for the synthesis of an arginase for which the cells have no control mechanism. The arginase depletes cellular arginine and, in turn, the synthesis of arginine-rich nuclear histones (2), thereby freeing the nucleus for greater synthetic activity (4, 35), which results in more rapid growth of the papilloma cells. Domestic rabbit papillomas induced with purified virus derived from wild rabbit papillomas were shown to contain an antigen which reacts immunologically like a wild rabbit antigen. It was distinct from the arginase, the protein coat of the virus, and the Vx-2 antigen. The presence of the wild rabbit antigen seems indicative of integration of the virus DNA in the host genome of the wild rabbit. Other incidental findings include the description of a simple method, accurate to within 1 per cent, for determining the partial specific volume of a protein, and the description of a modification of the Richards and Schachman method (9) enabling the determination of the molecular weight and homogeneity of a protein with as little as 0.25 mg and with an accuracy of 2 per cent.

Antiviral and Antitumor Immunologic Mechanisms Operative in the Shope Papilloma-Carcinoma System

In 1933 Shope described an infectious papilloma of wild cottontail rabbits. The tumor was regularly transmissible to domestic rabbits on primary inoculation but was not transmissible in indefinite series. He noted that the tumors of 2 animals regressed, whereas in most cases the papillomas persisted indefinitely. During the following 10 years, Rous, Kidd and Beard published extensive studies of these papillomas and of squamous cell carcinomas originating in them. Shope (1937) was the first to note that, although papillomas of domestic rabbits were commonly free of virus as judged by transmission experiments, they nevertheless contained viral antigen as judged by their capacity to stimulate the development of neutralizing antibody and resistance to reinfection following intraperitoneal injection of tissue suspensions. He spoke of “masked” virus and suggested that this might represent either virus rendered inactive by combination with neutralizing substance or by alteration to a non-infective phase. Carcinomas were likewise shown...

Methyl alcohol purification of the rabbit papilloma virus.

SummaryThe methanol precipitative technic of Cox, et al., when applied to the purification and concentration of papilloma virus from naturally occurring cottontail rabbit papillomas, domestic rabbit papillomas and neutral virus-antibody mixtures readily yielded active papilloma virus from the cottontail papillomas but it did not yield virus from either domestic rabbit papillomas or from papilloma virus and its antibody in neutral mixture.

THE RECOVERABILITY OF VIRUS FROM PAPILLOMAS PRODUCED THEREWITH IN DOMESTIC RABBITS

By preliminary preparation of the skin in ways that render it hyperplastic the presence of infective virus can be demonstrated in extracts of domestic rabbit papillomas which yield no growths when inoculated by the ordinary methods and which for this reason have been supposed to contain no virus. The amount of virus recovered by the method outlined in the present work, however, is small when compared with the yield obtained in most instances from comparable cottontail rabbit papillomas. The yield is greatly influenced not only by the virus strain used to produce the growths but by the individual rabbit host. Although virus has been obtained from papillomas produced in domestic rabbits by all of the virus strains tested, a total of 21 thus far, only about one-fourth of these strains are readily to be procured again from the growths they cause and the others are demonstrable only in hosts in which the conditions are favorable for reasons unknown. An experimental comparison of the capacity of suspensions of papilloma tissue from domestic and cottontail rabbits to elicit specific antibodies has shown that the titers attained are approximately proportional to the amount of infective virus demonstrable in the suspensions. The findings as a whole indicate that far less virus exists in infective or antigenic form in the papillomas of domestic rabbits than in those of cottontail rabbits.

Electrophoretic Analysis of Extracts of Rabbit Papillomas.

The serum of rabbits bearing the growths of infectious papillomatosis and of rabbits immunized with purified virus or wart extracts has been studied1 by the electrophoretic procedures of Tiselius. Differences from the normal electrophoretic patterns were seen only in the serum of animals actually carrying growths. In the present work studies were made on saline extracts of the virus-induced growths of domestic and cottontail rabbits.The growths had been present for 39 to 115 days and, except for retrogressing warts in one animal, were exuberant and fleshy. Growths were removed from rabbits killed by air injection and, freed of connective tissue, were pulped in a Waring Blender and extracted for 24 hr at 2-8°C in 20% suspension in 0.9% NaCl solution. The extracts were filtered with celite and dialyzed against buffer prepared as in the serum studies.1In Fig. 1 is shown the electrophoretic diagram of a filtered extract of domestic rabbit papillomas showing 3 distinct migrating peaks, the mobilities of which ...

THE DETECTION OF A "MASKED" VIRUS (THE SHOPE PAPILLOMA VIRUS) BY MEANS OF IMMUNIZATION

A study has been made of the immunization procedure described by Shope, with particular reference to the detection of "masked" papilloma virus by means of it. Papilloma extracts were frequently encountered which, though non-pathogenic, elicited the specific antiviral antibody and induced resistance to the virus upon injection intraperitoneally into normal rabbits. The results of the immunization experiments were often complicated, however, by the effects of extravasated antibody, which had accumulated in various amounts in many of the papillomas and was consequently present in extracts of them together with "masked" virus. The extravasated antibody was often sufficient to render extracts of domestic rabbit papillomas non-antigenic; and sometimes, when present in excess, its passive transfer conferred resistance to reinfection with the virus. The conclusion seems warranted that only positive immunization findings can be interpreted with certainty. Negative results provide no decisive evidence as to whether "masked" virus is or is not present in the injected material, unless the amount of extravasated antibody also present is known. The findings may have a bearing on the negative outcome of immunization experiments with extracts of the cancers deriving from the natural papillomas of cottontails. Crude suspensions of domestic rabbit papillomas, which contain little or no virus demonstrable by ordinary methods, are far less antigenic than extracts of the natural growths of wild rabbits, which contain virus in quantity. In explanation of the finding the possibility seems worthy of attention that domestic rabbit papillomas may contain much less virus than the growths of cotton tails, the natural hosts of the virus.

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.

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.