STUDIES ON THE ISOLATION OF AGENTS FNOM TUMORS. By Leek Tanasugarn A THESIS Submitted to tbe School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOGTON OF PHILOSOPHY Department of Bacteriology and Public Health 1955 Tanasugarn -ABSTRACT Many tumors of* bo t h animals and man have been report« "to be caused by viruses, fox* example, Rous sarcoma (Rous 1910), mouse mammary carcinoma (Bittner 1 936 ), human pap­ illoma (Wile and Hingery 1919)? and infectious myxomatosis (Rivers 1926). In this experiment 17 tumors were obtained from both animals and man. Both, tissue culture and serological mett ods were used in an attempt to isolate the causative agent of these tumors. Nine tumors grew well in tissue culture. There was r correlation between origin or type of the neoplasm and growth in vitro. Concentrated tumor allantoic fluid, representing the fifth passage of tumor extracts through embryonated chicks eggs, retarded the growth of normal chicken heart tissue d cultures. One exception was encountered in which exceptic growth occurred. /Then such concentrated allantoic fluids were tested for hemagglutination w i t h normal chicken cells, no re­ action occurred. Concentrated tumor allantoic fluid in many cases produced hemagglutination of chicken erythro­ cytes previously sensitized with Newcastle disease virus or modified with, trypsin. When the concentrated allantoic fluid was tested for hemagglutination with sensitized or modified cells between each passage, hemagglutination was frequently observed. None of the 17 tumors studied con­ tained an agent that wa s capable of producing hemagglutina tion through all five consecutive serial passages. No interference, in vitro or in vivo, could be detect ed between concentrated allantoic fluid tumor passage material and Newcastle disease virus. ACKNOWLEDGMENT '1’h.e author wishes "to express his sincere appreciation bo Dr. Walter H. Mack, Department of Bacteriology and Public Health, under whose lofty inspiration, constant sup­ ervision, encouragement, and unfailing interest this inves­ tigation was undertaken and to whom the results are here­ with dedicated. He is also greatly indebted to Dr. Bobert P. Langham, Professor of Pathology, for his help in identifying the tumors. Greatful acknowledgment is also due to Dr. Wade Oberlin Brinker, Professor of Surgery and Medicine, for his kindness in supplying of canine neoplasms, and to Dr. Don m. LeDuc and Dr. Leo V/. Walker for che human neoplasms. The writer deeply appreciates bhe financial support of the Thai Government, the United Stabes Government and Michigan Sbate College for Scholarships. He wishes to express his sincere appreciation to Dean Thomas H. Osgood, Dean of School of Graduate Studies, for his kind recommen­ dation for the Michigan State College Scholarship, and to Jr. Henrik J. Stafseth, Head of Department of Bacteriology and Public Health, for his kind recommendation for the Thai Government Scholarship, without which this investi­ gation would not have been possible. TABLE OF CONTENTS Page I Introduction g II Literature Reviews — — — III Materials and Methods: _ — 3 - - - - - - - - - 30 (1 ) Ultracertrifuge concentration of materials fox*: - 33 sue cul tux*e wo r k . A. Ti 3 B. Hemagglutination reaction. G. Tests on interference between tumor passage allantoic fluid followed by Newcastle disease. (2) Tissue culture work: - - A. -- -- - 34. Normal growth of tumor tissue. B. . The effect of tumor jja-ssage allantoic fluid on the growth (in vitro) of normal tissue. (3) Hemagglutination reaction after pas­ sage of tumor material in cnicken em­ bryos : - - - - - - - - - - - - - - A. Tests with normal chicken red cells . B. Tests with chicken red cells sensitized with Newcastle disease 42 virus. C. Tests with, chidten red cells / modified with trypsin. (4) Tests for interference between tumor passage allantoic fluid followed by Newcastle disease virus:- - 4-7 A. In vivo E. I n .vitro IV results - - - - - - - - - - - - - - - - - 50 V Discussion 86 VI Summary - - - - - - - - - - - - - - - - - 93 VII rieferences 95 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ INTRODUCTION I INTRODUCTION Most of th.e theories that have been offered regarding the cause and the nature of cancer fall into one of the fol­ lowing categories: (1 ) embryonic, (2 ) biochemical, (3 ) gene­ tic or (4) infectious agents. (Ackerman and delhegato, 1947) Cohnheim postulated (Ackerman and delxtegato, 1947) that neoplasms arise from embryonal cells which have per­ sisted and which retain a special erative potency. Aibbert1s modification of the theory was that differenti­ ated but embryologically displaced cells serve as foci for the genesis of neoplasia (Ackerman and delAegato, 1947). The biochemical theories assume that certain specil'ic biochemical or biophysical alterations in the environment of the cells cause them to acquire neoplastic properties, for example, those caused by chronic irrication, carcino­ genic hydrocarbons and other carcinogenic agents. According to Ackerman and delhegato, "There is ad­ equate proof that genetic background influences suscepti­ bility to neoplastic reactions. Neoplasms are characters and not genic factors, and susceptibility to neoplasms is expressed in degree. The view that cancer is a single Mendelian factor, either dominant or recessive, is no longer tenable. The continued reproduction of cells in neoplasia and the transmission of characters from one cell "bo another for an almost limitless number generations are in agreement with the view that cancer is a manifesta­ tion of a genetic difference from normal cells, or a gene­ tic alteration of normal cells." In this investigation we are interested in the in­ fections agent theory of cancer. There are several types of neoplasia which are associated with a virus-like agent. The virus, being a self-reproducing entity, is thus tran­ smitted through subsequent generations of the cell. Can­ cer, according to this theory, is an infectious process and the result of a virus-cell symbiosis. ibioads1 (1 9 5 2 ) opinion was that it may nob be enough to invoke virus production if a new neoplasm is caused by the injection of a cell-free extract from an old one. He considered thab there is an important distinction to be drawn between cancer and an infectious process of the con­ ventional type. He also suggested that neoplasia may be a process of aggressive cellular growth due to the inheri­ tance of new characteristics, consequent to the modifica­ tion, by mutation, of components filterable, or non—filter­ able, which are responsible for old characteristics, Pinkerton (1952) certain properties if suggested that a virus must possess it is to be successful as a cause of cancer: (1) It must be well adapted to life within its host cells. This means that its reproduction must be gear­ ed to that of the cells, but not necessarily as closely as that of genes, which, in normal mitosis, multiply precisely once for each cellular division. (2) It must be of low virulence and incapable of inducing a high degree of cellu­ lar immunity in its host. this suggests that it must be rather closely related to normal cytological constituents, so that it does not behave as a "foreign 11 protein. (3) It must stimulate its host cells to continuous aggressive pro­ liferation. Pinkerton finally concluded "Until then, I see nothing unscientific in accepting tentatively the hypothesis that cancer may be caused by self-duplicating virus-like agents." LI\i'j.,.x.Vl?U alii xffiVIM/S II LITERATURE xiEVIEWS The Infectious Sarcomata of Chickens hems in 1910 described the first of a series of trans­ plantable, malignant sarcomas of the chicken. In 1911 he showed that the turnor could be Induced with dried cells, with cells that had been destroyed with glycerol, and with cell-free filtrates. sarcoma. The tumor is now known as the nous It is a spindle—cell sarcoma which metastatizes freely and usually destroys its nost within one monah. In 1912 nous, Murphy, and fytler described another chicken turnor transmissible with filtrates, an osteochrondrosareoma, and a. third, a spindle-cell angiosarcoma. The active principle of these tumors, particularly of ho us sarcoma, has been extensively studies by Murphy and co-workers (1931 * 1932) and by Sittenfield, Johnson, and Jobling (1 9 3 1 )* : lh-e active agent has not been isolated in a pure state, but relatively pure extracts have been made by chemical precipitation. They possess the power of stimulating neutralizing antibodies when injected into rabbits, and in other respects do not differ in behavior from viruses that cause infectious diseases. Mouse Mammary Carcinoma Since Bittner's discovery (1936) of the "nursing influence ,11 it; lias been possible to define mouse maaiaary carcinoma as a disease of the adult; female, acquired in infancy through, an agent transmitted in the mother's milk. The definition is neither complete nor exclusive, but it is a consequence of Bittner's demonstration of the "nursing influence," With this in mind, many laboratories attempted to isolate the agent from milk. Graff et al. and Passy et al. (194-7)* with an electron microscope, found small spher­ ical particles having a diameter of from 20 to 200 mu. in samples of milk obtained from nursing mice known to carry the mammary carcinoma. Graff e_fc al. (194-7) and Dossing et al. (194-9) reported that similar spherical particles have also been found in tumor extracts. Porter and Thompson (194-8) also reported that they found these particles in cells cultured from mouse mammary carcinomas. It is difficult, at the present time, to form an opin­ ion concerning the nature of these particles. One has to take into consideration that particles similar in shape and size have been found in extracts prepared from presum­ ably normal mouse tissue, and in milk samples collected from mice apparently free from the mammary carcinoma agent. The differentiation of the various particles with the elec­ tron microscope is, at the present time, quite difficult. Many particles which may be of an entirely different origin may appear alike to the critical observer. This is partic­ ularly true when particles are observed afcer they have been shadowed with, heavy metals. It is true that the exact nature of the particles de­ tected in mouse milk have not yet been determined. Their consistent presence, however, in mouse milk known to con­ tain the mammary carcinoma agent, and their only occasional presence in samples of milk collected from mice Known to be free from the tumor agent, suggests that they may represent the mouse mammary carcinoma agent. Such an assumption was strengthened by the results of G-raff e_t al. (194-9) and tassey et a l . (1990). Milk samples containing the particles v/ere fractionated by centrifugation. Upon injection of this material into susceptible mice, mammary carcinomas evere produced. Graff e_t al. (1992) found that these prep­ arations from milk obtained both ultracentrifugally and electrophoretic ally, contain two components and all com­ ponents produced tumors in high yield. Immunological specificity is an important consider­ ation in cancer and considerable work has been reported by some laboratories on the immunological behavior of one of the milk agent preparation. Andervont and Aryan (194-4-) and Green and Bittner (194-5) showed that the agent was able to stimulate the production of neutralizing anti­ bodies, and that the agent is antigenically different from mouse tissues. These particles transmitted the disease in high dilu­ tion in characteristic manner in mice, and elicited anti— bodies in the rabbit. They were shown by iumru.nochemical techniques to be antigenically distinct from normal protein of the mouse or mouse milk. They concluded that these parti­ cles constitute the virus responsible for mouse mammary car­ cinoma. Kidney Carcinoma in the Leopard Frog Lucke (1934-) found that in the leopard frog (kana pipiens) , the kidney is frequently the site of a malignant tumor, an adenocarcinoma. In 1938 he made a survey of over 10,000 frogs and found the incidence of this tumor to be 2.7 per cent. The frog tumors are ivory-white in color, contrasting well with the brownish renal tissue. most of them are some­ what firmer than the surrounding normal kidney. They range in size from tiny, early tumors to large masses that have destroyed all but small portions of the kidney, displacing the neighboring organs and nearly filling the coelomic cavity. Lucke (1952) showed that the outstanding characteris­ tic of this frog tumor is the frequent presence of acidophi­ lic intranuclear inclusions wnich, in general appearance, are like those found in herpes and certain other diseases known to be caused by viruses. in their typical, fully developed form, the inclusions are readily recognizable and they were observed in over one—half of the series. It is obvious that there must be developmental stages of the in­ clusions, but the appearance of the early stages is still a matter of uncertainty. The inclusions are invariably confined to the neoplascic cells. They have never been observed in normal renal epithelium of tumor-bearing kid­ neys , or in bhe normal cells of other organs. The number of inclusions in the neoplastic tissue varies greatly; sometimes, relatively few are present; sometimes, nearly every cell in some portion of bhe tumor is affee bed. Lucke performed bransmission experimenbs on tumor ma­ terial containing no living cells. lie prepared the tumor desiccates from ten frog tumors by freezing bhe minced tumors ab approximately —80°C. in a mixbure of cellusolve and solid GO ^ , and then drying them by high vacuum distil- ation from a frozen state. The container’s were sealed under vacuum and stored at refrigerator temperature for' at least two or three weeks. The dried material was then ground to a fine powder under aseptic conditions and sus­ pended in sterile water. This maberial was used as in­ oculum in frogs. Anocher small group of ben frogs received an emulsien of glycex'inated tumor. olices of the tumor nad been stored for 20 days in 50 per cent glycerin at refrigerator temperature. They were wasned repeatedly in amphibian -Linger’s solution, ground bo an emulsion, and 0.5 ml. of the emulsion was injected. Two hundred and thirty—four frogs received. (0.5 ml.) intra-abdominal inoculations of tumor desiccates, and ten frogs received emulsions of glycerinated tumor. Tne results of both series corresponded closely with, those obtained from intra—abdominal injection of living tumor material. increased with time. The incidence of kidney tumor Twenty-one per cent of frogs sur­ viving the injection (of both dried and glycerinated tumor) for more than six months developed renal tumors. It is unlikely that the tumor desiccates contained living cells. Hence, the conclusion is warranted that the tumor-inducing agent is separable fx’om cells. These experi ments, and the i'requent presence of intranuclear inclusions within the neoplastic cells, make it very probable that the carcinogenic agent has the attributes of a virus. Avain Leucosis The agent of fowl leucosis can be carried from bird to bird indefinitely by intravenous inoculation into susceptible birds. Ellermann and hang (1 9 0 8 ) found that from 20 to 40 per cent of chickens were susceptible to in­ oculation. When the virus is passed from oird to bird by blood inoculation, the virulence often increases as indi­ cated by a shortening of the incubation period and sometines by an increase in tne percentage of birds in which 11takes" are obtained. In the beginning of a series of pas­ sages the incubation period may be several months in length This may be shortened, to less than one month alter a lew serial passages. Furth and Miller (1932) found that the agent oi leu­ cosis passes all siliceous filter's. Collodion membrane filtration is uncertain, but they thought that their re­ sults indicated that the particulate size ol the virus was some what less than 100 mu. Furth (1932) found that as little as 0.000,001 ml. of plasma was sufficient to produce the disease by intravenous inoculation. He also found that the infectious agent re­ tained its potency f o r .at least 3 d days wnen dried, and for at least 104 days wnen preserved in glycerol. Avain Lymphomatosis Lymphomatosis is an extravascular form of leucosis in whic.n the proliferating tumor cells are primitive, un­ differentiated lymphocytes. Several forms of lymphomatosis are recognized: neural, ocular, visceral, and osteopetrotic. Cell—free transmission experiments have been attempted with great difficulties, not the least of whicn is that the disease appears spontaneously. Many uninoculaued controls frequently develop the disease. Baber (1943) found that when hatches of cnickens were separated, some being reared where chickens had not previously been kept, the incidence of lymphomatosis varied according to the age which such chicks were brought back to infected premises. Hutt and co-workers (1944) found that chicks raised at great distance from infected birds showed a deminished in— cidence compared with chicks raised in an infected environ­ ment . Lee and Wilcke (1941) found that the incidence of lymphomatosis was much higher* among the progeny of birds suffering from the ocular form of the disease, especially when the female was affected. Waters (1947) and Waters and Prickett (1944), working in experimental fI o c k s kept under rigid quarantine, believe they have shown that the agent may be transmitted by infected eggs. Cottral and co-workers (1949) and Gottral (1950) found that the causative agent of visceral lymphomatosis is con­ tained in the chick embryo and embryonic fluids. The pre­ sence or absence of the agent in embryonic material was determined by inoculating this material into groups of 15 to 50 chicks. These cnicks were of a line known to be highly susceptible to this disease, although relatively free from infection when hatched, as indicated by the low incidence of the disease when the birds were raised in isolation. Gottral (1949-50) also performed egg transmission experiments in the production of tumors with material arising from appearently normal individuals. All embryos used wei*e normal and all dams were clinically normal when the eggs were laid, and they remained so for variable periods. dhe embryonic material was made into a suspension •and then inoculated into susceptible birds. During a per­ iod o± approximately one year alter tiie use of the embryos, three cases of visceral lymphomatosis developed in the 59 birds, 38, 136 and 14-4- days respectively following inocula­ tion * Burmester (1 9 3 2 ) mentioned that when passages were made with filtrates prepared from tumors of early develop­ ing cases, some of the bix*ds died with suggestive lesions of leukemia in less than 100 days. The Infectious Papillomas Papillomas, or common warts, occur in many sxjecies of animals. They seem to be most frequent in man, cattle, dogs and rabbits. All these tumors contain filterable agents with which the tumors may be induced in other in­ dividuals of the same species. Ihey appear to have a high degree of host specificity, and some of them have specificibies for1 particular kinds of epithelium within a single host. Warts occur in epizootic form in herds of cattle and in kennels of dogs. All varieties are most prevalent in bne young of the species. (Hagan and Brunet, 1951) Bovine Papillomatosis Bovine papillomatosis frequently occurs in calves and young stock less than 2 years old. Very little is known about tne causative agent of“ bovine warts except that it is filterable. Greech (1929) inoculated eleven calves with uniiltered minced wart material and eleven with filtrates of the same material. sterile. fhe filtrates were bacteriologically Eight tumors were produced with the unfiltered material and seven with the filtered. fhe inoculations were made by scarification and intradermal injections. darts affecting animals usually regress spontaneously after some time. 'There are indications that animals showing regress­ ing tumors are thereafter resistant to this agent. Canine Papillomatosis In dogs, the warts begin around tne lips, as a rule, are smooth wnitish elevations, wnicn later develop a rough­ ened surface and appear as typical papillomas. McFadyean and Hobday (1898) showed that tne warts were infectious by rubbing pieces of tumors on the scarified mucous membranes of other dogs. DeMonbreun and Goodpasture (1932) were also able to prorjagate the tumors in this way. McFadyean and Hobday stated that the incubation period is from 28 to 9-2 days. DeMonbreun and Goodpasture found the incubation per­ iod to be about 30 t 0 32 days as a. rule, but was somewhat longer" in dogs showing malnurishment. The latter workers passed turnon? suspensions through Berkefeld N and W filters and found that the virus was present in abundance in the filtrates. DeMonbreun and Goodpasture were unsuccessful in pro­ ducing warts on the vaginal mucous membrane, on the mucous membrane of the conjunctiva, and on the skin of bhe abdomen, both with filtrates and with fresh unfiltered wart tissue. They also failed to infect the mouths of cats, rabbits, guinea pigs, and rats. Clinical experience indicates that dogs wnich recover­ ed from an attack of warts seldom ox'1 never are infected again. McPadyean and Hobday, DeMonbreun and Goodpasture found that it was impossible experimentally to re-infect dogs that had recovered. Papillomatosis of Babbits In 1933 Siiope showed that the common wart of bhe western wild cottontail rabbit was infectious and that the infectious agent'was a virus. Experimentally, the disease may be transmibted by inoculating scarified skin areas with filtered or unfilbered tumor pulp. these bumox's can be serially bransmicted to biie cottontail rabbit, Put not in domestic rabbits, dhope (1 9 3 3 ) was successful bo ini ec b bhis virus to bo bh cottontail and domestic x'abiaits. xious and Beard (1933) showed that, if the tumor-bearing domesticabed rabbits were kept fox1 long periods, considerable numbers of the benign papillomas become transformed into malignant carcinomas. Kidd and kous (1940), studying the matter furth­ er, showed that the same thing was true of such humors px'o— duced by inoculation the non-domesticated rabbits. They believed that in tne r*abblt species in which the virus is foreign, there is virus variation which leads to malignancy, by gradual change in morpnological characteristics, the malignant tumors arise from cells which are already neo­ plastic as a result of virus action. bhope (1 933 ) found that the virus was readily filterable through Berkefeld filters of all grades of porosity but usually not through oeitz filters. The first detectable evidence of epithelial proliferation is seen from the sixth bo the twelfth days, averaging 8 days, after inoculation. Tne filtrates often produce tumors after shorter incubation periods than those of bhe same suspensions unfiltered. This is interpreted as meaning that some inhibiting agent nad been removed by filtration. Inoculation of the scarified skin is the only way by which the disease can be transmit bed with a high degree of regularity. Subcutaneous, nitraciascular, intra— peritoneal, and intravenous inoculations usually fail to produce tumor's. Persons matosis of and hidd bhe rabbit. epithelium w h i c h were domestic rabbits (194-3) d e s c r i b e d an oral p a p i l l o ­ These found are b e n i g n g r o w t h s of in a h i g h p e r c e n t a g e in the h e w f o r k C i t y area. bhe of n o r m a l They are readily transmitted by filtrates to other domestic rabbits. Tne virus readily passed berkefeld V and N candles. The average incubation period when unfiltered virus is used is 15 days; with .berkefeld V filtrates, 19 days; and with berkefeld N filtrates, 23 days. one. The virus is a very stable Tissues stored in 30 per cent glycerol at 4°G. retain their pathogenicity undiminished for 2 years and more. Those scored in the frozen state remain potent for long periods, and material dried, while frozen, remains potent for many months. able . The resistance to heat is rather remark­ Heating at 65^d. for 30 minutes appeal’s to exert little injury. Intranuclear Inclusions were found by Parsons and Kidd in about 10 per cent of the tumors in domestic rabbits. ?/hen present, they were located in the outer six to ten layers of epithelial cells. They varied greatly in size and shape. Home were hyaline and others showed a striped structure. They were basophilic and located near the center of the nucleus, the chromatin being marginabed. Such bodies were not found in normal oral epithelium of domestic rabbits. Human Papilloma file and Kdngery (1919) and'Kingery (1921 ) discovered the viral etiology of warts by the production of verrucae on human skin by inoculation of a filtrate of wart sus— pension. This evidence was supported by the observations of Strauss and co-v/orker (1949, 1950). Melnick and co-worker (1952) took material from the papillomas in which the elementary bodies were observed. They also used control specimens of papillomas in which elementary bodies were not found. Both bhe papillomas in which tne elementary bodies were observed and the control specimens were grounded with alundum and distilled water, centrifuged at 2,000 r.p.m. for five minutes, and the re­ sulting supernatant fluid subjected to further centrifu­ gation at 6,000 r.p.m. for 45 minutes. The sediment was resuspended in a small volume of distilled water (about 1 ml.). For electron microscopy, a small drop was placed on a collodion screen and shadow-cast with chromium or palladium before examination. Although isolated particles may be found in all specimens, the particles ax’e arranged in crystalline-like clusters with such regularity that this arrangement appears- to be characteristic. The particles are spherical and, when in crystalline array, average 52 mu. in diameter, with a range of 50 to 54 mu. When these particles are not in crystalline array, they average 68 mu. in diameter with a range of 50 to 80 mu. The papilloma virus particles are morphologically stable when stored in distilled water at 4^0. for 10 rnonnhs, and they nave been recovered from tissue stored in the frozen state for one month. Preparations from other warts (without intranuclear inclusion bodies) and from normal Jdunian skin hs.ve revealed no uniform, particles, but only amorphous scattered clumps of matter, collagen fibers, and spherical particles of varying diameter. Infectious Myxomatosis Infectious myxomatosis of rabbits is a highly con­ tagious and almost always fatal disease of domesticated rabbits which was first recognized in South America, later in ivlexico and California. whole rabbitries. The disease often destroys It was first described in 1898 by Sana— relli wno ascribed the disease to a virps since he could not see or cultivate any organisms in the lesions. divers (192S) was the first to call attention to another character­ istic of these virus tumors, that is, a peculiar type of degeneration of the epithelial coverings. Ihe epithelial cells are greatly swollen and vacuolated, and acidophilic bodies rapidly develop in their cytoplasm. contain blue-staining coccoid elements. These bodies The whole struc­ ture resembles the Bollinger bodies of fowlpox. diver and Y/ard (1 9 5 7 ) found it possible to obtain suspensions of these elementary bodies, which they regard as the virus, in a relatively pure form. hot only are such suspensions highly pathogenic, but the bodies are specifically agglutinated by the serum of recovered or immunized animals. Virus can be obtained from the tumors, internal organs, blood, and the discharges from the body openings. It is filterable through Berkefeld filters. It is believed that the elementary bodies, mentioned above, actually constitute the virus. Shope Fibi^oma of Rabbits Shope (1932) described a type of fibrous tumor of the cottontail rabbit which proved to be transmissible to other* cottontail rabbits and to the domestic species by the injection of cellular suspensions and of Berkefeld filtrates. The tuiaor occurs subcutaneously in naturally infected cases. animal. There may be one or several in the same They are firm, spherical masses which can be moved about under the skin because they are only loosely attached. Sections show that the masses are made up of spindle-shaped, connective tissue cells, without evidence of inflammatory reaction. Filtrate of tumor tissue when injected into bhe tes­ ticles of raboits regularly cause the formation of sim­ ilar tumors. Subcutaneous and intramuscular inoculations frequently, but not always, succeed. intracerebral inoculations fail. in the tumors. Intraperitoneal and The virus is found only It has not been demonstrated in ine bloom, visceral organs, or any of the secretions. In sasceptible animals it stimulates a proliferation of the connective tissue at the point where it is deposited. There is no evidence of inf lamination or of necrosis in the lesions, fxie virus is readily filterable through Lerkefeld V and N filters. It remains viable in glycerol for long periods of time. The mode of natural transmission of the virus is not known. It is not transmitted from animal to animal by simple contact. Hyde and Gardner (1939) found that it was not ti'ansmitted from mother to young either through the placenta or through the milk. Shope (1938) showed that in domestic rabbits in which fibroma tumors had formed and retrogressed, reinfection did not occur. He also found that these rabbits also had a high degree of resistance to the virus of myxomatosis. Berry and .uedrick (1936) and Eexn?y (1937) indicated that fibroma virus xurobably is an attenuated form of myxoma. The virus of myxomatosis was heated bo 75°0., which completely inactivated it. Mixed with fibroma virus, however, the mixture produced typical myxomatosis when injected into rabbits, and this disease can be transmitted to other animals indefinitely. It was suggested that something in the heated myxoma virus had acted as a sort of hapten to lend greater virulence and malignancy to the fibroma virus. Hodgkin's Disease Gordon et a l . (193^h were the fix's b bo scarb a system— atic search for a virus in Hodgkin's disease. Before that cime, others had speculated on the possible etiological role of virus in tumors in general. Their significance in certain tumors from animals was well known. Based mainly on clinical, inductive and some experimental evi­ dence, several authors had considered that Hodgkin's disease might be caused by a virus. Since then, many in­ vestigators have searched fox* a viral agent in Hodgkin's disease. Gox'don (1934-) reported that tissue from Hodgkin's disease, when injected into rabbits, produces am encepha­ litis. At first, tne material whicn caused this response seemed to possess many viral characteristics. 'The lymph node extracts contained no bacteria; the agent was Seitzfilterable; it was inactivated by heat (80°G. for 30 min­ utes); and the encephalitis produced had an incubation period 01 from two to six days after inoculation. The encephalitis, however, could not be passed from rabbit to rabbit. friedman (1934-) demonstrated that the encepha­ litis agent could be isolated from certain normal tissues and has clearly demonstrated that the factor is not a virus, that it is doubtlessly an enzyme, and that ic is probably associated with the eosinophilic leucocyte. Bostick (194-8) reported an increased mortality in embryonated chicken eggs inoculated with Beitz-filoered extracts of Hodgkin's disease lymph nodes. Tne controls were normal tissues inocul3.ted into the embryonabed eggs, iiie xiodgkin's disease material was inoculated into the amniotic sac of 7— day incubated eggs and the lethal effect was recorded during the following ten days. Grand (194-4-) noted the formation of small vesicles on chicuen egg choi'io-allantoic membranes infected with supernatant fluid from xiodgkin’s disease tissue cultures. In 194-9 he found an abnormal amount of cellular degener­ ation and liquefaction in tissue cultures of Hodgkin's disease tissue. He prefers to interpret this as being evidence in favor of the presence of an injurious agent specific for Hodgkin's disease cultures. hotbino (194-9) also noted a .Liquefaction tendency that was greater in Hodgkin's disease than in other tumor tissue cultures. Although greater in Hodgkin's disease tissue explants, ne preferred to consider it probably nonspecific and re­ sulting from fibrinolytic enzymes liberated by the •many reticular cells in Hodgkin's disease. xfeiman et al. (1 950 ) studied in detail the effect of Hodgkin's disease tissue and normal serum on different types of normal, abnormal, xiodgkin’s disease a ad neoplas­ tic tissue cultures. I’ney concluded th/b Hodgain’s disease cells and serum give rise to abnormal cissue cultures. Ann n t h e _0 changes are increased fat granules in cells, 5 -— more liquefaction and numerous free cell forms, also, larger numbers of nuclei in cue giant cell formations and a decreased span of life of dodgkin's disease cells. Gordon (1934) was the first to propose that Hodgkin's disease tissues contain elementary bodies. Elementary and inclusion bodies were next described by Grand (1944, 1949 ). In tissue cultures, brilliant cresyl blue was used for staining inclusion bodies which were irregular in size and shape in the Hodgkin's disease lymphocytic cells and Reed-Eternberg cells. Similarly stained cultures of normal, inflamed and lymphosarcomatous lymph nodes did not snow such inclusions. In fixed tissues, Giemsa's and Seller's stains showed the inclusions in the Hodgkin's disease material and not in the controls. In cultures of normal lymph nodes, Grand noted that inclusions began to appear in the cells within 15 minutes after the addition of the supernatant fluid from a Hodgkin's disease tissue culture. Ey 24 hours, these inclusions were even more striking and resembled those found in Hodgkin's disease. Supernatant fluid from other tissues than Hodgkin's disease did not cause inclusions to develop in the normal lymph node cultures. Hoster et al. (1950) studied the macromolecular particles from various types of lymph nodes. They used a ten-step differential centrifugation procedure and then examined the particles under the electron micro­ scope. Lymph nodes that were normal or non—neoplaotic were compared with Hodgkin's disease lymph nodes and lymphosarcoma lymph nodes. They studied the frequency of che various sizes of particles in bliss© tissues. In Hodg kin's disease, the predominant particle size was 10 to 20 mu. which differed significantly from the particle sizes found in non-neoplastic tissues. Gordon (1937) sbudied a flocculation reaction with tne elementary bodies from Hodgkin's disease tissue and 18 anti-Hodgkin's disease antisena made by injection of rabbits. He obtained only variable results. Grand (1930) inoculated rabbits wibh purified sus­ pension of Hodgkin's disease lymph nodes. The nodes were prepared for inoculation by differential centrifugation. The final purified suspension was injected intravenously inbo normal rabbits for 9 to 10 days. Later, rabbit serum was collected and inactivated at 53 °0 . for 30 min­ utes. It was tested for bhe presence of agglutinins for purified Hodgkin's disease lymph node extract. The read­ ings were made under the darkfield microscope. Agglutin- abion of very small particles was nobed in the Hodgkin's disease material. Agglutination was absent in similar tests using normal, lymphosarcoma and leukemia lymph node extracts. Bostick (1950) employed as an antigen from amniotic fluid harvested from eggs in which filtered Hodgkin's disease extract had been serially passed. aoniplement- fixation properties were sbudied from two aspects. In the first instance, Hodgkin's disease amnio tic xluid was ased as the experiment;al antigen, and., as a control anti­ gen, non-Hodgkin's disease serially-passed amniotic fluid, under these conditions, 56 per cent of the Hodgkin's disease patient sera fixed complement with the Hodgkin's disease ainniotic fluid, whereas, only 55 per cent of the control patients' sera produced the same result. In the second instance, the Hodgkin's disease amniotic fluid was inoculated into rabbits. Their sera were later tested for complement-fixation properties by comparing the results oboained with normal amniotic fluid antisera. Hot all rabbit sera possessed complement-fixing properties. In some rabbit sera however, the ability to fix complement was consistently demonstrated when using as antigens the serially passed and filtered amniotic fluid derived from cases of Hodgkin's disease. Bostick (1950) passed serially (at least 4 passages) Seitz-filtered extracts of Hodgkin's disease lymph nodes in the amniotic sacs of embryonated chicken eggs. ‘ The harvested Hodgkin's disease amniotic fluid v/as tested for virus interference properties. This was done by inoculat­ ing Hodgkin's disease amniotic fluid into 7 day incubated chicken eggs, and after three more days of incubation, a challenging dose of influenza Lee virus was inoculated into the amniotic sac. After 18 hours additional incuba­ tion, the amniotic fluid v/as harvested separately from each egg and tested for che amount of Lee viruo present by means of the hemagglutination test. 'The amniotic fluid derived fxom Hodgkin* s disease showed intei‘f erence capacity on many occasions hut not on all. virus inhibition was complete. Sometimes, the influenza More often, it was simply decreased as compared to of carefully tested control mat­ erial. In tests with Hodgkin's disease amniotic fluid, 60 per cent showed an ability to interfere. There was also some degree of reversal, i.e. greater hemagglutinative titers in the Hodgkin's disease series than in the control. Lundback and Lofgren (1950) inoculated ground lymph node emulsions from Hodgkin's disease, Hodgkin's sarcoma, ana a lymphoma into the amniotic sac of '7 day incubated chicken eggs. Serial allantoic passages were maintained. The harvested allantoic fluids showed hemagglutination titers of from 2.51 to 5.11 (log units). did not react. Control material The hemagglutinating agent fixed complement with mumps antisera. In 1952 they found that the hemagglu— tinating agent was mump contamination. Finally Bostick (1952) concluded that Hodgkin's disease is caused by an infectious viral ageno, which it— self is undetectable by the usual methods. It mighu however, have a demonstrable effect on the growth of known viruses. Plant Tumours There are several virus diseases of plants in which leafy outgrowths from the veins are produced, such as Kroepek disease of tobacco, Smith's rosette of tobacco, and tobacco mosaic in Nicotiana paniculata or N. tomentosa, Fiji disease of sugar cane, Wallaby-ear of corn, Glubroot of tobacco and 7/ound-tumor disease. (Black, 19!?2) iv IA T E iil A.LS A liD METHO-DS Ill MATERIALS AHD METHODS The tumors used in this work were obtained from both animals and man. The tissues were removed at operation and held at refrigerator temperature for periods up to 24hours. Representative samples of each tissue was placed in fixative for histological sections, the remaining tissue was used for egg inoculations and tissue culture. The tumors and their origin are listed below Tumor I Fibroma from human ovary Tumor II Papillary adenocarcinoma from human rectum. Tumor III Scirrhous carcinoma from human mammary gland. Tumor TV Leiomyoma from human uterus. Tumor V Squamous cell carcinoma from human vulva Tumor VI Fibrosarcoma from human Tumor VII Serous papillary adeno­ carcinoma from human ovary. Tumor VIII Melanoma from canine soft palate. Tumor IX Sweat gland adenoma from canine subcutaneous tissue. Tumor X Adenocarcinoma from canine anal region. Tumor XI Squamous cell carcinoma from external canthus of the eye of cow. Tumor XII Pibrosarcoma from canine subcutaneous tissue. Tumor XIII Lyphomatosis from fowl. Tumor XIV Identify lost. Tumor XV Carcinoma of breast from human. Tumor XVI Osteochondro-ade.no-carctnoma from canine. Tumor XVII Adenocarcinoma from canine subcutaneous tissue. (1) uETRACENTrUPUGE C0NO E1TTHATIG H OE MA’ JDEEIAL POP: a. Tissue culture work. b. Hemagglutination reaction. c. Virus interference. In this work each tumor was washed bhree times with sterile physiological saline solution. Penicillin and streptomycin were added in concentration of 500 u. per ml. and 50 mg. xoer ml. respectively. After adding the anti­ biotics, tne turnor was kept in the refrigerator at least three hours. fragments of the tumor were used for tissue culture, the remaining tissue was then masserated by grind­ ing with 90 mesh alundum. Hank's solution was added to make 20 per cent suspension. The suspension was centri­ fuged in a refrigerated centrifuge (5 ° 0 .) for 20 minutes at 2,000 r.p.m. After centrifugation the supernatant fluid was removed and used to inoculate the allantoic sac (0.1 ml.) of 6 to 7 day old ambryonated chicken eggs. After Lv to 5 days incubation allantoic fluid was harvested from these eggs and stored in the deep freeze (—25 C.). Just before use the allantoic fluid was thawed and clarified by refrigerated centrifuge fox' 15 minutes at 5,000 r.p.m. The supernatant fluid was then concentrated one-third by volume by ultracentrifugation (Gpinco model E.) at 115,000 X gravity for* one hour. The concentrated allan­ toic fluid was inoculated into the allantoic sac of embryo— nated chicken eggs and constituted the second passage. H'ive such passages were made each time with concentrated material. Normal allantoic fluid from eleven-day old chick embryos, was used for the control and it was also concen­ trated one-third by volume by ultracentrifugation as mentioned a b o v e . (2) TISSUE CULTURE WOHK V. Normal Gxnwth of Tumor Tissue in Tissue C u l t u r e . Tissue culture consi.sts of removing tissues under sterile conditions from a living organism and incubating them in an environment (nutriment, conducive to growth. aeration, temperature) Variations in the technique have been developed in accordance with the type of cellular activity desired. The most successful methods deal with the multiplication of individual cells, and with the main­ tenance of cells in organized gixmps for a study of certain phases of their function. After the tumor was washed with physiological saline solution and refrigerated with antioiotics for three hours, it was then removed from the cold and cut into pieces about half to one mm. mesh. Pieces of tumor were embedded into the chicken plasma coagulum and liquid nutrient medium was added. Erleimeyer flask and slide culture teciiniqa.es ere used. Cbicken plasma was prepared by aseptically bleeding cnicken from tiie Heart. Heparin solution (1:2,000 in hysiological saline solution) in 0.4- mi. amount per 20 ml i blood was used as anticoagulant;. Tne blood v/as centri- uged at 2,000 r.p.m. for lp minutes and tne plasma v/as re owed by a capillary pipette. A sterility test v/as per- ormed by dropping a few drops of plasma into nutrient roth. Tbe plasma v/as kept in a frozen stare at -25°C. an as tnawed and clarified by refrigerated centrifuge at ,000 r.p.m. for 20 minutes for use. dextrose 5 gin. distilled IL^O added to 250 ml. Both stock solutions were sterilized by autoclaving at 10 lb. pressure for half an hour. Both stock solutions were stored at refrigerated temperature. The phenol red solution in4 per cent concentration was made by dissolving 4 gm. of phenol red in 1.3 ml. of B/2 NaOH solution and finally making up the volume to 100 ml. with distilled water. The indicator solution was ster­ ilized by autoclaving at 10 lb. pressure for half hour. Sodium bicarbonate solution, sterilized by Seitzfiltation, was a 1.4 per cent solution. Hank's solution (stock solutions A and B) was made up just prior to use as follows: Stock solution A 1.0 ml. Stock solution B 1.0 ml. Distilled Water 18.0 ml. Phenol Bed (4%') 0.02 ml. The mixture was autoclaved at 10 lb. pressure for 10 minutes. After the solution was cooled, the following solutions were added. Penicillin (500 U. per ml.) 0.2 ml. Streptomycin (50 mg. per ml.) 0.1 ml. About 0.4 ml. of NaHCO^ (1.4^) was added to adjust the pH to 7.4 to 7*8. Embryo tissue extract was prepax*ed by using elevenday old chicken embryos. embryos removed. The eggs were opened and the The eyes and legs were removed from the bodies which were then minced with a pair of scissors. The pieces of tissue were crushed by a pestle-and mortar. Fifty-per cent Hank’s solution was added as a diluent and the mixture was then stored over night in the refrigerator. The following day the suspension was centrifuged in a re­ frigerated centrifuge at 2,000 r.p.m. for JO minutes. The supernatant fluid was removed by caxaillary pipette and stored at -2J°C. Just prior to use the embryo tissue ex­ tract solution was thawed and clarified by centrifugation in the cold at 2,000 r.p.m. for 1J minutes. The liquid nutrient medium consisted of two parts of cnicken serum, two jjarts of Hank's solution and one part of embryo tissue extract. Method of Flask Culture 'Twenty—five ml. Erlenmeyer flasks were used in this work. In the pr'epax’ation of solid medium, l.J ml. of Hank’s solution and O.J ml. of cnicken plasma In the flask. 'were placed Then 0.2 ml. of embryo tissue extract v/as adued to produce coagulation. The flask v/as then allowed to stand on bhe flat surface. After the plasma was clotted, pieces of tumor tissue //ere embedded into tnis solid mediu.u with a sterile pointed needle. The flask v/as allowed to sband, "ben minubes and then one ml. of liquid nufcrient medium was added and the culture v/as incubated at 57 °C. 'The liquid nutrient medium was changed every 3 to 4 days by removing the old medium with capillary pipette. One ml. of Hank's solution was then added and allowed to stand for 10 minutes. This Hank's solubion was then re­ placed by 1 ml. of fresh'liquid nutrient medium. The growth of the tumor can be observed by an invert­ ed microscope eye piece or by dissecting microscope. If the gx’owth is luxurious a halo zone surrounding the tumor tissue can be seen by naked eyes. Slide Culture Method The slide culture method was the double coverslip method as described by Maximov/ (1925)• Briefly it consis­ ted of using a 75 by 45 mm. micro-concavity slide with a spherical concavity of 55 mm. in diameter and 5 mm. deep. Large Ho. 2 cover glasses (70 by 45 mm.) and small Ho. 1 (20 by 20mm.) cover glasses v/ere combined. The small and lax'ge cover slides were held bogether by placing a drop of distilled water on their intersurfaces. The culture was prepared on the small square cover glass by placing one drop of Hank's solution, one drop of cnicken plasma and one drop of embryo tissue extract in the middle of the small cover glass. The tumor tissue was placed upon the clotted medium and was embedded with a sterile poinbed needle. A drop of liquid nutrient medium was placed upon the culture. The coverslip bearing the culture was inverted and pressed down over the depression in the slide and was seal­ ed in place with paraffin and the culture was incubated at 37°C. The liquid medium was changed every 2 to 3 days by dip ping the small square coverslip with culture into Hank's solution and letting it stand about 10 minutes. The cultur was then taken out and put on the large micro cover glass. One drop of fresh .Liquid medium v/as dropped on to the cul­ ture. The culture was then inverted and pressed down over the depression in bhe slide and was sealed in p>lace with paraffin. By the cover slide method the growth of tumor tissue can be examined under the microscope and photomicro­ graphs made. B. The Effect of Tumor Passage Allantoic Fluid on the Growth In Vitro of Normal Tissue. Normal chicken heart tissue from one day old chicks was used for this experiment. The heart was removed aseptically from the chick and the blood was washed off with sterile physiological saline solution and finally stored in Hank's solution in the refrigerator for further use. Carrel c flask and slide culture methods were used for the cultivation of bhe heart tissue. After a few experiments, the slide culture method was omitted in favor of the Carrel flask method. The flask technique for the cultivation of tissues in plasma depended on the prepara­ tion of two-phase system within the flask, one being a Ijermeable, and semi-solid plasma coagulum in which the tissues were embedded, the other, a fluid phase that may be introduced and removed at intervals of 3 bo 4- days. The semi-solid plasma coagulum was made by placing 1.3 ml. of Hank's solution, 0.5 ml. of normal chicken plasma and 0.2 ml. of embryo tissue extract into the flask. The flask was shaken to mix the content and then bhe flask was allowed to stand on a flat plane for the clotting of the jjlasma. The plasma obtained from normal chickens was found to have diffextent degrees of growth stimulating abilities for che normal tissue. Therefore chickens was pooled before use. the plasma from several This pooled plasmas was used on all tissue in this experiment so that conditions remained identical. Three pieces of heart tissue from a one-day old chick were embedded in the plasma coagulum one cm. agart. tested. Une flask was made for each tumor to be The liquid-phase consisted of one part of the concentrated passage allantoic fluid and one part liquid nutrient medium. The nutrient medium consisted of two parts of Hank’s solution, two parts of chicken serum and one part of embryo tissue extract. The liquid-phase for the control consisted of one part of the concentrated normal allantoic fluid and one part of liquid nutrient medium. The growth of the fibroblasts from the heart tissue was observed daily under the microscope. The eye piece of the microscope was fitted with a disc with 5 mm. squares enscribed upon it to record the area of growth occuring in each piece of tissue. The counting was done using a dis­ secting microscope with 15 X magnifications. The growth index (Ludford and Barlow, 194-4) of each piece of tissues v/as calculated as follows: The growth index = Total area of culture - Area of original e x p l a n t _______ Area of original explant• (3) HEMAGGLUTINATION REACTION AFTER PASSAGE OF TUMOR MATERIAL IN CHICKEN EMBRYOS. A. Tested with. Normal Hemagglutination, for detecting viruses, Chicken Red Cells the Hirst phenomenon (194-1) as used was used in this work in an attempt to identify any agent recovered from egg passage of tumor material. Buffered saline solution was prepared by the method of Mheeler, Luhby and Scholl (1950). One volume of M/15 phosphate ouffer solution (pH 7*5) was added to nine vol­ umes of 0.85 per cent sodium chloride solution. M/15 pnosphate buffer solution pH 7*3? was made by adding 7-86 ml. M/15 Na 2 HP 0 ^ solution to 2.55 ml. M/15 KH^PO^ solution. This buffered saline solution was used in all tests des­ cribed . The concentrated passage allantoic fluid of each tumor and each passage was tested for hemagglutinative properties, with 0.5 per cent suspension of normal chicken red cells. The red cells were washed three times with buffered saline solution then 0.5 per* cent suspension was made for the test. The passage allantoic fluid was diluted serially in two—fold dilution with physiological saline solution. Twenty—five hundredths ml. of each dilution and 0.25 ml. of physiological saline solution were placed into a tube (12 by 75 mm.) and then 0.25 ml. of 0.5 per cent suspension of cnicken red cells was added. Tne tubes were shaken to mix the content. the test was read at 1 5 , 30 and 4-5 minutes intervals try examining the patbern of cells formed on bhe botbom of the tubes. Concentrated normal allantoic fluid was tested in bhe same way and served as a control. i'he red cells from one-day old chicks were also used but no significanb differences in hemagglutinating properties was found in che different ages of chickens. * Tes bed with Chicken Red. Cells Sensitized with New- castle .Disease Viruses. Certain viruses and Vibrio cliolerae filtrates were found by Burnet, McCrae, and otone (1946) to modify the virus hemagglutination of human red cells. Normal chicken red cells 'were washed one with buffered saline solution and made up to a 10 per cent suspension. The source of the Newcasble disease virus was allantoic fluid. The virus was used for sensitizing bhe chicken erythrocytes. Two parts of 10 per cent suspension of chicken erythro­ cytes were added to one part (having a hemagglutination citex1 of 1:320) of Newcastle disease virus. This mixture was kept in the refrigerator (4-°C.) for one hour. „'fter refrigeration, the red cells were washed by cenbrifugation in the refrigerated centrifuge at 1,300 r.p.m. for 15 minutes. The supernatant fluid was discarded. The cells were resuspensed in buffered saline solution making a 10 per cent suspension by volume. The red cells were bhen washed until the supernatant fluid failed to agglu­ tinate a 0.5 per cent suspension o± chicken red cells, finally a 10 per cent suspension, by volume, was mane inbuffered saline solubion and the suspension inc-ubaued at 37^0. in a water bath for three hours. elubed the virus from the red cells. Tne incuoabion Afber incubabion the red cells were wasned by centrifugation in bhe ordinary centrifuge at 1,500 r.p.m. for 15 minutes. The red cells suspension was washed until the supernatant fluid failed to agglutinate a 0.5 per cent normal chicken erythrocytes suspension. The sensitized chicken red cells jere then made to a 0.5 per cent saline suspension and were ready for use. The passage allantoic fluid from tumors was diluted serially in two-fold dilution with physiological saline solution. 'Twenty-five hundredths ml. of each dilution and 0.25 -ml. of physiological saline solution were placed into a tube (12 by 75 mm.) and then 0.25 ml. of 0.5 per cent suspension of sensitized chicken red cells was added. The cubes were shaken to mix the content. The test was read at 15, 50 and 4-5 minutes intervals by examining the pattern of cells formed on the boctom of the tubes. Con­ centrated normal allantoic fluid was tested in the same way and served as a control. C. Tested with Chicken red Cells Modified with Trypsin. Morton and Picket (1949) found that red cells treated with, crude and crystalline trypsin solutions increased tneir sensitivity to the incomplete anti—hh antibody. Therefore trypsin modified erythrocytes were used with the bumor passage allantoic fluid. Thole chicken blood was washed with buffered saline solution. jline volumes of four pel" cent red cells suspension were added to one vol­ ume of one per cent crude trypsin (Difco 1:250 brand) in pnysiological saline solution. The mixture was incubated at 57°C. in a water bath for 10 minubes. The cells were centrifuged by refrigerated centrifuge 1,500 r.p.m. for 15 minutes and washed once with physiological saline solution. A 0.5 per cent red cells suspension was used for the test. The passage allantoic fluid from tumors were diluted serially in two-fold dilution with physiological saline solution. Twenty-five hundredths ml. of each dilution and 0.25 ml. of physiological saline solution were placed into a tube (12 by 75 mm.) and then 0.25 ml. of 0.5 por cent suspension of modified chicren red cells was added. tubes were shaken to mix the conbent. The The best was read at 1 5 , 50 and 45 minute intervals by examining the pattern of cells formed on the bottom of the tubes. Concenbrabed normal allantoic fluid was test-ed in bhe same way and served as a control. (4) TESTS FOE INTERFERENCE BETWEEN TUMOH PASSAGE ALLANTOIC FLUID FOLLOWED BY NEWCASTLE DISEASE VIAUS: In Vivo . It has been established by many workers, Price (1940) Vilches and Hirst (1947), Ginsberg and Horsfall (1949) that in certain circumstances the presence of one virus will interfere with the growth of a subsequently inoculat­ ed one. Although the usual laboratory test may not indi­ cate the passage of an agent in the allantoic fluid, an • in vivo test may show a difference between passage allan­ toic fluid and normal allantoic fluid. Concentrated passage allantoic fluid from tumors was first inoculated into 6 to 7 eggs. old embryonaled chicken This was followed in 3 to 4 days by sin injection of 0.05 nil* of Newcastle disease virus. given into the allantoic cavity. Both injections were The Newcastle disease virus used was adjusted so that a hemagglutination unit titer of 1:40 was contained in 0.05 ml. Twenty-four hours following the virus injection, the allantoic fluid was har­ vested. This fluid was then titrated by using the hem­ agglutination test and the titer of the tumor passage allantoic fluid was compared to the titer obtained in the controls. The controls consisted of concentrated normal allantoic fluid which was injected into embryonated eggs prior to Newcastle disease virus. B. In V i t r o . The purpose of tnis part was bo try bo demons bra be interference by concentrated passage allantoic fluid from rumors with, the absorption of Newcastle disease virus onto •guinea-pig erythrocytes. The guinea-pig erythrocyte was found to absorb Newcastle disease virus similarly to chicken red cell however, it was eluted very slowly when compared to the chicken red cell. Guinea-pig red cells were washed once with buffered saline solution. One part of concentrabed passage allan­ toic fluiu from tumors was added to two parts of 10 per cent buffered saline suspension of guinea-pig erythrocytes, fse mixture was held in the refrigerator (4-°C.) for one hour. After refrigeration the mixture was centrifuged in a refrigerated centrifuge at 1,500 r.p.m. for 15 minutes and the supernatant fluid was removed and discarded. The treated red cells 'were resuspeused to make a 10 per cent suspension in buffered saline solution. 'Two parts of tills suspension 'were added to one part of Newcastle disease virus (titer 1:1280). fne mixture was held in the refrigerator (4°G.) for one hour. cells were recentrifuged. After refrigeration the The supernatant fluid was then bested for the presence of Nev/castle disease virus by the regular hemagglutination reaction. The Newcastle disease virus hemagglutination titer in the supernatant fluid was then compared bo the control. Concentrated normal allantoic fluid and untreated red cells served as controls. IJLTS IV RESULTS Tissue Culture Work A. Normal Growth. of Tumor Tissue. i\line of the tissues from neoplasms grew well (Tumor I, Tumor II, Tumor V, Tumor VIII, Tumor IX, Tumor X, Tumor XI, Tumor XIII, and Tumor XVIII) and were maintained for several months in vi bro. Figures 1 through 4- show the tissue culture growth of these tumors. completely to grow. Eight tumors failed There was no correlation when comparing the type of neoplasm or its ability to grow in vitro. Cultivation of the tumor tissue was more often successful when the tumor tissue could be obtained immediately after removal, however, some tissues were capable of growth after periods of storage at refrigeration temperature (4-°C.). L. The Effect of Tumor Passage Allantoic Fluid on the Growth (in vitro) of Normal Tissue. When normal chicken heart was grown in tissue culture the rate of growth of the tissue could be estimated by making daily counbs of the area of growth. When using normal he art tissue and growing this tissue in a medium containing ulbracentrifuged concentrated tumor passage allantoic fluid, the results obtained varied a great deal. Table I gives the average growth index with the 1? tumors studied. Graphs I through IV show a composition of these average growth indexes. It may be seen from Graph ± ohat concentrated allantoic material, after five egg passages was capable of stimulating the growth of normal heart tissue culture cells. i’he growth index of Tumor II was nearly twice that of the control culture of cells. Allantoic fluid poassage material from tumors III and XII also gave growth indexes greater than the control cul­ ture however, the indexes are so close to that of the control that it is doubtful if they could be considered significant. The majority of allantoic fluid material adued to the normal tissue produced a retardation upon the growth of the tissue. fro m Lower than average gx’owth indexes were obtained egg passage allantoic fluid from tumors I, IV, VI, VII, VIII, IX, X, XIII, XIV. Figure 5 shows a photograph of the normal chicken heart to -which ultracentrifuge concentrated normal allan­ toic fluid has been incorporated into the medium. When comparing figure 5 and figures 6 and 7} it can be seen that a great deal of variation exists. Figure 6 illustrate normal chicken heart culture to wiiich passage allantoic .fluid from tumor II was used as nutrient in the medium. This tumor material gave the greatest average growth index (Graph I). Figure 7 illustrates the results obtained when passage allantoic fluid from tumor XVI was used in the nutrient medium. Figures 5 j 5 and 7 were photographed at the same magnification to better illustrate these results. 53 Table I Average Growth. Index of One Day Old Chicken Heart In Tissue Culture When Tumor Passage Allantoic Fluid Were Added • ——* Time In Days 4 3 6 Tumors 1 T .67 3.55 3-73 4.62 II .81 8.36 16.62 24.64 33-45 37.29 40.36 1.05 8.00 15.9 18.2 22.4 23.9 24.4 ITT 2 7.31 5 ... 5.41 7.66 5.31 7 6.45 8.48 8.38 22.58 23.23 7.44 7.49 3.1 5.85 1.53 6.45 11.44 16.2 VI .77 3.99 5.44 5.8 VII .8 4.4 9.8 12.9 17.2 19.7 19.6 VIII •75 6 .07 9.18 11.76 14.36 16 .8 17.62 IX .91 2.8 3.73 5-01 5-79 5.57 5.26 1.25 7.0 9.25 10.41 12.25 13.33 14.91 4.0 5.33 10 .6 16 .65 20.5 25.0 18.0 23 .06 25.6 28.98 14.5 TV •53 \r X 19.0 6.83 XI 0 XT! .88 7.91 10.58 XITI .98 3.08 7.15 8.34 9.97 11.87 XIV .5 2.1 1.76 1.3 1.5 3*6 9.26 v>r 1.05 3.61 3.30 6.33 6.54 6.87 10.20 AVI .44 3 .14 1.75 2.18 2.29 2.55 4.11 4.66 .38 lorma}. Concentrated. ■'-llantoic 5.28 •t'Tn.id .92 (Control) 5.89 5.88 6.0 7.11 8.17 10.04 16.05 20.78 24.39 25.61 IVII FIGUiSES 1 AND 2 . Figure 1 Tumor-I in tissue culture, 23 days old (Magnification about 1200X) Figure 2 . Tumor-II in tissue culture, 20 days old (Magnification about 1200X) 'r Figure 2. 55 / FIGUiiES 3 AND 4 5/ Figure :5. Figure 4. AI GOT ‘III ‘II ‘I SHrilF-D T- II Control Growth. InJ T-in T-V Grapli I 4-0 Lh. IruUs 30 H4- 10 Grapli II T - XII H Control -cs T-'XI si T - X III t - xrv 40 30 AO T-xvir T-XV T-XVI FIGUxiAS 5, 6, AND 7 Figure 5* Growth of formal Chicken Heart in 7 day old Tissue Culture to which Normal Concentrated Allantoic Fluid ( Control ) was added. Mag. 14-0 X. Figure 6. Growth o±* x^ormal Chicken Heart 7 d.ay old. Tissue Culture to which Concentrated Tumor Passage Allantoic Fluid fx'om Tumor-II was added. Mag. 140 X. Figure 7* Growth of I'lormal Cnicken Heart 7 day old Tissue Culture to which Concentrated Tumor Passage Allantoic Fluid from TumorXVI was added. Mag. 14-OX. Hemagglutination Isaction after r'assags of 'Timor us serial. Tested with Normal Chicken ned Ceils. Tnen a henagglutinacion test was made on allantoic fluid after the firs a passage, several produced hemagglutin­ ation, especially when the undiluted allantoic fluid was used. most of the tumor passage material nowever, failed to produce hemagglutination and 7.’as consistently negative. Tables II through VI give tne results of testing all tumor ■passage allantoic fluid through the five serial egg passages Tumor sassage maoerial from tumor Id (Table IV) gave hopeful results after the firs a egg passage in thee tne unuiluted fluid and a 1:5 dilution were capable of agglutinating normal chicken erythrocytes. gave a doubtful reaction. A 1:10 dilution of t m s maseri A second passage in eggs with uloracentrifugaoion coneentrated material from tne first pass a e , also gave doubtxul results mien one m h i d iantoic fluid. jso . <=>.1— Occasionally, nenaggluoination ..'as observed during tne serial passage, however the reaction was never stroiia and was not consistent in one i oil owing egg passage. Table II emag^lutination Tests with Uormal Chicken Erythrocytes ]-] Amo rs Pas­ sages Undi­ Tumor Passage Allantoic Pluid Dilution luted 1/5 1/10 1/20 1/40 1/80 Control 1 T 2 3 4 5 II 1 2 3 4 5 :n 1 2 3 4 3 IV 1 2 3 4 3 4" strong hemagglutination no hemagglutination 4- weeak h e m a g glutinati o n Table III Hemagglutination Tests 7/ith Normal Chicken Erythrocytes Amors Pas­ sages Undi­ luted. Tumor Passage Allantoic Fluid Dilution 1/10 1/5 V 71 VII 1 — — — — 2 - — — — 3 - — — 4 + 5 + 1 — 2 1/40 1/80 Control — - — _ _ — — — — — — — — — — - - - _ _ — — — — - — - - 3 - - - - - - — 4 — - — - - — - 5 — — — - - — - 1 — — - — •— — — - - — — - — — - — — — — — - — — 2 X 4 + - + ■ 4* + 5 VIII 1/20 l 4* 4~ + 58 Table XV Hemagglutination Tests With Normal Chicken Erythrocytes iiors Passages Undiluted Tumor Passage Allantoic Pluid Dilution 1/5 ix. l ± - 3 - - - - - - 2 - - 3 - - 4 - - 5 _ _ . 3 x xi l. l - - 2 - “ 5 “ - 4 5 XII i 2 4 1 2 5 4 1/10 + - - 1/20 1/40 1/80 Control 69 Table V Hemagglutination Tests 7/ith. Normal Cnicken Erythrocytes 'Limors Pas­ sages Undi­ luted Tumor Passage Allantoic Fluid Dilution 1/5 11 I 1 1/10 1/20 1/40 1/80 Control + 2 — — — — — — _ _ _ — _ _ _ — - - - - _ _ - - _ — - — _ _ — - _ _ 3 4 5 XIV 1 2 XV — + 3 — - — 4 - - — 5 - 1 - 2 + - — — - — — — — — — - — — _ — - 3 — - — 4 — — - — — — Table VI Hemagglutination Tests .Yitii Formal Chicken Erythrocytes P-'S Passage s Undi­ luted Tumor Passage illantoic Fluid Dilution 1/5 1/10 1/20 1/40 1/80 Control B. Tested with. Chicken fied Cells Sensitized with. New­ castle disease virus. When Newcastle disease virus was used to sensibize the chicken red cell prior to addition of uitracentrifuge con­ centrated tumor passage allantoic fluid, the results gave increased hemagglutination activity. Here, as with normal unsensitized cells the hemagglubination reaction was strong­ er with undiluted tumor passage allantoic fluid. The re­ sults of besting the 17 tumors with allantoic fluid from the 7th serial passages is shown in 'Tables VII through X I. Strong reactions were obtained when passage ma berial from tumor VIII was tested for hemagglu bination. Although the second passage allantoic fluid gave negative results lor hemagglutination, the 3rd, 4th and 7th passages produced strong reactions. The 3rd and 4th passages were posibive when tested undiluted and in 1:5 dilution. However, by the 5th passage only the undiluted material was capaole of producing hemagglutination. 72 Table VII biiagglutination Tests With Newcastle Disease Virus Sensitized Chicken Erythrocytes mors Pas­ sages * Tumor Passage illantoic Fluid Dilution 1/5 1/10 1/20 1/40 1/80 Control o o o o o o 1 o 2 o o o o o o o 3 o o o o o o o 4 o o o o 0 0 o 5 — - — - - — - 1 o o o o o o o 2 o 0 o o o o o 3 o o 0 0 o o o UL o + 0 o o o o o - - - - - — 1 o o o 0 o o o 2 o o o o o o o 3 o o o o o o o 4 o o o o o o 0 5 + — — — — — — 1 — - - - - - — 2 — - — — - — — — - - - — 4 + — + — 3 o o o 5 + . strong hemagglutination — = no hemagglutination o 0 o o I II 5 III IV Undi­ luted. ± - performed, 73 Table VIII hemagglutination Tests With. Newcastle Disease Virus Sensitized Chicken Erytnrocytes Tumors V vI VII VIII Pas­ sages Undi­ luted Tumor passage Allantoic Pluid Dilution 1/5 1./10 1/20 1/40 1/80 Control 1 o 0 o o o o o 2 o o o o o o o 3 o o 0 o o o o 4 + — - — — — - 5 o o o o o o o 1 o o o o o o o 2 o o o o o 0 o 3 - - — — — — — 4 o o o o o o o 5 - - - - — — — 1 — - - - - — — 2 — — - - — — — 3 — - - - - — 4 i— 2 — - — — — — — — - - — - — — 1 o 0 o o o o o 2 — — - - - — 3 + + — + + — — - — - — — 4 3 + — - — — ■ “ 74Table. IX :>a;•••1u bin at n on Tests With Newcastle .Disease Virus Sensitized Chicken Erythrocytes amors PasUndi- Tumor Passage Allantoic 'Fluid Dilution __ ___ sages luted__________ ________ ______________ ________ ________________________ 1/5 1/10 1/20__1/AO__1/80__ Control 1 o o o o o o o 2 + - — — — — — 3 + — - - — - - A + + — - — — - — - - — — — + — - - - - - a - — - - - - 3 + - - - - - — A + + + + — — - — — — + + — - - — + — + - — - — + — - — - - — + — - - - — — + - — - — — — + + - - — — — + - - - — — — — ““ - - — - - — 5 1 2 3 1 2 3 A 3 1 2 + ■y + + + + - - _ 0 A 5 - .. — — 75 Table X i.eaag lutination Tests With. Newcastle Disease Virus .Sensitized Chicken Erythrocytes !uaors PasUndi- Tumor Passage Allantoic l'Tuid Dilution _______sages luted_________________________________________ ______________ 1/5 1/10 1/20 1/40 1/80 Control + + 1 — 2 + — - - — — — 5 - — - - - - - 4 - - - - - — 5 + — — — — - — 1 — - _ - - - - 2 — + - - - - - — — — - - - - - _ - — - — - — - — - — - ■- - - - 5 4 5 1 + — 2 + - - - - - - 3 — — — - - - - 4 — - - - - — — 5 — — — — — — - 1 _ - - - + — + — 2 — + - - - — 3 — — - — - — — 4 - - — - - - — _ — - - - - 5 76 Table XI emagglutination Tests with Newcastle Disease Virus .Sensitised Chicken bry.bhrocytes amors Passages X V II Formal Doric encrated Allantoic fluid i Undi- Tumor Passage Allantoic Fluid Dilution l u t e d ______________________ ___________ ___ 1/6__1/10__1/20 1/40__1/30 Control - - _ _ _ _ _ - 9 Table XII Hemagglutination Tests Vith Trypsin Modified Chicken Erythrocytes mol's T Pas­ sages Undi­ luted Tumor Passag e illantioc Fluid Dilution 1/5 1/10 1/20 1/40 1/80 Control 1 o * o o o o o o 2 o o o o o 0 o 3 o o o o o o o 4 o o o o o o o 1 o o o o o o o 2 o o o o o o o 3 o o o o o o o 4 o o o o o o o 3 - — - — - - — 1 o o o o o o o 2 o o o o o o o 3 o o o 0 o o o 4 o o o o o o o 5 + + - — - — - - - - - - — — 5 II III IV 1 - — - - - — 3 - — — — - — 4 - - — - — — o o o 5 * _ = strong hemagglutination = no hemagglutination o o o I II +1o '2 * o weak hemaggl'utination The test was not perform 79 Table XIII Hemagglutination Tests With. Trypsin Modified Chicken Erythrocytes tlUIOX'S V VI VII VIII Pas­ sages Undi­ luted Tumor Passage Allantoic Fluid Dilution 1/3 1/10 1 0 0 0 2 0 0 3 0 4- 1/20 1/4-0 1/80 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + — - - — — 5 0 0 0 0 0 0 c 1 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 + — - — — - - 4- 0 0 0 0 0 0 0 5 — - - — - — - 1 — - - - - — - 2 - — - - - - — 3 — — — - - - — 4- + + 5 — — - - — — - 1 0 0 0 0 0 O O — — — - — — _ 2 — — — - — — —■ 3 4- - — - — — —• 3 + + + + - , + — Table XIV Hemagglutination Tests With. Trypsin Modified Chicken Erythrocytes Tumors IX Pas­ sages 1/10 1/20 1/40 1/80 Control o o o o o 0 2 + + — — — - — — — — — — — — 4 + - — — — — — 5 4- + + — - — - + - — - — — — 2 + + + - — - - 3 + + + + — - - 4 + + -U — + — - - - - — - — + - - - - 1 . 1 + — — 2 + + + + 3 + + + + + + - — — + - - - - — — — - - — + + - — - — — 2 + + + + — — 3 + + + + + - — — — - — — — - — - - — 4 5 XII 1/3 o 3 XI Tumor Pass age Allantoic Fluid dilution 1 3 X Undi­ luted 1 4 5 + — — Table XV Hemagglutination Tests With Trypsin Modified Chicken Erythrocytes rumors Pas­ sages Undiluted Tumor Passa ge Allantoic fluid Dilution 1/5 XIII + 1/10 — - + — — 4 + - - 5 + - - 1 — - — - — 1 2 1/20 _ 1/40 1/80 Control _ _ _ — — — _ _ - _ _ _ - _ _ - — _ _ - — _ _ _ - - — _ — — — _ — - - — -7 3 VTV 2 + + 3 4 XVI - — — + — — — — — 1 — — — 2 - — — - — — - 3 — - — 4 + - - - — — ~ + — — — 5 — — — — — — _ — 1 — - — — — - ~ — - — — - — “ — + - — — — ■* ~ 5 XV — 2 3 4 + ft; Table Xv'I iieniagglii'cina'feion lesbs Xiun Trypsin '•.lodif’ied Jbacicen Irytaro cyt es Pas­ sages ' /.0ITS Jndi- Tumor Passage Allan aoic Tuuid ^ilueian luted 1/ft — H :r~rT T 1/10 — 1/20 — l/“0 - 1/30 Con.~rol ~ - C\. ft + .iornai 3 one en~rS. CL .--l_ajricoic fluid m Tests for Interference between Tumor Passage Allantoic Fluid. Followed by Newcastle disease virus in vivo. A. When the ultracentrifuged concentrated tumor allantoic fluid was injected into embryonated eggs and fol­ lowed by Newcastle disease virus, there was no indication that any interference had occured. Table XVII gives results of two—fold dilution titration of the Newcastle disease virus found in eggs that had previously been in­ jected with tumor passage allantoic fluid and finally Newcastle disease virus. Slight differences of one dilution tube occured but are not significant. Tests on Interference between Tumor Passage Allantoic Fluid followed by Newcastle disease virus in vitro. B. Table XVIl'I gives the results of tests done to de­ termine if there was any interference between the absorption of tumor passage allantoic fluid and Newcastle disease virus upon the washed guinea pig red cell. as can be seen by the results, concentrated tumor passage ailanooic fluid or concentrated normal allantoic fluid botn reduced uhe original titer of the original Newcastle disease virus. There was no difference, however, between tue tumor passage fluid and that obtained by normal concentrated allantoic fluid. 'Table XVII Tests For: Interference Between Tumor Passage allantoic Fluid Followed By Newcastle Disease Virus, In Vivo. 'luors Undi­ luted Dilution Titer'S of Newca.stle J)iseass Virus 1/5 1/10 1/20 l A o 1/80 1/160 1/320 1/640 1/1280 Control I + * 4 4 + 4 * 4 II 4 4 4 4 4 4 7TT 4 4 4 + 4 4 4 IV 4 4 4 4 4 4 4 V 4 4 4 + 4 4 VI 4 + 4 + 4 4 4 VII 4 4 4 + 4 4 4 VIII + 4 4 + 4 4 IX 4 4 4 4 4 a + 4 4 4 4 4 XI 4 4 4 + 4 4 XII 4 4 4 + 4 4 4 XIII 4 4 4 4 4 4 * 4 4 XIV 4 4 4 4* 4 4 4 XV 4 4 4 4 4 4 XVI + 4 4 4 4 4 XVII 4 4 4 4 4 4 4 4 4 a - 4 4 4 Normal — — — — 4 - - - — - - - - - - — - - - - 4 _ _ - 4 4 — - 4 - - - - - - - — - - - - _ _ - 4 _ - - 4 _ 4 - - - 4 4 4 4 — — 4 4 _ - — 4 _ - 4 _ - 4 4 4 4 - 4 4 (Control) = strong - = no nemagglutinafcion 4 - Concentrated allantoic fluid 4 4 ': T r r / v r-\ l r * hoim o • 1it44 nation. Table XVIII Tests lor: Interference Between Tumor Passage illantoic fluid followed by Newcastle Disease lirus » In Vi bro. Tamers Undi­ luted Dilution Titers of Newcastle Disease Virus 1/5 1/10 1/20 1/40 1/80 1/160> 1/320 1/640 1/1280 Control + + + — + + — 4+ — — 4* + — — — + + + + 4* — + . + — — — — 4“ + . 1 . + * II + III + IV + 7 + 4* + + 4- ii + + + + 4* in -r + -t- + VIII T r. j.~ fi + 4- + + 4“ + — + + + + 4" Pi. + 4" + + 4* fvJL + 4* + + -A.-LI + 4 - + + 44* A.III + 4- + + -A.J.I + 4“ + + jLV + 4 - + -r XVI + 4- + + + + J4IV11 Normal -Cone entrated .dlanfcoic + +• + fluid + (Control) Newc astle Disease Virus (orig inal') 44+' + = no hemagglutination S-oo - - — - - _ - — - - — _ _ - — — — — - — — + - — - - — - — - - — - - - - - - — — — — - — + — - _ _ - — — + — — _ _ - — — - — — - - 4 ~ — _ _ — — 4* + + - — - - 4* — - _ ~ - - + -t- 4 - — + T -r t U ^r r tr ->rr>cl11f*4U .CJ1linn — * DISCUSSION V 'DISCUSSION The experiments described here were primarily designed to tesc the theories of Bostick (1952) and Londback and Lofgren (1950) ? who had descx*ibed agents isolated from tumors or neoplasms by embryon&ted chicken egg passage. However, the methods used in this work were sucn that any agent capable of multiplication or existing in an extract from tumor tissue, was given greater opportunity to do so. for instance, if the total allantoic fluid from an embryonated chicken's egg contained one infectious or hemagglutinating unit the material used for a second- passage into embryonated eggs would therefore contain less than one unit, the inoculum (0.1 mm.) being only a fractional amount of the total allantoic fluid contained in an egg. To concentrate all possible infectious material contained in the allantoic fluid, a pool of allantoic fluid from each passage was made in the ultracentrif'uge. This concentrated allantoic fluid, then, constituted the inoculum for the next embryon­ ated egg passage. It was essential that a more sensitive indicator system be used to test the presence of hemag^lucinj.oing agents since normal chicken erythrocytes failed to snow agglutination in the preliminary tumor passage .,-.11ancon c fluid material. Therefore, normal cnicken red cells were sensitized by contact with. Newcastle disease virus op modified with, trypsin prior to use in the hemagglutination tests. This has been shown to enhance their reaction in the presence of small amounts of viral agents. Attempts were made to use the interference phenomenon co detect any agent contained in the material used for egg passage. Newcastle disease virus was used primarily be­ cause of the work of Lundback and Lofgren (1950) in v/hich they showed that this virus was similar to the agent that they had isolabed from tumors. ultracentrifuged, concen­ trated tumor passage allantoic fluid was injected into embryonated eggs to be followed by Newcastle disease virus. Neoplastic tissues were shov/n to be capable of growth in vitro. Nine cultures of these tumors could be maintained almost indefinitely; eight failed to grow. Although eight of the 17 tumors failed to grow in tissue culture, they were not excluded from the serial egg passages. It was felt that failure to grow in tissue culture did not ex­ clude the presence of an agent, nor did it prove thab the neoplastic cells were dead cells. Therefore, all tumors received at the laboratory were subjected che same experi­ mental process. When the c o n c e n t r a t e d tumor passage allantoic fluid was incorporated into the nutrient medium of normal chicken heart cultures, the effect was a retardation of the growth of the normal tissue. However, one outstanding exception to this retardation, (Graph I) was found in allantoic fluid originally from tumor II. was from human rectum. This neoplasm Therefore, there' seems to he doubt as to the origin of bhe agent since fecal virus might well have contaminated the tissue. On the other hand, there must be some reason for the marked stimulation given to the normal heart tissue by the concentrate of allantoic passage material from this tumor. This same allantoic passage material did not produce a hemagglutin­ ation reaction (Table II, VII, XII) wnen tested with normal or altered indicator cells. Even when considering the variation in growth area with normal cells, the effect produced in this one instance does not possibly seem to be due to chance alone. The results obtained in the various experiments with normal and altered red cells in the presence of concen­ trated tumor allantoic fluid have one thing in common. It was frequently found that hemagglutination occurred when the undiluted concentrated fluid was used. This was especially true when sensitized or modified red cells were used in the hemagglutination reaccion as illustrated in tables IX and XIV. Occasionally a hemagglutination re­ action was found occurring in the concentrated material which could be diluted 1 to 5 and still produce ex. p-*.e— ceptible reaction, when controls were negative (Tumor ^.11, Table IX). These results were never found bo persist throughout the five serial passages in the egg. Ihe strongest hemagglutination reactions were per­ sistently found when trypsin .modified red cells were used with the allantoic passage fluid. quite frequently, strong reactions were found in 1:40 dilutions of the allantoic fluid. is is shown in table XIV, tumor XI and XII. This was to be expected since trypsin modified cells are very sensitive to agglutinating agents (morton aud Pickel, 1949 ), viral or others. Control material (Table XVI) for this experiment con­ sisted of concentrated normal allantoic fluid. Ho evidence of agglutination occurred when the normal allantoic fluid was combined with the modified cells. It is impossible to answer the question as to why the joassage allantoic fluid material produced hemagglutination whereas normal allantoic fluid did not. Both were concentrated by ultracentrifugation before use in the hemagglutination test. It might be that tumor XI (Table XII) contained an agent which increased in titer from the first through the third passage but had been diminished or lost when the fourth concentrate was bested. It also might be that serial passage of concen­ trated allantoic fluid carried with it, some component, normal to the fluid or its membrane, whicn would produce hemagglutination whentested with che modixied erythrocytes. The evidence found herein does not warrant a claim of positive isolation of an infectious agent as such. In an at tempi bo find a m o m sensitive means of test­ ing for agents as causes of neoplasms, on interference in vh-VQ test was devised. In theory, the concentrated pas­ sage allantoic fluid, provided it contained an agent cap­ able of multiplying, may interfere with the growth of Newcastle disease virus in the allantoic sac. However, as table >0/11 indicates, there was no significant differ­ ence in Newcastle disease virus titers obtained from allantoic fluids whether or not the tumor passage material preceded the introduction of Newcastle disease virus. There is a possiblity, however, that a tumor agent was capable of multiplication in only select tissues or fluids of the embryonated egg, wnile Newcastle disease virus is capable of propagation or, at least, existing in all tissues and fluids of such eggs. An experiment was also devised to test for the inter­ ference that might occur when concentrated tumor passage allantoic fluid was allowed to be in contact with eryth­ rocytes followed by Newcastle disease virus. The fact that the virus may be eluted vex’y slowly from guinea pig cells, when compared to what happens in the case chicken red cells are used, it seemed advisable to use guinea pig cells as an indicator. As can be seen from Table XVIII, the presence of tumor concentrated allantoic fluid did not alter the release of Newcastle disease virus from guinea pig cells. There were indications that considerable amounts of Newcastle disease virus had been retained by the guinea pig cells. The original Newcastle disease virus heinagglutinating was 1:1280, while in the presence of the control material, containing normal concentrated al­ lantoic fluid, the Newcastle disease virus tiber was 1:20. The normal concentrated allantoic fluid and tumor passage allantoic fluid were comparable in titer. Throughout these experiments, all the known methods available were used to enhance the growth and detection of any agent derived from neoplasms which would survive five serial passages in the embryonacing hen's egg. In several experiments, results were obtained suggesting that the concentrated tumor passage allantoic fluid reacted when tested by the hemagglutination test, whereas concentrated normal allantoic fluid did not. However, no evidence was found that an agent such as a virus, was present or sur­ vived five passages in embryonating hen's egg. SUMLvIARY VI 1. SUMMARY Using tissue culture methods, nine of a total of seventeen tumors were cultivated, in vitro. tissue was from animals and man. r J?iie neoplastic There was .no correlation between origin or type of neoplasm, and growth in vitro. 2. Concentrated tumor allantoic fluid, after five serial passages in embryonatea cnicken eggs, retarded the gx'owth of normal chicken heart tissue in cultures. One exception was encountered in which exceptional growth occurred. 3. When the concentrated allantoic fluid was tested for hemagglutination with normal chicken cells, no re­ action occurred. Sensitizing cells with Newcastle dis­ ease virus or modification with trypsin produced hem­ agglutination in many cases. 7/hen the concentrated al­ lantoic fluid was tested for hemagglutination with mod­ ified or sensitized cells between each passage, hemagglut­ ination was frequently observed. None of the seventeen tumors studied contained an agent that was capable of pro­ ducing hemagglutination through all five consecutive serial passages. 4. No Interference, in vitro or in vivo, coula be detected between concentrated allantoic fluid tumor passage material and Newcastle disease virus. 4 i l ; m >] i h j f s * rj 0 <, * 4 r?Hi? 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