canine oral papillomatosis ii. immunologie aspects of the disease*

Canine Oral PapillomatosisII. Immunologie Aspects of the Disease*

VELMAC. CHAMBERS,CHARLESA. EVANS,ANDRUSSELLS.

(Department of Microbiology, University of Washington, Seattle 5, Wash.)

SUMMARY

The subcutaneous and intramuscular injection of canine oral papilloma virus preparations with adjuvants into young dogs 2-3 weeks before inoculation of virus into theoral mucosa prevented the development of papillomas.

In dogs given an initial inoculation of virus into a single site in the oral mucosa followed by subsequent inoculations at other sites, papillomas developed at sites inoculated during the first 2 weeks of the incubation period but not at sites that were inoculated 3 or more weeks after the initial inoculation. The papillomas became visible in5-10 weeks and continued to grow for an additional 8 weeks even though the host became immune to reinfection before the tumors appeared.

All areas of the oral mucosa of young dogs were susceptible to infection with thÃ¨-virus. In a few instances papillomas developed in the skin around the nose and mouthand at the muco-cutaneous junction of the eyelid. Most old dogs were resistant to infection with this virus, presumably as a result of a previous natural infection. Dogswhose papillomas had regressed resisted a second challenge inoculation of virus into theoral mucosa. The virus-neutralizing antibody titer of the serum of five dogs whosetumors had regressed ranged from < 1:10 to 1:500.

Attempts to accelerate or delay the onset of regression of tumors by actively immunizing dogs to tumor extract and to living tumor cells gave no clear evidence of asignificant effect. Injections of immune serum may have delayed the onset of regressionof tumors in two of four dogs and in addition may have prolonged the regressionperiod in one of the animals. Transfer of "immune" lymph node and spleen cells from

dogs in which tumors had regressed to dogs with developing papillomas appeared toaccelerate regression in two of eleven dogs.

The course of infection with the virus of canine that the oral mucosa of young dogs was uniqueoral papillomatosis as observed in 90 experimen- in its marked susceptibility to infection with thetally infected dogs was described in a previous papilloma virus. The skin and the mucosa of thepublication (4). The present investigations were nose, genitalia, and alimentary tract below theundertaken for the purpose of obtaining additional glottis including the intestinal mucosa were re-knowledge concerning susceptibility and immunity sistant. Occasionally the cornea of the eye, thein canine oral papillomatosis and to gain some muco-cutaneous junction of the eyelid, and theunderstanding of the mechanism of regression outer hairy surface of the lip could be infectedof these tumors. (8). Attempts to infect guinea pigs, rabbits, rats,

DeMonbreun and Goodpasture (7) reported mice, monkeys, and kittens were uniformly un-*ThiS investigation was supported by a grant from the successful (7). Most attempts to infect old dogs

National Cancer Institute of the National institutes of Health, were likewise unsuccessful (7).U.S. Public Health Service. The mechanism which brings about the regres-

tThe authors gratefully acknowledge the assistance of sion of canine oral papillomas in a rather predict-Kenneth S. W. Kim and Spencer W. Shaw. able manner is not known. The tumors never

Receivedfor publication .January27, 1960. persist indefinitely and never become malignant.

1083

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108J. Cancer Research Vol. 20, August 1960

Studies of the Shope skin papilloma of rabbitssuggest that regression is brought about by ahost resistance directed toward the papilloma cellrather than toward the virus (11, 19). A hostresistance directed toward the papilloma cell issuggestive of the immune mechanism which is involved in the rejection of homografts of trans-plantable tumors and normal tissues.

MATERIALS AND METHODSVims.â€”The Seattle A strain of canine oral

papilloma virus was obtained from spontaneouspapillomas found in a dog in Seattle, Washington,in 1955. It was originally known as the N-211strain (4). Extracts of the papillomas and ofgrowths of the first three passages were used asa source of virus for producing the experimentaldisease in dogs. The ID5o of the stock virus preparations ranged from 10~3-5to 10~5-4.The methods

used for preparation of the extracts and for virusassay have been described in an earlier publication(4).

Experimental animals.â€”The dogs used in the

present experiments were mongrels of uncertainage and unknown background unless stated otherwise. Papillomas were produced in young dogsby injecting an appropriate dilution of the stockvirus into the oral mucosa or by applying it tothe scarified oral mucosa. Tumors appeared 4-8weeks after inoculation.

Collection of serum,.â€”Blood was collected from

the dogs by heart puncture. In some instancesthe animals were anesthetized with pentobarbitalgiven intraperitoneally. The blood was allowedto clot at room temperature and the serum removed, centrifuged at 2000 r.p.m. for 10 minutes,and stored in the frozen state.

Antigen preparations used for active immunization of dogs.â€”The antigen preparations were either

extracts of tumor tissue that had been frozen andthawed or suspensions of finely divided fresh papilloma tissue. Freund's adjuvants were sometimes

incorporated with one or the other of these preparations. The adjuvant mixture, which usuallyconsisted of paraffin oil and Arlacel1 in the ratio

of 9:1, was combined with an equal volume of10 per cent tumor extract in Ringer's or Hanks'

solution or with finely minced papilloma tissuein a ratio of 20:1. On one occasion a 10 per centtumor extract containing 7 per cent heat-killedmycobacteria was combined with paraffin oil andArlacel in the ratio of 2:3:1. The preparationscommonly contained 100 units of penicillin and100 yug.of streptomycin and occasionally 25 units

1The Arlacel was obtained from Atlas Powder Company,Wilmington, Delaware.

of Mycostatin2 per ml. All mixtures were homogenized in a Waring Blendor at low speed for 8-10minutes at 4Â°C. or at room temperature. The dose

of antigen used for immunizing dogs was 0.5ml. injected subcutaneously into each axillaryand inguinal region and 0.5 ml. given intramuscularly into each thigh.

Procedure for hyperimmunization of dogs.â€”DogsN-216 and N-182 were hyperimmunized after theyhad recovered from papillomatosis. The antigenfor hyperimmunization consisted of 10 per centcanine oral papilloma tissue, paraffin oil, andArlacel in the ratio of 10:9:1. The antigen wasgiven as described above, and the inoculationswere repeated 2 weeks and again 5 weeks afterthe first injections. The dogs were bled from theheart 7 times between the 16th day prior to andthe 53d day after the third injection. The serafrom the seven bleedings from each dog werepooled and stored in the frozen state until used.

Assay of virus-neutralizing antibody.â€”The se

rum specimens were thawed, inactivated by beingheated at 56Â°C. for 30 minutes, and appropriatedilutions were made in Locke's solution. Five-tenths of a milliliter of either a 10~2 or a 10~3

dilution of supernatant fluid from a stock canineoral papilloma suspension in distilled water orin Locke's or Ringer's solution was added to 0.5

ml. of each serum dilution. After incubation atroom temperature for 1 hour, 0.1-ml. doses ofthe serum-virus mixtures were inoculated at different sites in the oral mucosa of pups.

Complement-fixation tests.â€”The complement-

fixation procedure employed was the outcome oftrials in which variations in volumes and concentrations of reactants, length of incubation periods,and temperature of incubation were tested. Theantigen used for complement-fixation tests consisted of the supernatant fluids from 10 to 20per cent suspensions of homogenized papillomatissue in CF saline (0.85 per cent sodium chlorideplus 0.01 per cent magnesium sulfate). Variousdilutions of serum were used. Each mixture of0.2 ml. of canine oral papilloma antigen, 0.2 ml.of diluted serum, and 0.2 ml. of complement wasincubated in a water bath at 37Â°C. x\fter 1 hour

0.5 ml. of sensitized sheep erythrocytes was addedto each mixture, and incubation was continuedfor another hour. The extent of hemolysis wasrecorded at the end of the 2d hour.

Preparation of suspensions of cells from spleensand lymph nodes of immune dogs.â€”The immune

dogs were anesthetized with sodium pentobarbital, and samples of blood were withdrawn from

2Mycostatin was obtained from E. R. Squibb and Sons,Xew York, X.Y.



CHAMBERSet al.â€”Canine Oral Papillomatosis 1085

the heart to provide serum. Usually the animalswere then killed by exsanguination to reducethe amount of blood in the tissue. This was doneby severing the vessels entering the heart. Lymphnodes and spleens were removed aseptically, theformer from one or more of the following areas:submandibular, cervical, axillary, inguinal, mesen-teric, and paravertebral. The fat was dissectedfrom the lymph nodes and spleens. Cells werethen extricated from the tissues by mincing withscissors, by teasing with a dissecting needle, orby pressing minced tissue through one or twowire mesh sieves by means of a sterile spatulaor pestle. The cells were collected either in serumfrom the dog from which the cells were obtainedor in a diluent consisting of Hanks' solution with

1, 2, or 5 per cent normal dog serum or 5 percent serum from the donor dog. Cells were usuallywashed 1 or 2 times in the diluent. After thefinal centrifugation in a graduated centrifuge tubeat 200 to 1700 r.p.m. for 5-10 minutes, the packedcell volume was determined, and the appropriatediluent was added. The final concentration ofcells injected into the recipients ranged from 10 to50 per cent.

Cell counts made on aliquots of cell suspensionsshowed that between 65 and 85 per cent of lymphnode cells in suspensions from dogs T-9, T-18,and T-16 were viable as determined by the trypanblue test (9). Differential cell counts on samplesof the same suspensions were as follows: lymphocytes, 90-98 per cent; polymorphonuclear neutro-phils, 0-3 per cent; monocytes, 2-9 per cent. Two

per cent plasma cells were found in one cellsuspension. Between 40 and 50 per cent of cellswere viable in the spleen suspension from dogT-16. The differential count on this sample was:92 per cent lymphocytes, 3 per cent polymorphonuclear neutrophils, and 5 per cent monocytes.

RESULTSSusceptibility as related to age and site of inocu

lation.â€”Although the exact age of dogs was not

known, an attempt was made to use young dogsbetween 2 and 6 months of age for most of theexperiments. Of 128 dogs that were estimatedto be in this age group, 125 were susceptible toinfection with canine oral papilloma virus afterinoculation of the virus into the oral mucosa. Thethree dogs that were resistant to infection wereobserved for periods of 66-155 days after inocula

tion. One pup that received a virus inoculationat 18 days of age was also susceptible. In contrastto the high susceptibility rate among young dogs,four of seven adult dogs that were given inoculations of papilloma virus were resistant.

The susceptibility of various regions of the oralmucosa following experimental infection is illustrated by data on dog N-315, which was giveninoculations of virus by the scarification method.Papillomas occurred in all inoculated areas including the floor and roof of the mouth, the gingivalmucosa, the under surface of the tongue, the innersurface of the cheeks and the lips to the hair line.Figure 1 shows the numerous papillomas thatdeveloped on the under surface of the tongue.

Goodlow (8) inoculated papilloma virus intovarious tissues of six dogs by applying the virusto the scarified tissue. He reported that tumorsdeveloped in the hairy portion of the outer lipin one of the six dogs, in the upper eyelid ofanother, in the cornea of another, and in the rectalmucosa of still another dog. All tumors exceptthe one on the rectal mucosa produced at leastone tumor on reinoculation.

In the present study, the inoculation of virusinto the scarified skin of the face of one pupfailed to produce papillomas. However, papillomaswere observed in the skin around the mouth ornose in three of the many dogs that were giveninoculations in the oral mucosa. Two of the threedogs also developed tumors in the conjunctivaor muco-cutaneous junction of the eyelid. Severalof these papillomas are shown in Figures 2 and3. Regression of the tumors in the mouths of thesethree dogs occurred at 8, 9, and 21 weeks, respectively. It is possible that the conditions responsiblefor the long growth periods between appearanceand regression of tumors may also favor the growthof tumors in tissues other than the oral mucosa.

Tests for virus in tumors of the oral mucosa,skin, and muco-cutaneous junction of the eyelidof dog N-367 were carried out by inoculatingsuspensions of each of these tumors into the oralmucosa and into the skin and/or conjunctivaof two susceptible dogs. Suspensions of papillomatissue from inside the mouth and from the lipinduced tumors in the oral mucosa but not in theskin or conjunctiva. Suspensions of tumor tissuefrom the skin and the muco-cutaneous junctionof the eyelid failed to induce tumors in the oralmucosa. The suspension of skin tumor also failedto induce tumor formation upon injection intothe skin, and the eyelid tumor material likewisefailed to produce tumors upon injection into theconjunctiva. It is possible that the skin and eyelidtumors contained a small amount of virus whichwas not detected by the means employed.

Artificial immunization.â€”Three pups were giv

en subcutaneous and intramuscular injections of10 per cent canine oral papilloma tissue incorporated in Freund's adjuvants. One week after the



1086 Cancer Research Vol. 20, August 1960

second inoculation of the above material a challenge dose of virus contained in 0.05 ml. of thesupernatant fluid from a 10 per cent suspensionof papilloma tissue in distilled water was inoculated into each of twelve sites in the oral mucosaof each pup. No papillomas occurred in any ofthe three pups during a Sf-month period of observation. Another pup, which received an intra-peritoneal inoculation of virus without adjuvantshortly after birth, was resistant to oral infection2 months later. It was concluded from these observations that pups may be readily immunizedagainst canine oral papillomatosis by subcutaneous, intramuscular, and intraperitoneal injectionsof the virus containing fluid prepared from suspensions of the tumor tissue.

on the 21st day after the initial inoculation demonstrates that immunity to reinfection had developedby this time. The persistence of this immunitythrough the remainder of the incubation periodand during at least the first 7 weeks of the growthperiod of tumors that developed at the site of theoriginal inoculation was shown by the failureof tumors to develop at the sites of the laterinoculations.

The immunity of dogs to reinfection after recovery from canine oral papillomatosis was determined by challenge inoculations given at intervals following regression of the tumors. No furtherpapillomas developed in five dogs that receiveda second virus inoculation from 2 to 16 weeksafter regression of their papillomas. These results

TABLE1THKDEVKLOPMKXTOFIMMUNITYINDOGSASSHOWNBYTHERESPONSETOSUCCESSIVE

INOCULATIONSOFCANINEORALPAFILI.OMAVIRUSINTOTHEORALMUCOSA

DenX-317X-318X-319X-355X-358X-359DEVELOPMENT

OFPAPILLOMASATPITESOFSUCCESSIVEINOCULATIONSDay

ofinoculation0

t-4 5-7 8-10 11-13 11! al 28 3.54Ã¤+

++++

++ + +++

++-L._ _ ___|_

_ _ __+

_

+ = An inoculation site at which papillomas developed. The incubation period ranged from 5 to 10 weeks.â€”= An inoculation site at which papillomas failed to develop.Dogs X-355, X-358, and X-359 were reinoculated 21 days after the first inoculation and every week thereafter

for 10 weeks. Tumors appeared at the site of the first inoculation after an incubation period of 7 weeks and regressed4-8 weeks later. Tumors failed to develop at any of the other sites of inoculation in these three dogs.

Resistance to reinfection.â€”Inorder to determinethe time at which immunity to viral infectiondevelops after inoculation of virus into the oralmucosa, each of three pups was inoculated byapplying increasing amounts of virus to differentscarified oral sites at intervals of 2-6 days. A2.5 per cent suspension of stock virus was inoculated into the oral mucosa of three other pups.Three weeks later and at weekly intervals thereafter for 10 weeks, each of these three pups wasgiven another inoculation of virus in a differentsite in the oral mucosa. The results of the inoculations in the six dogs are shown in Table 1.

In the first three dogs, no immunity to rechal-lenge with virus was observed during the periodscovered by the successive inoculations after thefirst infective dose of virus was given. Theseperiods were 7, 12, and 16 days, respectively.In the last three dogs the failure of tumors todevelop at the sites given inoculations of virus

strongly indicate that a prolonged immunity results from canine oral papilloma infection as suggested by DeMonbreun and Goodpasture (7).

Testsfor neutralizing and complement-fixing antibody.â€”Antibody studies were carried out on theserum of several dogs whose tumors had regressed.The level of the virus-neutralizing antibody wasdetermined by injecting appropriate serum-virusmixtures into the oral mucosa of susceptible dogs.Failure of papillomas to develop at the inoculatedsites indicated that the virus had been neutralizedby a specific antibody in the serum employed inthe test. The capacity of these serum specimensto fix complement in the presence of papillomaantigen was also determined. The combined results of two neutralization experiments and theresults of one complement-fixation test aie presented in Table 2. The serum of six of the sevendogs whose papillomas had regressed had a neutralizing antibody titer of 1:50 or higher. A 1:4



CHAMBERSet al.â€”Canine Oral PapillomatosÃ¹ 1087

dilution of the same sera fixed complement. Thecapacity of these sera to fix complement in dilutions greater than 1:4 was determined only forserum from hyperimmunized dog N-216. Twofolddilutions of this serum to 1:160 fixed complementunder the conditions employed. The neutralizingantibody titer of this serum was 1:500.

The results of complement-fixation tests presented in Table 2 compare favorably with theresults of the neutralization tests. However, inmany instances the complement-fixation resultswere unreliable. Tests for anti-complementary ac-

papillomas 7-46 days old, three dogs whose papil-lomas had begun to regress 2-3 days previously,six dogs 16-18 days after regression, and sevendogs 23-59 days after regression. No reactionwas observed in the conjunctiva of any of the dogs.

Attempts to delay or accelerate the onset of tumorregression by active immunization.â€”In order to

stimulate the early production of antibody to thetumor extract which was used as source of virusfor producing tumors, the extract was combinedwith adjuvants and injected subcutaneously andintramuscularly into eleven dogs at various inter-

TABLE 2

TKSTSFORANTIBODYIN THF,SERUMOFHYPKRIMMUXIZKDDOGSANDDOGSFOLLOWINGTUMORREGRESSION

SERUMCOLLECTEDFrom

dogX-442Ã•

X-302X-312X-315X-316X-319X-216X-182Days

afterDays after papillomasinoculationappearedBefore

inoculation (control)7846126

81126951269511474Hyperimmunized

HyperimmunizedDays

afterregression

begun2418591824VIRUS-

NEUTRALIZINCANTIBODYTITLK*<Ã¯;Ã®o1:501:501:501:5001

: 500> 1: 5000RESULTS

OKCOMPLEMENT

FIXATIONTESTf--j_-\--|--|-Ã®

* Xeutralizing antibody titer is recorded as the highest tenfold dilution of serumthat neutralized a 10"5-3 or 10~3-3 dilution of stock papilloma virus suspension. Neu

tralization of virus was determined by the failure of papillomas to develop at oralmucosal sites that received injections of the serum-virus mixture.

t The complement-fixation results were from one experiment. Dilutions of serumfrom dog N-216 were twofold from 1:10 to 1:160; all other sera were diluted 1:4 only.Additional results, not presented in the table, showed fixation of complement by severalsera from susceptible dogs.

Ã•The pooled serum from ten susceptible control dogs failed to neutralize virus inanother neutralization test.

Dogs N-216 and N-182 were hyperimmunized after recovering from papillomato.sis.Serum was collected from seven bleedings between the 16th day prior to and the 58dday after the third injection of antigen.

tivity of the antigen indicated that the range ofsuitable antigen dilutions was too narrow to givereliable results. Whereas concentrations higherthan this range were anticomplementary, concentrations below this level were not consistent inreacting with antiserum to fix complement. Itis possible that papillomas produced by anotherstrain of virus might yield more suitable antigenpreparations.

Tests for hypersensitivity to papilloma antigen.â€”

Dogs bearing canine oral papillomas and dogs inwhich papillomas had regressed were tested forhypersensitivity to papilloma extract. A dose of0.05-0.1 ml. of a 2 per cent extract in Locke's

solution was injected into the conjunctiva of theupper eyelid of the following dogs: five dogs with

vals before and after the appearance of papillomasin the oral mucosa. The results of these experi-ments are presented in Table 3.

A comparison of growth periods of tumors inthese dogs with the growth periods of tumorsin 57 untreated dogs (4) shows little difference.A tumor growth period of 2-3 weeks occurred in18 per cent of treated dogs as compared with 14per cent of untreated dogs, of 4-8 weeks in 55

per cent of treated dogs as compared with 65 percent of untreated dogs, and of 9 weeks or longerin 27 per cent of treated dogs as compared with21 per cent of untreated dogs. It is possible, butnot at all certain, that the extremely late onsetof regression in dog T-15, 140 days after appearance of tumors, may have been caused by the




injections of tissue extract. With this possibleexception the injections of tumor extract incorporated with adjuvants did not appreciably alterthe course of the disease. It is realized, however,that even if the treatment had accelerated ordelayed regression in one or two dogs this wouldnot have been detectable because of the widerange of the tumor growth periods that occurredin control animals.

In another approach to the problem of stimulating an active immunity which will cause papillomaregression, fresh papilloma tissue was used asantigen. The tumor tissue was obtained from four

dogs between the 15th and 78th day after appearance of tumors and between the 8th and 62dday before regression began. The procedure forpreparing the tissue for injection is described under"Materials and Methods." Between 0.2 and 2.0

ml. of a 10 per cent tissue suspension with orwithout Freund's adjuvants was injected subcu-

taneously in each axillary and inguinal regionof recipient dogs at various intervals before andafter the appearance of papillomas. The resultsof these experiments are presented in Table 4.

Tumor regression began in three of the fivedogs after a growth period of 34-42 days, which

TABLE3THEEFFKCTOFSUBCUTANEOUSANDINTRAMUSCULARINJECTIONSOFTUMOREXTRACT

WITHADJUVANTS*ONTHETIMEOFREGRESSIONOFPAPILLOMAS

DnaÂ°KX-431N-432N-433X-436X-437N-438\-442T-8T-15T-24T-31Days

betweeninjectionofvirusinto

tlieoralmucosaandfirstinjectionof

extract(10039393939(122022-27,-34,-34,+

12,+12,+1,0,-26,-26,-29,-12,-13-20-20+22,+22,+11,+

10+13+

1-14,+

5Days

before ( â€”) or after (+)tumorsappeared

that extracts wereinjected+28,

+36, +43, +49, +56. +64.+71+28. +36, +43, +49,+5Â«+

17+

10Days

betweentheappearanceof

tumorsandtheirregression28142678562514341404561

* Five-tenths ml. of tumor extract with adjuvants was injected subcutaneously into each axilla and each groinand intramuscularly into each thigh at the times indicated. The papilloma extract in the subcutaneous and intramuscular inocula was the same as that inoculated into the oral mucosa for producing papillomas. The adjuvantsconsisted of paraffin oil and Arlacel. Heat-killed mycobacteria were included in inocula for dogs T-8, T-15, T-24,and T-31.

TABLE 4

THE EFFECTOFSUBCUTANEOUSINJECTIONSOFFRESHPAPILLOMATISSUEONTHE REGRESSIONOFPAPILLOMAS

T-6.5T-69T-74T-76T-85TlME

OF INJECTIONS IN TERMS OF DAYS BEFORE (â€”)Tissue

withoutadjuvants-20,

-15-33,-28-:!4,

-29,

- 7, 0, + 8,+13.-20, -13, - 5, 0Tissue

with+

8,-5,+8,0,-34,adjuvants*+

130+13+

5-89DAYS

HETWEKNAPPEARANCEBe^inniiiK

regression4234231535Completeregression70237332558

* One part minced tissue was combined with 19 parts Freund's adjuvants.Dog T-65 received a subcutaneous injection of 0.5 ml. of his own papilloma tissue on the 13th day after tumors

were first observed.Regression of tumors in dog T-69 was unusually slow after regression had begun.Dogs T-74 and T-76 were from a litter of five pups, all of which liad received inoculations of virus at the same

time. Tumors regressed early in all five litter-mates.



CHAMBERSet al.â€”Canine Ural Papillomatosis 108!)

was within the normal range. The tumors of dogsT-74 and T-76 exhibited early regression aftergrowth periods of 23 days and 15 days, respectively. These dogs were given inoculations of papillomavirus at the same time as their three litter-mateswhich did not receive injections of fresh papillomatissue. Since the papillomas regressed early inall five dogs, no significance was attached to thetreatment that they received. It is not knownwhether the treatment had any bearing on theextremely long regression period in dog T-69.In untreated dogs the oral mucosa usually resumesa normal appearance between 1 and 4 weeks afterregression begins.

The fact that the papilloma cells used for attempted immunization were from dogs unrelated

causing an inflammatory response in the areaof tumors, might stimulate tumor regression. Theoral mucosa of pup N-287 was given an inoculationof papilloma virus and, on the first day after tumors appeared, trauma was produced by removingseveral small pieces of adjacent un inoculated oralmucosa. This procedure was repeated 4 times at7-14-day intervals except that on the last twooccasions a portion of papilloma tissue was alsoremoved. Regression began 42 days after appearance of tumors and was complete by the 52dday. Oral trauma and removal of tumors apparently did not accelerate regression of the remainingtumors in this dog. It has been our experiencewith biopsies of oral papillomas of many dogs,often with extensive excision of tumor tissue,

TABLE5THEEFFECTOFPASSIVETRANSFEROFIMMUNEANDHYPERIMMUNKSERAONTHEREGRESSIONOFPAPII.I.OMAS

DOGX-258T-10T-60T-62AGE

OF TUMORSnrniNGTIMEOF

SERI'MINJECTIONS(DAYS)1515-297-107-165-85-14SERATotal

amt.

(ml.)39171431214612No.injections19;j10310Route

ofadministration*I.

P.LocalI.

P.Ix>calI.P.LocalREGRESSION

OK TOMOHRiv THE HE<IPIKNTDays

fromtumorappearanceto:Beginning

regression25197472Completeregression>495893133Days

fromfirstserum

treatmentto:Beginning

regression104(Â¡707CompleterÃ©gression344386128

* I.P. = intraperitoneal. Local = injection of serum into the tumors and into the oral mucosa surrounding the tumors.Dogs T-fiU and T-62 received five injections of 0.1-0.3 ml. of histamine into the lower right oral mucosa during the period

of serum injections. This was intended to promote absorption of the serum.

to the recipient and hence genetically and anti-genically different from the recipient's tumor cells

may have been an important factor in the failureto stimulate early regression. Therefore, autol-ogous tumor tissue, which was removed fromdog T-65 on the 13th day after appearance oftumors, was minced and a suspension of the tissuewas injected subcutaneously into the same dog.Tumor regression occurred 29 days later, whichwas not early enough to indicate that it wasaccelerated by the injections of autologous tumortissue.

It has been observed that papillomas frequentlyregress soon after removal of a portion of thetumor (7, 8). This observation was reconfirmedin the course of the present investigation. Thepossibility was considered that oral trauma, by

that, when this treatment was performed latein the tumor growth period, regression of the remaining papillomas commonly followed, whereas,when the treatment was performed during theearly phases of tumor growth, it had no appreciableeffect on the time of regression of the remainingtumors.

Attempts to alter the time of regression by passiveimmun izat ion.â€”

1. Transfer of Serum: Dog N-258 was givenan intraperitoneal injection of pooled "immuneserum" obtained from three dogs between the

13th and 73d day following regression of theirtumors. The tumors of two of these donor dogshad been produced by a strain of virus differentfrom that used to produce tumors in dog N-258.Dog T-10 was given local injections, and dogs




T-60 and T-62 were given local and intraperitonealinjections of "hyperimmune serum" obtained fromdogs N-182 and X-216 following treatment asoutlined under "Materials and Methods." The

results are presented in Table 5.There was no apparent alteration in the time

of regression of tumors in dog N-258. Regressionoccurred at approximately the same time in alitter-mate which was inoculated with virus atthe same time as dog N-258 and which was givenan intraperitoneal injection of serum from a normal dog. The apparent early onset of regressionof treated tumors in dog T-10 was probably adirect result of the trauma caused by the multipleinjections of relatively large amounts of seruminto the base of selected tumors. One of thesetumors had begun to grow again on the 19thday after the last serum injection and continuedto grow for a period of 2 weeks, after which timeall tumors regressed.

The possibility that the late onset of regressionin dogs T-60 and T-62 and the extended 61-dayperiod between the onset of regression and completeregression in dog T-62 may have been the resultof an enhancing effect of the serum injected wasconsidered. However, late regressions also occurredon occasion in untreated dogs. In an earlier study(4) the tumors in nine of 57 untreated dogs regressed later than 9 weeks after the appearanceof tumors. Other factors which might have contributed to the unusual course of tumor regressionin dogs T-60 and T-62 were the local injectionsof histamine into some tumors, the removal oftumor tissue during the course of the experiment,and intercurrent diseases. Both dogs were sickwith distemper during most of the incubationperiod and received hepatitis vaccine, penicillin,and Chloromycetin during this time. They alsodeveloped severe dermatitis which was only partially controlled and persisted throughout the experiment.

2. Transfer of whole blood: The effect of transfusion of whole blood from immune dogs to adog with papillomas in the early growth stagewas investigated. Papillomas were first observedin the recipient dog 33 days after virus was inoculated into the oral mucosa. Eight days after theappearance of tumors the dog weighed 7 kg.At this time 150 ml. of blood was removed byheart puncture. The dog was immediately givensuccessive intravenous transfusions, totaling 160ml., of citrated whole blood derived from threeimmune dogs whose tumors had regressed 38-64days earlier. The papillomas in the transfuseddog continued to grow for a period of 5 weeks

before regressing. The transfusion apparently hadno accelerating effect on tumor regression.

3. Transfer of cells of lymph nodes and spleens:The possibility that the regression of canine oralpapillomas is mediated by cell-associated antibodywas considered. To test this hypothesis, cells oflymph nodes and spleens from immune dogs weretransferred to dogs whose tumors were in an earlygrowth phase. In some experiments serum wastransferred together with the cells; in others thecells were washed and transferred without serum.

Regression of tumors began in four of the sixrecipient dogs between 2 and 13 days after thecell transfer. The tumor growth period from thefirst appearance of tumors to the onset of regression in these four dogs was between 9 and 23days. The early regression of tumors in two ofthe four dogs, T-73 and T-75, cannot be regardedas being significant, since these two dogs werefrom a litter of five pups all of which exhibitedearly regression of their tumors. These pups weredescribed earlier, and two of them, T-74 andT-76, are listed in Table 4. All five pups of thelitter were given inoculations of virus at the sametime; only pups T-73 and T-75 received immunecells. The fifth litter-mate, T-72, received no treatment other than inoculation of virus.

The cell transfer may have induced regressionin dog T-108 and very probably did so in dogT-110. Two intravenous injections of lymph nodecells were administered to dog T-110 in additionto the intraperitoneal injections. In a later experiment, not included in Table 6, two additional dogs,T-113 and T-115, were given intravenous andintraperitoneal injections of immune lymphocytessuspended in Hanks' solution containing 5 per

cent donor dog serum. The injections were given5 and 18 days, respectively, after tumors appeared.Regression in the respective animals began 38and 10 days later. However, regression progressedvery slowly and was not complete until 47 and43 days, respectively, after onset was first observed.

In another experiment five dogs with papillomas11-25 days old were given intraperitoneal injections of 22-28 ml. of spleen cells suspended in81-125 ml. of donor dog serum. The onset ofregression did not appear to be accelerated ordelayed in this group of dogs.

The results of the passive immunization experiments described in the preceding paragraphsshow several instances of a prolonged intervalbetween the onset of regression and the completionof regression when all tumor tissue disappeared.Whereas in untreated dogs this phase of the disease



CHAMBERSet a!.â€”-CanineOral Papillomatosis 1091

usually ranged from a few days to 3 weeks andrarely to 4 weeks (4), in six of seventeen treateddogs it ranged from 5 to 9 weeks. Two of thesedogs had received immune serum, two had receivedimmune cells, and two had received both immuneserum and cells. The significance of this apparentrelation of treatment to prolongation of the regression phase is not known.

In an attempt to gain evidence of the adequacyof transfer of cell-associated antibody, trials weremade to passively transfer tuberculin sensitivity

slightest indication of reactivity to tuberculin.Since the cutaneous tuberculin reaction is de

pendent on the local persistence of tuberculinat the test site (15), the donor dogs were alsosubjected to a more sensitive test with preparationsof "depot tuberculin" in oily emulsion containing

concentrations of tuberculin similar to those usedfor the regular tuberculin tests described above(10). Mild inflammatory reactions resulted fromcontrol tests with depot preparations in normaldogs, and oils alone in the donor dogs. Although

TABLE6THEEFFECTSOFTHKADMINISTRATIONOFIMMUNELYMPHOCYTESox THKREGRESSIONOFPAPII.LOMAS

DONORDOG*T-9,

T-lÃŸ,T-18T-9,

T-1C,T-18T-9,

T-Iti,T-18T-89,

T-93,T-101T-89,T-9:i,T-101,T-105T-89,

T-93, T-101DONOR

CELL PREPARATIONS

ADMINISTERED TORECIPIENTSVolume

of cellsuspensiont(ml.)Lymph

node31

4335:u52451724Spleen111011125112Route;I.P.LiÂ«':ilI.

P.LocalI.P.

LocalI.P.I.P.

I.V.I.P.DATA

ONRECIPIENTSDogT-66T-73T-75T-108T-110T-lllDays

from appearance of tumorsto:Cell

transfer2020710410Beginningregression4Â«239201052Completeregression843334382184

* In donor dogs the period from beginning of tumor regression to transfer of their cells to the recipient was 32-72 davs indogs T-9, T-16, and T-18; 4 days in dogs T-89, T-93, and T-101; and 20 days in dog T-105. Dogs T-89, T-93, T-101, and T-105had received multiple subcutaneous injections of live Mycobacterium tuberculosis, BCG strain, in Freund's adjuvants in anattempt to sensitize them to tuberculin.

t The diluent for cells was Hanks' solution with 1, 2, or 5 per cent normal dog serum. The final concentration of cells for intra -peritoneal and local injections ranged from 10 to 50 per cent. The supernatant fluid after centrifugation of the lymph nodesuspension at 500 r.p.m. for 5 minutes was used for the intravenous injection.

Ã•I.P. = intraperitoneal. Local = injection of the lymph node suspension into the tumors and oral mucosa surrounding tumorsof the right side of the mouth. I.V. = intravenous.

from some of the donor dogs to the recipients.As shown in Table 6, four of the donor dogswere vaccinated with BCG. At the time of celltransfer, tests for cutaneous sensitivity to tuberculin were conducted on these dogs, the recipients of their cells, and normal control animals by the injection of a dose of 0.1 ml. of 1:50or 1:20 OT or 0.025 to 0.5 mg/ml PPD given intothe skin of the ears and back. The reactions of thedonor dogs to tuberculin tests conducted at thetime of cell transfer were equivocal. None of therecipients of cells or normal animals gave the

the stronger reactions to the depot preparation,which appeared in several of the donors, probablyrepresented positive tuberculin reactions, theywere weak and irregular at best. The failure ofthe donor dogs to give uniform and clear-cutcutaneous reactions to tuberculin is not surprising,because tuberculous dogs are notorious for theirweak and irregular responses to tuberculin tests(1).

It is obvious that these trials failed to providepositive evidence of either the adequacy or inadequacy of cell transfer of cell-associated antibody.




DISCUSSIONThe present attempts to elucidate the mecha

nism of regression of canine oral papillomas werebased on the probability that the regression ofthese tumors is induced by an immunologicalreaction. It was shown that, by the end of the3d week after young dogs were given an inoculation of canine oral papilloma virus at a single sitein the oral mucosa, immunity was ample to prevent reinfection of the oral mucosa at other sites.Presumably this immunity resulted from the development of virus-neutralizing antibody. However, since the tumors resulting from the primaryinoculation grew well in dogs that were immuneto reinfection with the papilloma virus, it wasassumed that humoral antibody directed towardthe virus does not cause regression. Furthermore,immune and hyperimmune sera transferred fromdogs that had sloughed their tumors to dogsbearing tumors were not effective in hasteningregression. This suggested that regression wasnot caused by any kind of humoral antibody tothe virus or to the tumor itself. Indeed, hyper-immune serum administered to three dogs duringthe growth phase of their tumors appeared to havean enhancing effect on the tumors of two of thedogs as judged by the delay in the time of regression.

The reports which indicate that cell-associatedantibody is commonly the most important mediator of regression of homografts of normal tissue(2) and of transplantable tumors (12-14, 16-18)prompted our efforts to demonstrate a cell-associated antibody in the regression of canine oralpapillomas. It has been shown that this type ofcell-associated antibody is directed toward a labilecellular antigen in homotransplanted cells (3).In the present study, living papilloma cells transferred from one dog to another dog that hadreceived an inoculation of papilloma virus apparently failed to produce an antibody which couldreact with the recipient's tumor cells to cause

regression. However, one would expect this procedure to succeed only if the cellular antigen in

question were common to the tumors of bothdonor and recipient dogs. On the other hand,if the antigen is determined by each host's genetic

specificity, it may vary in the tumors of differentdogs. In that case only tumor cells from dogswhose genetic complement for histocompatibilityis the same as that of the recipient would beexpected to produce cell-associated antibody thatmight conceivably react with the tumor antigenof the recipient to bring about regression of tumors.

One of the proofs of the immune nature of abiological reaction is the capacity to produce theimmune state by transferring antibody or antibody-producing cells from an immune animal toa susceptible one. Thus, homograft immunity isconveyed by transfer of lymph node and spleencells from immune animals to others of the samegenetic strain (2, 12-14, 18). It was recognizedthat the lack of inbred strains of dogs in thepresent work might militate against successfulpassive transfer of papilloma immunity in thismanner. However, the fact that tuberculin sensitivity was readily transferred in guinea pigswithout regard to genetic differences betweendonors and recipient animals (5, 6) stimulatedour attempts to passively transfer immunity tocanine oral papillomas by transferring cells oflymph nodes and spleens of immune dogs to dogswith early tumors.

Only suggestive evidence for the role of cell-associated antibody in tumor regression was obtained by the passive transfer of "immune" lym

phocytes from dogs in which tumors had regressed.There is an appreciable likelihood that the earlyregression of the tumors in dogs T-108 and T-110was induced by the cell transfer. Moreover, theearly stages of regression of papillomas that beganin dog T-115 10 days after cell transfer and 28days after the appearance of tumors may havebeen initiated by the transferred cells. The greatlyextended period of 43 days from the beginningto the completion of regression of tumors in thisdog may be an indication that the number of ac-

FIG. 1.â€”Numerous papillomas on the ventral surface ofthe tongue of dog \-315 after inoculation of the scarified mucosa Â«ntha 10~3dilution of papilloma virus.

FIG. 2.â€”Arrows point to papillomas that developed onthe uninorulated skin in the region of the nose and mouth ofdog \-367. The mucosa inside the mouth was inoculated byinjecting a 2.5 per cent stock virus preparation into twelve tofifteen sites. Tumors that developed at several of the inoculatedsites are also shown in the photograph.

FIG. 3.â€”A small tumor (arrow) that developed at themuco-cutaneous junction of the eyelid of dog X-367.



CHAMBERSet al.â€”Canine Oral Papillomatosis 1093

live "immune" lymphocytes transferred was only

sufficient to initiate but not to complete the regression.

From the present studies it is not clear whetherthe failure in most instances to demonstrate anaccelerated tumor regression following transferof cells from lymph nodes and spleens of immuneanimals to dogs with developing tumors was dueto the genetic differences between donor and recipient animals or to some other factor such asinsufficient numbers of immune cells transferred.In future work on this problem it would be desirable to control the genetic factors by using inbredanimals or animals in which a state of tolerancefor the donor cells had been induced.

RKFKRKNCES1. BERG,0. A. Tuberculin Reactions in Dogs. Acta Tubere.

Scandinav. (Suppl.), 43:9-48, 1958.a. BILLJNGHAM,R. E.; BRENT, L.; and MEDAWAR,P. B.

Quantitative Studies on Tissue Transplantation Immunity.II. The Origin, Strength and Duration of Actively andAdoptively Acquired Immunity. Proc. Roy. Soc. London,s.B, 143:58-80, 1954.

3. . The Antigenic Stimulus in Transplantation Immunity. Nature, 178:5U-19, 1956.

4. CHAMBERS,V. C., and EVANS,C. A. Canine Oral Papil-lomatosis. I. Virus Assay and Observations on the VariousStages of the Experimental Infection. Cancer Research,19:1188-95, 1959.

5. CHASE,M. W. The Cellular Transfer of Cutaneous Hyper-sensitivity to Tuberculin. Proc. Soc. Exper. Biol. & Med.,69:134-35, 1945.

fi. . Immunological Reactions Mediated through Cells.In: A. M. PAPPKNHEIMEH,JR. (ed.). The Nature and

Significance of the Antibody Response, X: 156-69. NewYork: Columbia University Press, 1953.

7. DEMONBREUN,W. A., and GOODPASTURE,E. W. Infectious Oral Papillomatosis of Dogs. Am. J. Path., 8:43-56,1932.

8. GOODLOW,R. J. Canine Oral Papillomatosis. Thesis, University of Minnesota, Minneapolis, Minn., 1941.

9. HARRIS,S.; HARRIS,T. N.; and FARBER,M. B. Studieson the Transfer of Lymph Node Cells. I. Appearance ofAntibody in Recipients of Cells from Donor RabbitsInjected with Antigen. J. Immunol., 72:148-60, 1954.

10. JAMES,D. G., and PEPYS,J. Tuberculin in Aqueous andOily Solutions. Skin Test Reactions in Normal Subjectsand in Patients with Sarcoidosis. Lancet, 270:602-4, 1956.

11. KIDD,J.G. The Course of Virus-induced Rabbit Papillomasas Determined by Virus, Cells, and Host. J. Exper. Med.,67:551-74, 1938.

12. MITCHISON,N. A. Passive Transfer of TransplantationImmunity. Nature, 171:267-68, 1953.

13. . Passive Transfer of Transplantation Immunity.Proc. Roy. Soc. London, s.B, 142:72-87, 1954.

14. . Studies on the Immunological Response to ForeignTumor Transplants in the Mouse. I. The Role of LymphNode Cells in Conferring Immunity by Adoptive Transfer.J. Exper. Med., 102:157-78, 1955.

15. PEPYS,J. Effect of Local Adrenaline and Histamine onTuberculin Reactions. Acta Allergol., 6:265-85, 1953.

16. PREHN, R. T.; ALOIRE, G. H.; and WEAVER, J. M.The Diffusion Chamber in Homograft Research. Transpl.Bull., 2:147-48, 1955.

17. PREHN,R. T.; WEAVER,J. M.; and ALGIRE,G. H. TheDiffusion Chamber Technique Applied to a Study of theNature of Homograft Resistance. J. Nat. Cancer Inst.,16:509-18, 1954.

18. SNELL,G. D. The Homograft Reaction. Ann. Rev. Micro-biol., 11:439-58, 1957.

19. ZILBER, L. A. Specific Tumor Antigens. Adv. CancerResearch, 6:291-325, 1958.



1960;20:1083-1093. Cancer Res Velma C. Chambers, Charles A. Evans and Russell S. Weiser DiseaseCanine Oral Papillomatosis: II. Immunologic Aspects of the

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