[CANCER RESEARCH 37, 4150-4155, November 1977j

crude cell walls and purified endotoxins from C. parvumcell walls found that these preparations injected i.v. causedan increase in spleen and liver weights of female W-Swissmice infected with live albogeneic Sarcoma 180T/G cells.Their results are difficult to interpret because they neithermeasured a direct effect on tumor growth nor accountedfor the effects of histocompatibibity differences. Jolles et a!.(5) isolated water-soluble “active―substances after debipidation with a mixture of pynidine and acetic anhydnide;their criteria for activity was the enhanced appearance ofantibody-producing cells by the Jerne technique, increasedcarbon clearance, hypersensitivity to ovalbumin, and stimulation of rabbit anti-influenza antibodies. Less purifiedwater-soluble substances were thought to be active againstinfection of adult BALB/c x DBA/1 mice with encephalomyocarditis virus and adult BALB/c mice with Moboneysarcoma virus. Fractions injected i.v. 2 hr prior to viralinfection produced partial (dose-rebated) protection; watersoluble fractions were more effective than were the intactcell suspensions from which they were derived.

Dawes et a!. (3) described a soluble wall componentfound in culture broths of C. parvum and released fromsuspensions by acid and alkaline hydrolysis; it was anacidic polysacchanide with a molecular weight of 100,000to 150,000. It produced a precipitin reaction with antibodyto intact C. parvum and bound to human, mouse, andsheep erythrocytes. It was assumed that, since the component was extracted from a culture with antitumor activity, itwould possess the same property. Later, these investigators(7) extracted a lipid from C. parvum and Propionibacteriumfreudenreichii cultures with chloroform and methanol afteracid or alkali hydrolysis. In isogenic CBA mice the lipidcomponent had antitumor activity if given i.v. 1 day beforei.v. administration of fibrosarcoma cells or if given i.p. orS.C. , admixed with these cells. The lipid extracted from

alkali hydrolyzed cells, unlike whole bacteria, had littleimmunotherapeutic activity when given i.l.2 3 days afters.c. tumor implantation unless absorbed onto latex. Thelipid fraction isolated from acid-hydrolyzed cells had noactivity under the same conditions. These investigatorsconcluded that full antitumor activity depended on theintegrity of the whole bacterium.

We will present information regarding the activity of astrain of P. acnes and fractions thereof on our tumormodel, consisting of treating only 1 or 2 tumor-bearingfootpads; the tumor in the untreated foot pad we refer toas “pseudometastasis.―Our results show that tumor-inhib

2 The abbreviation used is: il., intralesional.

4150 CANCERRESEARCHVOL. 37

Antitumor Activity of Propionibacterium acnes (Corynebacteriumparvum) and Isolated Cytoplasmic Fractions1

I. Millman, A. W. Scott, and T. Halbherr

The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

SUMMARY

The tumor-inhibitory effect of an intralesional injectionof Propionibacterium acnes was of limited duration(“finite―).Our model was the DBA/2 syngeneic mouseinjected with P815 mastocytoma cells (5 x 10°)into eachrear footpad; only the left was treated, leaving the right asa “pseudometastasis.―The finite effect occurred at approximately 21 days after the first treatment. Subsequent i.p.treatments with P. acnes did not alter this effect, althoughthey increased mean survival time. With one footpad tumor,we achieved 22% cures with complete regression and nosign of metastatic growth. A RNA fraction from P. acnesproduced inhibition of tumor growth, but crude cell wallsand cell walls treated with Pronase had no effect. A P.acnes cytoplasmic fraction with tumor-inhibitory activitywas pebleted by high-speed centnifugation; this fractioninhibited P815 mastocytoma as fully as whole cells injectedin one-fifth the dose on a nitrogen basis and did not causea local inflammatory reaction. The activity of the pellet alsodiffered from whole cells in that it was equally inhibitory tothe pseudometastasis in the contralateral right rear footpad. The cytoplasmic fraction apparently contained at beasttwo activecomponentssinceactivitywasobtainedat twodilution levels. Such activity was relatively stable at 5°,butit was unstable at —30°.

INTRODUCTION

Certain anaerobic conyneform bacteria particularly Corynebacterium parvum (currently designated Propionibacterium acnes) have been extensively investigated for their

immunopotentiating antitumor properties (2). In treatmentof tumors, heat-killed suspensions of whole organismshave been used; they proved toxic, and side effects limitedtheir use (11). Several investigators have therefore begunto consider the merits of C. parvum fractions.

Most published accounts of C. parvum fractions havedealt mainly with the ability of C. parvum fractions tostimulatethe reticuboendothelialsystem as reflectedbychanges seen in carbon clearance, spleen and liverweights, and hypersensitivity to a variety of antigens. Prévotet al. (9) reported the bymphoreticubar stimulatory activityof cell wall fractions. Adlam et a!. (1) working also with

, This work was supported by USPHS Grants CA-i 7704, CA-06551 , RR

05539, and CA-06927 from the NIH and by an appropriation from theCommonwealth of Pennsylvania.

Received May 24, 1977; accepted August 16, 1977.

on March 30, 2016. © 1977 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

Antitumor Activity of P. acnes and Fractions

itory fractions can be separated from Inert and/or toxiccomponents of intact suspensions of P. acnes. These fractions are more effective than intact cells in inhibiting bothprimary and pseudometastatic lesions.

MATERIALS AND METHODS

Mice. Syngeneic DBA/2 males and females of approximately 17 to 24 g were earmarked and randomly housed 6/cage. When different preparations were to be compared,the preparations were injected into individual mice serially,by number, so thateach mouse in a cage receivedadifferent preparation. In this manner “cageeffect―andmouse weight differences did not contribute to changesregarded as results of treatment. Each treatment groupconsisted of a minimum of 12 mice.

Tumor Model. Mice were given injections in both left andright rear footpads with 5 x 10@P815 mastocytoma cells in0.05 ml Roswell Park Memorial Institute 1640 Medium. Thisinoculum resulted in a 100% tumor take, and the increasein footpad size was measurable at 7 days. Footpad measurements were made with Schnelltaster calipers, used tomeasure the diameter of the footpad, with an accuracy of0.1 mm. Measurements were made at 3- to 4-day intervals.Treatment was started at 7 days posttumor, the 1st injectionwas usually given i.l. into the left rear footpad; subsequentinjections were given i.p. 14 and 21 days posttumor. Sincethe tumor growing in the night footpad was not injectedwith P. acnes or fractions of P. acnes, the growth of thistumor was considered equivalent to a metastatic lesion(pseudometastasis).

Tumor. Methodsfor maintainingthe P815 mastocytomacells and the preparation of these cells for inoculation intomice were described previously (8).

P. acnes Suspensions.One strain of P. aCnes,ATCC11827, which showed the highest bevelofantitumor activity,was selected for these experiments. P. acnes was grown inActinomyces broth (BBL, Baltimore, Md.) at 37°for 7 days.The cells were harvested by centnifugation (2°)at 6000 rpmfor 30 mm. The pellet was resuspended in 0.85% NaCIsolution and heat inactivated at 65°for1 hr in a water bath.Thimerosal was used as a preservative at 0.01% concentration. All suspensions were adjusted to a concentration of14 mg (dry weight) per ml.

p. acnes Fractions. P. acnes was grown in 1-biter Enbenmeyer flasks containing 600 ml of Actinomyces broth. Theyields of 10 flasks were stoned at —30°as paste, withoutpreheating or preservatives. For disruption a single harvestof approximately 15 to 25 g (wet weight) was thawedovernight at 5°and homogenized. P. acnes cell walls andRNA were made by the procedure described by Youmansand Youmans (12) for mycobactenia. For all other fractionstested, P. acnes was resuspended in 0.1 M Tnis-HCI (pH7.5), instead of the sodium dodecyb sulfate-sucrose bufferused by Youmans and Youmans, at a concentration of 25 g(wet weight) per 400 ml. The suspension was first homogenized (VirTis 523 homogenizer) and then filtered through a60 mesh screen to remove clumps. During the entire procedurethe cellsuspensionwas keptinan icebath.Thesuspension was then passed through a French pressure

cell (Amicon Rapid Fill J4-3337 with a 1-inch-diameterpiston) at between 20,000 and 32,000 psi . It was difficult tomaintain a constant pressure due to occasional cloggingof the pressure cell by clumps of microorganisms. Thiscould be rectified by manipulation of the bleeding valve.Approximately 80% of the cells were disrupted by thisprocedure. The disrupted cell mass was centrifuged at6000 rpm in a refrigerated centrifuge (2°)for 1 hr. Thepellet was composed mainly of intact cells, partially disrupted cells, and cell wall debris. The opalescent supernatant was recentrifuged at 50,000 rpm (250,700 x g) for 4 hrin a Beckman L2 65B ultracentnifuge with a Ti 60 rotor. Thepellet from this was homogenized in 0.9% NaCI solutioncontaining 0.01% thimerosal. The final materials testedwere 0.25- to 4-fold concentrations, based on the originalvolume of disrupted material from the French pressurecell. These preparations were stored at either 5°or —30°(frozen). As a rube all preparations unless stated otherwisewere tested within 7 days.

Quantitation. Dry weights were determined by washing aportion of suspension in distilled water 3 times with centnifugation and by drying it in an oven at 60°overnight.Nitrogen values were determined by the procedure of Lannieta!. (6).

RESULTS

Antitumor Effect of P. acnes Preparation. Two groups ofmice are represented in Chart 1. The P. acnes-treatedanimals received an initial treatment of 0.7 mg i.l. into theleft rear footpad 7 days after inoculation of tumor. The 2ndand 3rd treatments of 0.7 mg i.p. were given 14 and 21days posttumor. The control group received comparableinjections of 0.9% NaCI solution. The tumor in the left rearfootpad of the P. acnes-treated mice showed a markedreductioningrowth rate,which was firstdetectableat17days and still evident at 28 days. Thereafter, the growthrate showed a sudden increase that paralleled that ofuntreated tumors in control animals. The reduction intumor size of the left footpad was significant at 17, 21 , 24,28, and 31 days posttumor as shown by the Mann-Whitney2-tailed probability test (p = 0.022, 0.0016, 0.00059,0.00083, and 0.0018, respectively). The untreated right rearfootpads were also affected although to a lesser extent;tumor inhibition was significant at 17, 21, 24, and 28 days(p = 0.0098, 0.036, 0.028, and 0.043, respectively). Almostall of the animals in this 2-footpad model eventually died asa result of metastatic spread, presumably from cells seededfrom the right rear footpad tumor. The mean survival timeof the P. acnes-treated animals was significantly extendedbeyond that of the control animals injected with 0.9% NaCIsolution; the former lived 54 ±0.96 days (S.E.), the latterlived 34 ±0.66 days.

Treatments in Mice Bearing Only 1 Footpad Tumor.Chart 2 shows a representative 33% of mice in an expeniment in which only the left rear footpads were inoculatedwith 5 x 10°P815 tumor cells. These animals were giveneither P. acnes or 0.9% NaCI solution i.l. 7 days later.Subsequent injections of 0.7 mg i.p. were given at 7-dayintervals up to 36 days. The group treated with 0.9% NaCI

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Similar results were obtained in 2-footpad experiments,regarding the variability of response in syngeneic animals.However, the effective period of the 1st i.l. treatment wasremarkably uniform in all experiments, suggesting that theantitumon effect of P. acnes is of limited duration (“finite―),lasting approximately 21 days from the 1st treatment withP. acnes.

Effectivenessof Addftionali.p. Treatments.These expeniments were designed to determine whether the 2 i.p.treatments given 14 and 21 days posttumon had any antitumon effect. One group of mice received the usual regimenof i.b. treatment at 7 days posttumor, followed by i.p.treatments at 14 and 21 days. Another group received thei.l. treatment only. The i.p. treatments had no effect on thesize of the tumor in either the left or the right footpad.However, there was a significant difference in survival time(Table 1). Those receiving additional i.p. treatments livedmuch longer. A 3rd group was treated with 3 0.7-mg i.p.injections 7, 14, and 21 days posttumor. Both left and rightfootpads were equally protected , and tumor inhibition wassignificant compared to controls given 0.9% NaCI solution;the inhibition was not as good as that produced in the leftrear footpad by i.b. treatment, but it was better than thatobserved in the contralateral right rear footpad. Twenty

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individual mice injected with tumor in the left rear footpad only. P. acnes[0.7 mg (dry weight) il.] was administered in the left rear footpad 7 daysposttumor, and 4 additional treatments were administered i.p. The resultsplottedshowa representative33%of the mice.Only1of the 4 curedmice isshown here. The finite effect occurs between 28 and 32 days when the rateof growth of the tumor changes. The additional i.p. treatments had no effecton this change. •,controls treated with 0.9% NaCI solution; 0. P. acnestreated mice.

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Chart 1. The inhibitory effect of heat-inactivated whole-cell preparationsof P. acnes (0.7 mg dry weight) injected into the left rear footpad on tumorgrowth. An il. treatment was given 7 days posttumor, and 2 additional i.p.treatmentsweregiven14and 21daysposttumor.x—x , injectedleft rearfootpads of controls treated with 0.9% NaCI solution; x - - - x , right rearfootpads of controls; 0—0, injected left rear footpads of mice treatedwith P. acnes; 0 - - - 0, right rear footpads of P. acnes-treated mice. Themeans (12 mice/group) of the left rear footpads show a reduction in size upto 28 days posttumor (21 days posttreatment). After this time the tumorgrowth resumes at a rate comparable to that of the controls (finite effect).The right footpads (pseudometastases) show a significantly decreased rateof growth.

solution showed a fairly uniform rate of development of thetumor, but the P. acnes-treated group showed a surprisingrange of responses. These fell into 3 categories. In someanimals the treated footpad regressed from measurementsof 3.0 mm or greater to a normal diameter of 1.6 to 1.8mm. In others there was a brief plateau in the tumor growthrate without regression. The 3rd and largest group showeda leveling oft of growth, starting at 15 to 17 days, followedby regression in tumor size until 27 to 32 days. Thereafter,if the size had not regressed to normal, there was a rapidacceleration of growth rate that paralleled that of the tumorin the control animals. The i.p. treatments given in thisexperiment did not delay the secondary growth spurt; thispattern was similar to that of other experiments when only3 i.p. treatments were given. Four of 18 P. acnes-treatedanimals (22%) showed regression to a normal-sized footpadat 32 days. These 4 animals were kept for 62 days aftertumor inoculation, sacrificed, and autopsied. There was noevidence of tumor at the primary site, nor were there anymetastases in 3 mice. The 4th mouse had a small recurrenttumor at the primary site, but there was no evidence ofmetastases.

p. i.p.

Days Post Tumor

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Four groups of mice received different treatmentregimens.Eachgroup comprised 12 mice, and treatment consisted ofP.acnes[0.7 mg (dry weight) administered either i.l. or i.p.asindicated].

The 1st treatment was administered 7 daysposttumor,andfurther treatments were administered 14 and 21 days posttu

mor.Treatment% surviving at 40daysi.l.,

i.p., i.p.58i.I.only17i.p.,

i.p., i.p.250.9%NaCIsolution 0

Antitumor Activity of P. acnes and Fractions

concentration produced a significant inhibition of tumorgrowth, which in some instances was as effective as theintact cell from which the fraction was produced. In contrast to what was seen in the whole cells, the cytoplasmicfractions produced an equal antitumor effect in both footpads.

A fraction pelleted at 250,700 x g (50,000 rpm) from thecytoplasmic fraction produced an inhibitory effect equal toor better than that seen with whole cells (Chart 4). The inhibition of tumor was equal in both footpads compared tothat seen with intact cells. This 50,000-rpm pellet fractionproduced no inflammatory response in the injected footpad,which uniformly followed injection of intact cells. The doseof the high-speed pellet (50,000 rpm) used in Chart 4 was0.019 mg nitrogen compared with that of 0.0925 mg for intact cells (approximately one-fifth the total nitrogen). A RNAfraction, isolated from this same batch of P. acnes by theprocedure of Youmans and Youmans (12), was also testedand plotted (Chart 4) for comparison; a 0.1-mg dose inhibitedtumor to a significant degree compared to that of controls.

DoseEffectof P. acnes HIgh-SpeedPelletand StabilIty.The high-speed pellet fraction of P. acnes was tested atvarying doses, from 0.25- to 4-fold concentration (Chart 5).The high-speed pellet produced significant inhibitory activity at 0.5- to 4-fold although not at 1-fold concentration.This finding is highly reproducible and has occurred with

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tumor growth. Treatments were administered il. 7 days posttumor and i.p.14 and 21 days posttumor. The greatest inhibitory effect is seen in micetreated with the 50,000 rpm pellet (@)by which both left and right footpadsareequallyinhibited.Eachpoint representsthe meanof 12 mice;solidline, right rear footpads;broken line, left rear footpad; + , controls treatedwith 0.9% NaCI solution; x , P. acnes-treated mice; 0, treatment with RNArich fraction.

five % of the animals in this group survived 40 days.Effectof P. acnes Cell Wall and RNA Fractions.Chart3

shows the results of fractions isolated from P. acnes by theprocedure used in other studies with mycobactenia (12).The yield of RNA was poor, only a fraction of that isolatedfrom tubercbe bacilli (12). Whether this resulted from harvesting P. acnes during the stationary growth phase is unknown at this time. The RNA fraction of P. acnes did, however, produce some inhibition of tumor growth. Graphs oftumor growth of the treated footpad tumors show that byDay 23 there is a significant decrease in tumor growth (p =0.02). Although the plot of results obtained with the cell wallfraction appeared to be inhibitory, none of the points weresignificantly different from the control group. Pronasetreated crude cell walls produced no antitumor effect. BothANA and cell wall fractions were tested in doses foundhighly effective with equivalent fractions isolated from tubercle bacilli.

p. acnes Cytoplasmic Fractions. To determine the bocation of the antitumor moiety(s), we used a 0.1 M Tnis-HCIbuffer (pH 7.5) rather than sodium dodecyl sulfate-sucroseto reduce the viscosity of the disrupted cell mixture. Thecytoplasmic fraction (minus 6000-rpm precipitate) at 0.5-fold

Table1Survivalof mice with different treatment regimens

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Chart 3. The effect of P. acnes RNA, crude cell walls, and Pronasetreated walls on tumor growth. The effectiveness of a P. acnes RNA-richfraction was limited . Crude cell walls and Pronase-treated walls wererelatively ineffective compared with that of whole cells. Each point reprosents the mean of 12 mice. The mice received an i.I. treatment at 7 daysposttumor.+, controlstreatedwith0.9%NaCIsolution;x ,P.acnes-treatedmice; 0, RNA-rich fraction; •,crude cell walls; V, Pronase-treated walls.

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!. Mi!!man et a!.

although not by the timing of the secondary growth spurt.If the treated footpad had regressed to normal size (1.8 mmor less) within 21 days of treatment, the animal could beconsidered “cured,―if no secondary rise in tumor growthwas seen. The untreated right footpad of P. acnes testanimals always showed a slower rate of growth comparedto the footpads of control animals and in some casesshowed regression. Scott (10) reported that i.v. administration of C. parvum resulted in antitumor activity of limitedduration. He found that multiple doses were less effectivethan a single dose and did not prevent this biphasic growthcurve. Our observations on the effect of multiple doses ofP. acnes (Chart 3) have indicated that 5 treatments given atweekly intervals did not change the time pattern of nesponse. The antitumon effect and the secondary growth oftumor were the same in those receiving 5 treatments as theywere in those receiving 1 i.l. treatment. There are 2 possibleexplanations ta consider: (a) the tumor cells change character with resultant change in their growth rate; (b) thereare limiting factors operating in the host, which determinethe period of effectiveness of P. acnes as an antitumonagent. If the secondary growth spurt was due to mutationsin tumor cells, it seems unlikely that this would occuralways at the same time as we have observed. We favor theexplanation that the rise is due to immunological changesin the host.

The experiments in which different treatment regimenswere compared revealed that the initial i.I. injection andnot subsequent i.p. treatments were the only factor affecting the size of the footpads. The i.p. treatments, however,caused increased survival time. It has been reported thatthe effect of P. acnes is primarily macrophage stimulation(4, 10). One can speculate that additional i.p. treatmentswould cause a relocation of activated macnophages thatotherwise might tend to localize at the 2 primary tumorsites. These activated macrophages could protect the animal from metastatic spread of the tumor. If this relocationdoes occur, it does not affect the antitumor effect inoperation at the primary sites.

The high-speed-pelleted fraction from P. acnes appearsto be different from any isolated tumor-inhibitory fractionthus far described. It appears to be different from cell wallfractions and their endotoxins or the RNA fraction equivalent to that isolated from tubercle bacilli (8). Unlike theRNA fraction the high-speed pellet appears to be stablewhen stored for 30 days at 5°;it is, however, not stablewhen stored frozen. The apparent lack of effect at mediandoses (Chart 5) could mean that there are 2 active moieties.

We observed consistently that i.b. treatment with wholecells produced local inflammation at the site of injection.An inflammatory reaction occurred only in the left P. acnestreated footpads. This local inflammatory response was nota significant part of the antitumor effect since fractions ofP. acnes with antitumor activity did not cause inflammation.The high-speed pellet from P. acnes did not cause aninflammatory response at the site of injection and, moreimportantly, produced equal inhibitory activity in both footpads, indicating that intimate contact between tumor andfraction is not essential for activity.

The value of immunopotentiation lies probably in prevention of metastases and not in elimination of primary tumor

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all other fractionated batches of P. acnes. The inflammatoryresponse observed on injection of intact cells is not observed with these fractions. Preliminary studies indicatethattheremay be atbeast2 inhibitorycomponents foundinthe high-speed pellet. This is based on the activity obtainedat 2 different concentrations.

Chart 5 also shows the effect of storage at —30°versus 5°of the P. acnes high-speed pellet. The latter did not undergoany change in antitumor activity. The material stored at—30°(1-step freezing and thawing only) lost most of itsantitumor activity.

DISCUSSION

The 2-footpad model was chosen to observe and measurethe effect of .1. treatment on both the directly treatedfootpads and the contralaterab footpads; the latter wereincluded to provide information on the probable effectiveness of treatment on metastatic growth . Therefore, thetumors implanted in the right footpads are consideredpseudometastases.

In our studies antitumor activity was first observable inthe treated footpad 17 days posttumor after an inflammatoryresponse, caused apparently from the injection of intactcell suspensions, had subsided (10 days posttreatment).Inhibition of tumor growth had lasted 21 to 23 days posttreatment (28 to 30 days posttumor) when a secondarygrowth spurt occurred. There was considerable variation inthe response of individual syngeneic mice to P. acnestreatment, shown by the extent of the antitumor response

20f 24 28 32 36i.p.

Post TumorChart 5. The effect of different doses of P. acnes fractions on tumor

growth. The 50,000 rpm fraction was active at 0.25-fold and 4-fold but wasless active at 1-fold, indicating the possiblepresenceof 2 active components. The stability of these fractions stored for 33 days at 5°or —30°isindicated. Each point represents the mean of 12 mice. Treatments wereadministered .1. 7 days posttumor and p. 14 and 21 days posttumor. Although mice received tumor in both footpads, only left footpad measurements are plotted for greater clarity. + , controls treated with 0.9% NaCIsolution; x , treatment with P. acnes whole cells; 0, 4-fold concentration(5°);V, 1-fold concentration (5u); L@,0.25-fold concentration (5°);•,4-fold concentration (—30°):V 1-fold concentration (—30°);A, 0.25-foldconcentration (—30°).

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Antitumor Activity of P. acnes and Fractions

mass. It is recognized that the value of nonspecific immunopotentiating agents such as Baci!!us Ca!mette-Guérinand C. parvum is as an addition to surgery and/or chemotherapy. The use of purified bacterial fractions that are freeof inflammatory on inert components would be preferablefor treatment of patients whose immune systems are alreadytaxed by immunosuppressive chemical and physical thenapy.

REFERENCES

1. Adlam, C., Reid, D. E., and Torkington, P. The Nature of the ActivePrinciple of Corynebacterium parvum. In: B. Halpern (ed), Corynebacterium parvum. Applications in Experimental and Clinical Oncology, pp.35-39.NewYork: PlenumPress,1975.

2. R. E. Buchanan and N. E. Gibbons (eds.) Bergey's Manual of Determinative Bacteriology, Ed. 8, pp. 639-640. Baltimore: Williams & Wilkins,1974.

3. Dawes, J., Tuach, S. J., and McBride, W. H. Properties of an AntigenicPolysaccharide from Corynebacterium parvum. J. Bacteriol., 120: 24-30, 1974.

4. Halpern, B. N., Prevot, A. R., Biozzi, G., Stiffel, C. Mouton, D., Morard,J. C., Bouthillier, Y., and Decreusefond, C. Stimulation de I'ActivitéPhagocytaire du Système Rèticulcendothdlial Provoqude par Corynebacteriumparvum. RESJ. ReticulcendothelialSoc., 1: 77-96,1963.

5. JoIles, P., Migliore-Samour, D., Korontzis, M., Floch, F., Maral, R., andWerner, G. H. Study on Soluble Substances Extracted from Corynebacterium parium. In: B. Halpem (ed), Corynebacterium parvum. Appplications in Experimental and Clinical Oncology, pp. 40-47. New York:Plenum Press, 1975.

6. Lanni, F., Dillon, M. L., and Beard, J. W. Determination of SmallQuantities of Nitrogen in Serological Precipitates and Other BiologicalMaterials. Proc. Soc. Exptl. Biol. Med., 74: 4-7, 1950.

7. McBride, W. H., Dawes, J., and Tuach, S. Antitumor Activity of Corynebacterium parvum Extracts. J. NatI. Cancer Inst., 56: 437-439, 1976.

8. Millman, I., Scott, A. W., Halbherr, T., Youmans, A. S., and Youmans,G. P. Mycobacterial Ribonucleic Acid: Comparison with MycobacterialCell Wall Fractions for Regression of Murine Tumor Growth. InfectionImmunity, 14: 929-933, 1976.

9. Prevot, A. A., Nguyen-Dang, T., and Thouvenot, H. Influence desParoisCellulairesdu Corynebacteriumparvum (Souche936B)sur leSystèmeRdticuloendothélial de Ia Souris. Compt. Rend., 267: 1061-1062,1968.

10. Scott, M. T. Corynebacterium parvum as a Therapeutic Antitumor Agentin Mice. 1. Systemic Effects from Intravenous Injection. J. NatI. CancerInst., 53: 855-860, 1974.

11. Woodruff, M. F. A., Clunie, G. J. A., McBride, W. H., McCormack, R. J.M. , Walbaum, P. R. , and James, K. The Effect of Intravenous andIntramuscular Injection of Corynebacterium parvum. In: B. Halpern(ed), Corynebacterium parvum. Applications in Experimental and Clinical Oncology, pp. 383—388.New York: Plenum Press, 1975.

12. Youmans, A. S., and Youmans, G. P. Factors Affecting ImmunogenicActivity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations.J. Bacteriol., 99: 42-50, 1969.

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and Isolated Cytoplasmic Fractions(Corynebacterium parvum)Propionibacterium acnesAntitumor Activity of

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