Vol. 27, No. 2INFECTION AND IMMUNITY, Feb. 1980, p. 643-6490019-9567/80/02-0643/07$02.00/0

Effects of Activated Macrophages on Nocardia asteroidesGREGORY A. FILICE,' BLAINE L. BEAMAN,2 AND JACK S. REMINGTON`*

Division of Infectious Diseases, Department ofMedicine, Stanford University School ofMedicine, Stanford,California 94305,1 Division ofAllergy, Immunology and Infectious Diseases, Palo Alto Medical Research

Foundation, Palo Alto, California 94301,1 and Department ofMedical Microbiology, University ofCalifornia School ofMedicine, Davis, California 95616Y

The mechanisms) of host resistance against Nocardia asteroides has not beenwell defined. Since disease due to N. asteroides frequently occurs in patients withimpaired cell-mediated immunity, we studied the interaction of N. asteroideswith activated and control mouse peritoneal macrophages. Activated macro-phages were from mice infected with Toxoplasma gondii or injected with Cory-nebacterium parvum. N. asteroides in the early stationary phase (>99% in thecoccobacillary form) was used for challenge of macrophage monolayers. Growthof two strains of N. asteroides was markedly inhibited in activated macrophages,whereas N. asteroides grew well in control macrophages. Quantitation of macro-phage-associated N. asteroides indicated that activated macrophages killed 40 to50% of N. asteroides within 6 h (P < 0.002). In control macrophage preparations,it appeared as if Nocardia filaments extended from within macrophages to theoutside, and many of these filaments appeared to have extended to and thengrown through neighboring macrophages. In activated macrophage preparations,Nocardia remained in the coccobacillary form in most macrophages. Controlmacrophage monolayers were almost completely overgrown with and destroyedby Nocardia 20 h after challenge, whereas activated macrophage monolayersremained intact. Nocardia that grew in control macrophages were not acid-alcohol fast or only weakly so, whereas the few Nocardia that grew in activatedmacrophages were strongly acid-alcohol fast. Our results indicate that activatedmacrophages may be important in host defense against N. asteroides.

Nocardia asteroides causes severe, often fatalinfection in immunocompromised patients (19,23) and Nocardia infection has been increas-ingly recognized in recent years (5). At StanfordUniversity Hospital, Nocardia infection had oc-curred in 21 of 165 patients who had receivedheart transplants as of December 1978 (G. L.Simpson, E. B. Stinson, and J. S. Remington,manuscript in preparation). In persons withoutdemonstrable evidence of a predisposing condi-tion, Nocardia infection occurs less commonlybut still with serious consequences (23).The mechanisms) of resistance to Nocardia

infection is not known. Prior studies in a mousemodel by Krick and Remington suggest thatactivated macrophages are important in resist-ance (18). To elucidate the role of activatedmacrophages in resistance to Nocardia infec-tion, we examined the interactions of N. aster-oides with activated and control mouse perito-neal macrophages in vitro.

(Portions of these results were presented atand appeared in the abstracts of the 11th Inter-national Congress of Chemotherapy and the19th Interscience Conference on Antimicrobial

Agents and Chemotherapy, 1 to 5 October, 1979,Boston, Mass.)

MATERIALS AND METHODSN. asteroides. Both strains of N. asteroides we

used had been isolated originally from patients withdisseminated nocardiosis. Strain GUH-2 is highly vir-ulent for mice; strain 14759 (American Type CultureCollection [13]) is less virulent for mice. Intravenousdoses of early stationary-phase organisms which kill50% of mice are 8.7 x 105 and 8.5 x 106 for strainGUH-2 and strain 14759, respectively (7). Thesestrains have been used extensively in experiments inmice (6, 7, 13), rabbits (13), and guinea pigs (13). Theeffect of rabbit alveolar macrophages on these strainshas also been reported (3, 4, 9).

Before use, the strains were maintained in brothand were passaged through mice. Infected kidneysfrom the mice were transferred into brain heart infu-sion broth, as previously described (7). To obtainpreparations in which the majority of Nocardia werein the coccobacillary form, the organisms were har-vested during the early stationary phase of growth (8).After the brain heart infusion broth cultures were

centrifuged at 60 x g for 5 min to remove clumps andfilaments, the supernatants containing coccobacillaryforms were centrifuged at 900 x g for 15 min and

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644 FILICE, BEAMAN, AND REMINGTON

suspended in phosphate-buffered saline, pH 7.2. Theconcentrations of organisms in the suspensions weredetermined by optical density at 580 nm. The organ-isms were brought to the desired concentration inmedium 199 (M199) plus 10% fetal calf serum (FCS).(M199 and FCS were obtained from GIBCO Labora-tories, Grand Island, N.Y.).

Sources of macrophages. Macrophages were ob-tained from the peritoneal cavities of Swiss Websterfemale mice (Simonsen Laboratories, Gilroy, Calif.).Activated macrophages (defined by their ability toinhibit the growth of Toxoplasma gondii [1, 21]) wereobtained from 13-week-old mice chronically infectedwith T. gondii (14) or from 6-week-old mice injected7 days earlier with Corynebacterium parvum (7 mg,dry weight, per ml; lot CA 592A; kindly provided byJohn Whisnant, Burroughs Wellcome Co., ResearchTriangle Park, N.C.), as previously described (29).Control macrophages were from age-matched litter-mates, except in one experiment in which activatedmacrophages from mice that were injected with C.parvum were compared with peritoneal macrophagesfrom mice that had chronic renal infection with N.asteroides GUH-2. Peritoneal macrophages from theNocardia-infected mice were not activated to inhibitthe growth of T. gondii.Macrophage culture. Peritoneal exudate cells

were harvested in Hanks balanced salt solution, aspreviously described (22), and suspended in M199 +10% fetal calf serum at a concentration of 4 to 5.7cells/ml. From 0.5 to 0.7 ml of the cell suspensions wasseeded onto glass cover slips (22 by 11 mm or 22 mm2)in plastic petri dishes (35 by 10 mm) or into eachchamber of four-chamber Lab-Tek slides (Lab-TekProducts, Naperville, Ill.). For all experiments, con-centrations and inoculum sizes were such that 8.3 x105 peritoneal exudate cells were seeded onto eachsquare centimeter of the cover slips or the floors ofthe chambers. After approximately 3 h of incubationat 370C in 5% C02, nonadherent cells were washed offwith saline, and the remaining cells were challengedimmediately thereafter. Approximately 50% of perito-neal exudate cells obtained in this way are adherent,and approximately 95% of the adherent cells have themorphology of macrophages and phagocytose heat-killed Candida albicans (29).

Challenge of macrophage monolayers with N.asteroides. Monolayers were challenged with Nocar-dia in a ratio of 2 to 8 organisms per macrophage andincubated for 1 to 3 h. The choice of organism-to-macrophage ratio for these experiments was based onthe earlier observation that a similar ratio causedinfection ofapproximately 50% ofnormal macrophageswith from one to five organisms in each infected mac-rophage (B. L. Beaman, unpublished data). The mono-layers were then washed twice with 1 ml of saline eachtime, fresh medium was added, and the preparationswere reincubated. At various intervals, the numbers ofviable Nocardia associated with macrophages and insupernatant mediums were determined as describedbelow. Lab-Tek slides or cover slips run in parallelwere stained with either Gram stain or Kinyoun mod-ified acid-fast stain.

For enumeration of viable non-macrophage-associ-

ated Nocardia, supernatants were removed from thecover slip cultures, and the cover slips were washedthree times with 1 ml of saline each time. The washeswere added to the supernatants, and the resultantsuspensions were dispersed by vigorous mixing in aVortex blender and then serially diluted. Portions ofthe dilutions were plated on brain heart infusion agar.In preliminary experiments to determine the efficiencyof this washing procedure, Nocardia were added tocell-free cover slips in petri dishes, the supernatantswere removed after 30 min, and the cover slips werewashed in the manner described. When these methodswere used, approximately 99% of Nocardia was re-covered.

For enumeration of viable macrophage-associatedNocardia, macrophages on the washed cover slipswere lysed by incubating the cover slips in 2 ml ofdistilled water for 15 min. The cover slips were thenscraped with a rubber policeman and washed twicewith 1 ml of distilled water each time. The washeswere combined with the lysates, and the resultantsuspensions were dispersed by vigorous mixing in aVortex blender and then serially diluted. Portions ofthe dilutions were plated on brain heart infusion agar.Suspension of these Nocardia strains in distilled waterfor 15 min does not affect their viability (unpublisheddata).

Challenge of macrophage monolayers with T.gondii. In parallel with Nocardia challenge, macro-phage monolayers were challenged with T. gondii, aspreviously described (1). Macrophages were consid-ered activated if they inhibited multiplication of T.gondii (1, 21).

Analysis ofthe effects ofmacrophages on mac-rophage-associated Nocardia. To analyze the ef-fects of macrophages on the Nocardia that were mac-rophage associated at the end of the 1- to 3-h challengeperiods, the numbers of macrophage-associated No-cardia at the end of the challenge periods were com-pared with the numbers of Nocardia in the wholepreparations (sum of Nocardia associated with mac-rophages and Nocardia in the supernatant medium)at later times. Immediately after the challenge suspen-sions were removed and the monolayers were washed,essentially all Nocardia in the preparations were mac-rophage associated. Therefore, Nocardia that wereisolated from the whole preparations at later timepoints originated from the populations of macrophage-associated Nocardia present immediately after chal-lenge.

Statistics. Analyses of the differences betweenmeans were performed by using the Student's t test.

RESULTS

The results of interactions of N. asteroidesGUH-2 with activated macrophages from micechronically infected with Toxoplasma and withmacrophages from age-matched controls areshown in Fig. 1. One hour after challenge, manyNocardia in control macrophages were in theform of short filaments (Fig. 1A), whereas almostall Nocardia in activated macrophages were in

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FIG. 1. Interactions of N. asteroides GUH-2 with activated macrophages from Toxoplasma-infected miceand with macrophages from controls (Gram-stained preparations). (A) At 1 h after initiation of challenge,filament formation has begun in Nocardia (arrow) within control macrophages (x200). (B) At 1 h afterinitiation of challenge, Nocardia within activated macrophages are in coccobacillary form (x200). (C) At 3 h,Nocardia in many control macrophages have grown extensively (x200). (D) At 3 h, Nocardia in most activatedmacrophages remain in the coccobacillary form (x200). (E) At 6 h, macrophages in control preparationsappear to have migrated toward cells containing Nocardia and form multicellular plaques (x35). (F and G)Higher magnifications (x100 and x200, respectively) ofpreparation shown in E reveal growth of Nocardiawhich apparently stimulated plaque formation. (H) At 12 h, growth of Nocardia in control preparations ismore extensive, and the plaques surrounding centers ofgrowth have enlarged (x80). (I) On higher magnifi-cation of preparation shown in H, Nocardia filaments intertwine extensively through and around macro-phages; some filaments clearly appear to be in vacuoles (arrow) (x200). (J) At 20 h, control macrophagemonolayers are overgrown and destroyed by Nocardia (x20). (K) At 20 h, although Nocardia have grown insome areas, the activated macrophage monolayers are still intact (x20). (L) On higher magnification ofpreparation shown in K, growth of Nocardia is still markedly inhibited in many activated macrophages(x200).

the coccobacillary form (Fig. 1B) and appearedas they did in the original inoculum. Most No-cardia appeared to be intracellular, althoughsome may have been adherent to macrophage

surfaces. At 1-, 3-, and 6-h time points in thisand the two experiments described below, thedensities of activated and control macrophagemonolayers were similar. By 3 h after challenge,

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646 FILICE, BEAMAN, AND REMINGTON

Nocardia filaments in control macrophages hadelongated, and branching was apparent (Fig.1C). In almost all activated macrophages at thistime, Nocardia were still in the coccobacillaryform (Fig. iD). At later time periods, extensivearborization ofNocardia filaments was observedin control macrophage preparations; it appearedas if Nocardia filaments had extended fromwithin the macrophages to the outside, andmany of these filaments appeared to have ex-tended to and then grown through neighboringmacrophages (Fig. 1F-I). In activated macro-phage monolayers, similar filamentous growthoriginated in a few isolated macrophages but, ingeneral, growth of Nocardia was markedly in-hibited (Fig. 1L). Control macrophages tendedto cluster around the growing filaments of No-cardia by 6 h (Fig. 1E, F, H); this phenomenonwas not observed in activated macrophagemonolayers. By 20 h, almost all cells in controlmacrophage monolayers were destroyed (Fig.1J), whereas most cells in activated macrophagemonolayers were still intact (Fig. 1K).

In the experiment depicted in Fig. 1, the num-ber of Nocardia recoverable immediately afterthe 1-h challenge appeared to be fewer fromactivated than from control macrophage prepa-rations (Fig. 2a), but the difference was notsignificant. After the supernatants were removedand the monolayers were washed at the end ofthe 1-h challenge period, the numbers of mac-rophage-associated Nocardia were similar incontrol and activated macrophages (Fig. 2b).Thereafter, the numbers of Nocardia in thewhole preparations (sum ofNocardia associatedwith macrophages and Nocardia in the super-natant medium) reflected the interactions ofmacrophages with the Nocardia that were as-sociated with macrophages at the end of the 1-hchallenge period (see Materials and Methods).Three hours after challenge, the numbers ofNocardia in activated and control macrophagepreparations had not changed from those ob-served at the end of the challenge period. By 6h, the numbers of Nocardia in activated mac-rophage preparations were significantly reducedwhen compared with the numbers in similarpreparations at 3 h (P < 0.0001) or with thenumbers in control macrophage preparations at6 h (P < 0.002) (Fig. 2b). By 12 h, the numbersof Nocardia increased in both types of macro-phage preparations, but the numbers in acti-vated macrophage preparations were still signif-icantly less than in control macrophage prepa-rations (P < 0.001). After 12 h, extracellularorganisms grew profusely in a pellicle on thesurface of the medium in both types of macro-phage preparations, precluding meaningful in-terpretation of colony counts.

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FIG. 2. Interactions of N. asteroides GUH-2 withactivated macrophages from Toxoplasma-infectedmice (E) and with macrophages from controls (0).For each time point after challenge, data representthe mean (± standard deviation) offour samples. (a)Mean colony-forming units of N. asteroides in acti-vated and control macrophage preparations 1 h after3.8 X 106 viable Nocardia were added. For eachsample, counts of Nocardia in supernatant mediaand Nocardia associated with macrophages weresummed; the means reflect effect of macrophages onthe inoculum. (b) Mean colony-forming units of mac-rophage-associated Nocardia in macrophage prepa-rations immediately after washing (1 h) and Nocar-dia in whole preparations at later time points (seetext).

A second experiment with N. asteroidesGUH-2 was performed to determine whetherthe inhibition of growth of macrophages acti-vated by Toxoplasma infection was unique tothis method of activation. The interactions of N.asteroides GUH-2 with macrophages from C.parvum-injected mice (macrophages activatedto kill Toxoplasma) were compared with theinteractions of N. asteroides GUH-2 with mac-rophages from Nocardia-infected mice (macro-phages which were not activated to kill Toxo-plasma and served as controls). Examination ofstained cover slips and results of cultures in thisexperiment revealed that growth of Nocardia inmacrophages from Nocardia-infected mice wassimilar to that in control macrophages in otherexperiments. Growth of Nocardia in C. parvum-activated macrophage preparations was mark-edly inhibited when compared with growth inmacrophage preparations from Nocardia-in-fected mice. The inhibition observed with C.parvum-activated macrophages in this experi-ment was similar to that observed with Toxo-plasma-activated macrophages in the experi-ment described above.To determine whether differences between in-

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ACTIVATED MACROPHAGES AND N. ASTEROIDES 647

teractions of control and activated macrophageswith Nocardia were unique to the GUH-2 strain,interactions of the less virulent 14759 strain ofNocardia with macrophages from C. parvum-injected mice and age-matched controls werestudied. Examination of stained preparations in-dicated that proliferation of Nocardia occurredin control macrophages but not in activatedmacrophages, as in the previously described ex-periments. Colony counts indicated that thenumbers of Nocardia decreased in the first hourin both control and activated macrophage prep-arations (Fig. 3a). However, immediately afterthe monolayers were washed at 1 h, there weresignificantly more Nocardia associated with ac-tivated than with control macrophages (P <0.005) (Fig. 3b). The numbers of Nocardia incontrol macrophage preparations remained thesame at 1, 3, and 6 h and then increased by 12 h.In contrast, there were significantly fewer No-cardia in activated macrophage preparations at3 h than at 1 h (P < 0.002). Although countsrose in activated and control macrophage prep-arations by 12 h, there were significantly fewerNocardia in activated than in control macro-phage preparations (P < 0.001) at this time.At 6 h and at later time points, the few No-

cardia that had elongated in activated macro-phages were strongly acid-alcohol fast. In con-trast, Nocardia that had elongated in controlmacrophages were not acid-alcohol fast or onlyweakly so. The Nocardia in the challenge sus-pensions and those that had remained in thecoccobacillary form in activated macrophageswere not acid-alcohol fast.

DISCUSSIONThese data provide evidence that activated

macrophages may be important in defenseagainst Nocardia infection. The ability of acti-vated peritoneal macrophages to inhibit thegrowth of N. asteroides as compared with thatof control peritoneal macrophages was apparentboth by microscopy and by quantitation of via-ble Nocardia in macrophage preparations atvarious intervals after challenge. The results ofthe quantitative analyses revealed that activatedmacrophages killed a significant portion of theNocardia in the challenge inocula. The effectsof activated macrophages were not limited toone method of activation or to one strain ofNocardia; this lends further support for theimportance of this line of defense. Additionalevidence that the effects of activated macro-phages upon Nocardia differed from those ofcontrol macrophages was the observation thatthe few Nocardia that grew in activated mac-rophage preparations were strongly acid-alcohol

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FIG. 3. Interactions of N. asteroides 14759 withactivated macrophages from C. parvum-treated mice(A) and with macrophages from controls (x). Foreach time point after challenge, data represent themean (± standard deviation) of four samples. (a)Mean colony-forming units of N. asteroides in acti-vated and control macrophagepreparations 1 h after7.2 x 106 viable Nocardia were added. Means arederived as described in the legend to Fig. 2a. (b)Mean colony-forming units of macrophage-associ-ated Nocardia in macrophage preparations immedi-ately after washing (1 h) and Nocardia in wholepreparations at later time points. Means are derivedas in Fig. 2b.

fast, whereas those that grew in control macro-phage preparations were not. The degree of re-sistance ofNocardia to decolorization with acid-alcohol depends upon the conditions of growth.Strains of Nocardia growing in tissues of ani-mals or humans are often acid-alcohol fast,whereas the same strains grown in cell-free me-dia are often not. Enhanced resistance of Nocar-dia to decolorization with acid-alcohol reflectsaltered lipid composition of the cell envelopeand may be related to enhanced ability to evadedestruction by host defenses. Further study ofsuch changes in the cell envelope of Nocardiamay lead to a better understanding of the path-ogenesis of Nocardia infection. Recent studiesin rabbits suggest that alveolar macrophagesmay also be activated to inhibit Nocardia (4).

In control macrophage preparations, it ap-peared as if macrophages migrated toward in-fected cells, and large plaques were formedwithin hours. Similar migration has been ob-served in studies of interactions of N. asteroides14759 with alveolar macrophages from normalrabbits (3) and in studies of interactions of Lis-teria monocytogenes with peritoneal macro-phages from mice that had previously been im-munized with L. monocytogenes (20).

In previous studies, a number of strains of N.asteroides were found to grow intracellularly tovarious degrees in vitro when incubated withmouse peritoneal macrophages (10), guinea pigperitoneal and alveolar macrophages (24, 25),and rabbit alveolar macrophages (3, 9). Bour-

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geois and Beaman (10) found that a strain of lowvirulence for mice was able to survive withinnormal mouse peritoneal macrophages. The No-cardia could not be detected by Gram stain, andonly low numbers were detectable by culture onbrain heart infusion agar. However, by 8 days ofincubation, L-form-like variants were observedwithin macrophages by electron microscopy andcould be isolated on hypertonic media. It ispossible that reductions in numbers of viableNocardia recoverable from activated macro-phages in the present study may have reflectedtransformation of Nocardia into L-form-likevariants. If this did occur, it still does not refutethe observation that growth of typical forms ofNocardia was markedly inhibited by activatedmacrophages.

Results of earlier in vivo experiments withmice (18) suggested that activated macrophagesare important in host defense against Nocardia.Immunization with N. asteroides resulted inprotection against subsequent challenge with N.asteroides injected intraperitoneally in hog gas-tric mucin. Marked protection was also observedin mice known to have activated peritoneal mac-rophages as a result of infection with other or-ganisms. Peritoneal macrophages from mice im-munized with N. asteroides 3 weeks earlier in-hibited uptake of tritiated thymidine by tumortarget cells, which in the model used correlateswith activation of macrophages (17). Transfer ofserum did not protect against subsequent chal-lenge with Nocardia in this model. To ourknowledge, transfer of resistance by serum hasonly been reported by Nelson and Henrici. In anabstract by E. Nelson and A. T. Henrici, (Proc.Soc. Exp. Biol. Med. 19:351, 1922), they statedthat transfer of serum from vaccinated rabbitspartially protected guinea pigs against challengewith Actinomyces gypsoides (renamed N. aster-oides [11]). Although the studies of Krick andRemington (18) provided evidence that perito-neal macrophages from Nocardia-infected miceare activated whereas peritoneal macrophagesfrom Nocardia-infected mice in the presentstudy were not activated, the data in these twostudies are not necessarily inconsistent. Differ-ent strains, doses, and routes of inoculation ofN. asteroides were used, and macrophages werestudied with different assays of activation and atdifferent intervals after infection.

Previous investigations (6, 12) have revealedthat athymic mice were more susceptible thantheir heterozygous littermates or normal mice tochallenge with Nocardia, suggesting a role forthymus-derived (T) lymphocytes in protectionagainst Nocardia infection. In the Krick andRemington model, attempts to transfer resist-

ance to Nocardia infection by transferringspleen cells from immune to normal mice wereunsuccessful when they used doses that hadbeen sufficient for transfer of resistance to otherinfections (18). Sundararaj and Agarwal (26-28)concluded from their studies in which a guineapig model was used that resistance to Nocardiainfection could be transferred by spleen cells,but the small numbers of animals employed intheir studies and the assays of resistance per-formed make the findings difficult to interpret.Since transfer of spleen cells was not betweenisogeneic animals, genetic differences betweendonor and recipient cells may have preventedoptimal immunological function. The availabledata suggest that resistance to experimental No-cardia infection is mediated at least in part byT-lymphocytes.

Nocardiosis in humans occurs most frequentlyin immunocompromised patients, most ofwhomhave had underlying conditions or therapyknown to result in marked defects in cell-me-diated immunity (5, 19, 23, 30; G. L. Simpson, E.B. Stinson, and J. S. Remington, manuscript inpreparation). Data in animals now suggest thatT-lymphocytes and macrophages play impor-tant roles. Nocardiosis has also been reported inpatients with chronic granulomatous disease(16) in which there is a defect in oxidative killingmechanisms in polymorphonuclear leukocytes,monocytes, and alveolar macrophages (2, 15).Which components) of the immune system inhumans is important in defense against nocar-diosis remains to be determined.

ACKNOWLEDGMENTS

This work was supported by grants Al 04717, CA 06341,and Al 13167 from the National Institutes of Health. G.A.F.is the recipient of an Edith Milo Fellowship Award.We gratefully acknowledge the excellent technical assist-

ance of Saundra Smith and Steve Scates.

LITERATURE CITED

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2. Babior, B. M. 1978. Oxygen-dependent microbial killingby phagocytes. N. Engl. J. Med. 298:659-668, 721-725.

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4. Beaman, B. L. 1979. Interaction of Nocardia asteroidesat different phases of growth with in vitro-maintainedmacrophages obtained from the lungs of normal andimmunized rabbits. Infect. Immun. 26:355-361.

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17. Krahenbuhl, J. L., L. H. Lambert, Jr., and J. S.Remington. 1976. The effects of activated macro-phages on tumor target cells: escape from cytostasis.Cell. Immunol. 25:279-293.

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Nocardia infection in heart transplant patients. Ann.Intern. Med. 82:18-26.

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21. McLeod, R., and J. S. Remington. 1977. Influence ofinfection with Toxoplasma on macrophage function,and role of macrophages in resistance to Toxoplasma.Am. J. Trop. Med. Hyg. 26:170-186.

22. McLeod, R., and J. S. Remington. 1977. Studies on thespecificity of killing of intracellular pathogens by mac-rophages. Cell. Immunol. 34:156-174.

23. Palmer, D. L., R. L. Harvey, and J. K. Wheeler. 1974.Diagnostic and therapeutic considerations in Nocardiaasteroides infection. Medicine 53:391-401.

24. Splino, M., V. Merka, and F. Kyntera. 1975. Phagozy-tose und intrazellulare Proliferation von Nocardia as-teroides (Stamm Weipheld) in Zellstrukturen in vitro.1. Alveolare Makrophagen von Meerschweinchen. Zen-tralbl. Bakteriol. Hyg. Parasitenkd. Infektionskr. Abt.1 Orig. A 232:334-340.

25. Splino, M., V. Merka, and F. Kyntera. 1976. Phagozy-tose und intrazellulare Proliferation von Nocardia as-teroides (Stamm Weipheld) in Zellstrukturen in vitro.2. Peritoneale Makrophagen von Meerschweinchen.Zentralbl. Bakteriol. Hyg. Parasitenkd. Infektionskr.Abt. 1 Orig. A 235:512-520.

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27. Sundararaj, T., and S. C. Agarwal. 1977. Cell-mediatedimmunity to Nocardia asteroides induced by its ribo-nucleic acid protein fraction. Infect. Immun. 18:253-256.

28. Sundararaj, T., and S. C. Agarwal. 1978. Relationshipof macrophages to cell-mediated immunity in experi-mental Nocardia asteroides infection. Infect. Immun.20:685-691.

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