INFECTION AND IMMUNITY, Feb. 1981, p. 716-722 0019-9567/81/020716-07$02.00/0

Vol. 31, No. 2

Effects of Muramyl Dipeptide Treatment on Resistance to Infection with Toxoplasma gondii in Mice JAMES L. KRAHENBUHL,' * SOMESH D. SHARMA,2 RODOLFO W. FERRARESI,3 AND JACK S. REMINGTON2 4 Leprosy Research Unit, U. S. Public Health Service Hospital, San Francisco, California 94118;' Palo Alto Medical Research Foundation, Palo Alto, California 94301;2 Syntex, Inc., Palo Alto, California 94304;3 and Stanford University School of Medicine, Stanford, California 943054

Studies were carried out to determine whether treatment of mice with the synthetic adjuvant muramyl dipeptide afforded any resistance to infection with the obligate intracellular protozoan Toxoplasma gondii. Marked resistance to lethal challenge infection was observed in CBA but not C57BL/6 mice pretreated with muramyl dipeptide. In CBA mice, a single muramyl dipeptide treatment administered 14, 7, or 4 days before Toxoplasma challenge did not afford protection, whereas mice treated at -1 day were highly resistant. Additional studies carried out to investigate the mechanisms underlying the enhanced resistance to Toxoplasma in muramyl dipeptide-treated mice failed to reveal either enhanced cytolytic antibodies to the parasite or evidence that peritoneal macrophages from treated mice were activated as determined in vitro by their microbicidal capacity for Toxoplasma or cytotoxic capacity for tumor target cells.

The synthetic compound N-acetyl muramylL-alanyl-D-isoglutamine (muramyl dipeptide [MDP]), when incorporated in oil, appears to possess the minimal chemical structure necessary to replace the mycobacterial cell wall component in Freund complete adjuvant (1, 7, 28). Moreover, in responsive strains of mice, administration of MDP or MDP analogs in an aqueous solution with the appropriate antigen can markedly enhance both in vivo (3, 23) and in vitro (8, 20, 21) antibody responses. In addition to these adjuvant effects, treatment of mice with MDP and certain MDP analogs and isomers stimulates remarkable resistance to infection with Klebsiella pneumoniae (5, 31), Trypanosoma cruzi (17), Pseudomonas aeruginosa (27, 30), Candida albicans (27) and Streptococcus pneumoniae (13). The goals of the present studies were twofold: to determine whether treatment of mice with MDP enhances resistance to challenge with the obligate intracellular protozoan Toxoplasma gondii and to determine whether treatment in vivo with MDP activates macrophages as measured by their in vitro microbicidal (for Toxoplasma) or cytotoxic effects for tumor cells. MATERIALS AND METHODS Mice. Swiss Webster (SW) mice were obtained from Simonsen Laboratories, Gilroy, Calif. C57BL/6J and CBA/J mice were obtained from Jackson Laboratories, Bar Harbor, Maine. All mice were female and weighed 16 to 18 g at the beginning of each experiment. MDP. MDP was obtained from Gordon Jones, Di716

vision of Synthetic Chemistry, Syntex Research, Inc., Palo Alto, Calif. The appropriate amount of MDP was dissolved in 0.85% saline and sterilized by filtration (Millex, 0.45 ,um, Millpore Corp.) before each treatment. Unless mentioned the dose of MDP employed was 80 mg/kg subcutaneously (s.c.) in the area of the groin. C. parvunL A killed suspension of Corynebacterium parvum (7 mg/ml, dry weight, lot CA 380) was obtained from John Whisnant, Burroughs Weilcome Co., Inc., Research Triangle Park, N.C. Mice were treated with a single intraperitoneal (i.p.) injection of 1,400 ,Ag of C. parvum 7 days before challenge with Toxoplasma. Toxoplasma challenge. Tachyzoites of the relatively avirulent C56 strain of T. gondii were obtained and employed as described previously (35). After i.p. challenge with various doses of C56 Toxoplasma, percent dead was recorded in each group of mice, and the significance of protection afforded by MDP treatment was evaluated by chi-square analysis. Macrophage cultures. At varying intervals after treatment with MDP, C. parvum, or saline, cells were harvested from the peritoneal cavities of 7 to 10 SW, C57BL/6, or CBA as described previously (34). Peritoneal cells were washed by centrifugation at 150 x g and resuspended at a concentration of 4 x 106 peritoneal cells per ml in the appropriate tissue culture medium containing 10%o fetal calf serum and antibiotics (100 U of penicillin per ml and 100 ,g of streptomycin per ml). For determination of the cytotoxic capacity of macrophages for tumor cells, Dulbecco modified minimum essential medium (GIBCO, Berkeley, Calif.) was employed, and 0.5-ml aliquots of each peritoneal cell suspension were seeded into wells of Linbro tissue culture plates (16 mm; Fb-16-24-TC; Linbro Chemical Co., Vineland, N.J.). For determina-

VOL. 31, 1981

EFFECTS OF MDP ON TOXOPLASMA INFECTION IN MICE

tion of the microbicidal capacity of macrophages, medium 199 (GIBCO) was employed, and 0.5-ml aliquots of each peritoneal cell suspension were seeded into Linbro culture wells containing round glass cover slips (15-mm diameter, Bellco Glass Co., Vineland, N. J. [27a]). After 2 h of incubation, nonadherent cells were removed by washing with warm (37°C) saline, and the macrophage monolayers were challenged with either 5"Cr-labeled EL-4 tumor celLs or Toxoplasma tropho-

zoites. Tumor target cells. EL-4 lymphoma cells, syngeneic for C57BL/6 mice, were maintained by twice weekly passage in Dulbecco-modified minimum essential medium-10% fetal calf serum. Cytotoxicity assay. The assay for in vitro release of 51Cr from target cells was adapted from the methods used by Cerottini and Brunner (2). Briefly, EL-4 cells (5 x 106/ml) were resuspended in tris(hydroxymethyl)aminomethane-phosphate-buffered saline (pH 7.4), labeled for 30 min with 100 ,uCi of 61Cr sodium chromate (specific activity, 100 to 300 Ci/mmol; New England Nuclear, Gardena, Calif.) and washed three times by centrifugation (150 x g) in 10 ml of Dulbeccomodified minimum essential medium-10% fetal calf serum. The cells were adjusted to 105 viable (trypan blue exclusion) cells per ml, and 1.0 ml of the cell suspension was immediately added to empty control wells or wells containing monolayers of control macrophages or macrophages from treated mice. Twenty-four hours after challenge, the amount of 5"Cr released into the supernatant in the presence or absence of macrophages was determined by counting in a Beckman Autogammacounter. The percentage of 61Cr released was determined by the formula (E - SI R -S) x 100, where E = experimental amount of 5"Cr released from EL-4 target cells cultured in the presence of normal macrophages or macrophages from treated mice, S = 61Cr released spontaneously from cultures of EL-4 cells alone, and R = amount of 51Cr released by suspension of EL-4 cells in distilled water for 6 h. Microbicidal assay. Tachyzoites of the RH strain of Toxoplasma were obtained from the peritoneal fluid of mice and processed for in vitro challenge of macrophage monolayers as described previously (27a, 32, 35). Macrophage monolayers, prepared as described above, were challenged with 2 x 106 tachyzoites and reincubated. After 1 h the monolayers were washed to remove extracellular organisms; representative infected and uninfected monolayers from each group were fixed in amino acridine hydrochloride (0.4% in ethyl alcohol), and the remainder of the cultures were reincubated for 24 h, at which time monolayers from each of the groups were fixed. After staining with Giemsa stain, the monolayers were examined microscopically, and the effects of the macrophages on the intracellular growth of Toxoplasma were determined by quantitating the percent cells infected and the number of organisms per infected cell (27a, 35). Tooplaksma antibodies. Blood was collected from mice sacrificed at various times after infection. Fourfold serial dilutions of heat-inactivated aliquots of serum were tested in the Sabin-Feldman dye test as described previously (12).

717

RESULTS Effects of treatment with MDP on resistance ofmice to challenge with Toxoplasma. To determine whether treatment with MDP enhanced resistance to challenge with Toxoplasma, a series of experiments was performed in which mice received single or multiple injections of MDP at varying times before challenge with Toxoplasma. In the initial studies depicted in Fig. 1, groups of SW mice were either treated with MDP on 4 consecutive days before Toxoplasma infection or received a single injection of MDP on the day before challenge. In addition, a control group treated with saline and a group treated i.p. with C. parvum 7 days previously were also challenged with Toxoplasma. As seen in Fig. 1A, neither C. parvum nor MDP treatment protected mice from the higher challenge dose of Toxoplasma (5 x 106). In the case of the lower challenge dose (5 x 106, Fig. 1B), only C. parvum treatment afforded marked protection (P < 0.001, day 12). Although both single and multiple treatments with MDP appeared to prolong survival after Toxoplasma chellenge, only the multiple treatments protected some of the animals from death. However, the level of protection did not differ significantly from that of controls (P > 0.05). In subsequent experiments on the effects of MDP treatment on resistance to Toxoplasma, inbred C57BL/6 and CBA mice were employed. The former strain has been shown to be only weakly responsive to the adjuvant effects of MDP, whereas the latter ranks among the most highly responsive strains of mice (46). In preliminary studies carried out to determine the relative sensitivity of each strain to Toxoplasma infection, C57BL/6 mice were clearly more susceptible to challenge than CBA mice (Fig. 2). In A

so0-70

60

50

40w

DAYS

FIG. 1. Effects of treatment with MDP or C. parvum on i.p. challenge of SW mice with 5 x 106 (A) or 5 x 105 (B) T. gondii trophozoites. Symbols: x, control; 0, MDP s.c. day -4, -3, -2, and -1; C, MDP day -1; A, C. parvum i.p day -7. Ten mice per group.

718

INFECT. IMMUN.

KRAHENBUHL ET AL.

Fig. 3 are shown the results of an experiment in which groups of mice were treated with MDP on the 4 consecutive days before infection with different doses of Toxoplasma. MDP treatment did not appear to enhance resistance of C57BL/ 6 to any of the three challenge doses (Fig. 3A) other than slight prolongation in timne to death. In marked contrast, MDP treatment appeared to enhance resistance of CBA mice to all three challenge doses of Toxoplasma (Fig. 3B). Resistance was most demonstrable against the lower challenge dose (5 x 10' organisms; P < 0.001). In the experiment depicted in Fig. 4, CBA mice were employed, and the timing of administration of a single dose of MDP was varied. Treatment 4, 7, or 14 days before challenge did not enhance resistance to challenge with 5 x 104 Toxoplasma. Only the mice which were treated on the day before challenge were significantly protected (Fig. 4, P < 0.01).

DAYS

FIG. 2. Effects of i.p. challenge with various doses of Toxoplasma trophozoites in C57BL/6 (A) and CBA (B) mice. Symbols: , 8 x 103; 0,4 X 104; 0,2 X 105; 0, 1 x 106 trophozoites. Twelve to fifteen mice per group.

Effects of treatment with MDP on antibody response to Toxoplasma infection. As part of the experiment described in Fig. 3B, sera were collected from three control and three MDP-treated mice on day 12 after challenge with-5 x 10' Toxoplasma and treated for neutralizing antibody in the Sabin-Feldman dye test. Although the protective effects of MDP were clearly evident (80% of controls were dead by day 12, but none of the treated mice had died by this time; P < 0.001), no differences in antibody titers were observed between the two groups-each of the six serum samples had a dye test titer of 1:1,024. An additional experiment, carried out to determine whether treatment with MDP enhances the humoral antibody response to Toxoplasma, confirned these findings. As shown by the data in Table 1, regardless of the interval between treatment with MDP and challenge with Toxoplasma or the time after infection, the level of dye test antibodies in treated mice did not differ from that in control mice. Cytotoxic and microbicidal capacity of macrophages from mice treated with MDP. At various times after i.p., intravenous (i.v.), or s.c. administration of MDP, peritoneal macrophages were removed from control and treated mice and tested in vitro for their cytotoxic or microbicidal capacity. As shown in Table 2, regardless of the strain of mouse employed or the timing or route of administration of MDP, no enhancement of the cytotoxic capacity of peritoneal macrophages was observed. Treatment i.p. with C. parvum was employed as a positive control to elicit activated peritoneal macrophages in SW and C57BL/6 (Table 2, experiments 2 and 3). C. parvum treatment had a similar effect on the peritoneal macrophages of

a cw)

I-I

z

w

w a-

DAYS

FIG. 3. Effects of treatment with MDP on i.p. challenge of C57BL/6 (A) and CBA (B) mice with various doses of Toxoplasma trophozoites. Mice treated s.c. on days -4, -3, -2, and -1 with saline (solid symbols) or MDP (open symbols). Symbols: *, 0, 5 x 104; A, 4, x 105; O, O, 5 x io5 trophozoites. Ten mice per group.

VOL. 31, 1981

90

EFFECTS OF MDP ON TOXOPLASMA INFECTION IN MICE

-m%

W-L

with a diverse selection of pathogens such as K. pneumoniae (5,31), T. cruzi (17), P. aeruginosa (27, 30), C. albicans (30), and S. pneumoniae (13). The present report extends these observations to include protection against challenge with the obligate intracellular protozoan parasite T. gondii. We found that MDP treatment afforded substantial protection to Toxoplasma infection in CBA/J mice, but not in C57BL/6 mice. Variation in the responsiveness of different strains of mice to the effects of MDP and MDP analogs has been reported previously (5, 6). Good correlation was reported between in vitro spleen cell blastogenic responses to MDP in different strains of mice and the in vivo adjuvant activity of these compounds (6). However, as new information on the biological effects of MDP treatment has accumulated, the evidence shows that

CBA

80

Wp

L

___

60-

0

z w

50

-

040w

0-30 20

X

10 0

2

4

6

8

10

12

14

16

DAYS

FIG. 4. Effects of timing of a single treatment with MDP on i.p. challenge of CBA mice with 5 x 104 with MDP at -14 days Toxoplasma. Mice treated (0), -7 days (A), -4 days (O), -I day (0) or at -1 day with saline (x). Ten mice per group. s.c.

TABLE 1. Effects of treatment with MDP on antibody response to Toxoplasmaa Group

A

Effect on day after infection: 3 6 9 12 16 Saline, day -1 Negb Neg 128c 512 *d Treatment

Neg Neg 256 1,024 * Neg Neg 256 1,024 * B MDP, day -1 Neg Neg 256 512 1,024 Neg Neg 128 2,048 1,024 Neg Neg 128 1,024 512 C MDP, day -8 Neg Neg 256 512 1,024 Neg Neg 256 256 * 512 Neg Neg 256 'Twenty CBA mice per group. MDP (80 mg/kg) was injected s.c. Three mice were sacrificed and bled on each of the designated days. b Negative in Sabin-Feldman dye test at 1:4 dilution. c titer. Reciprocal d* Did not survive. No animals in group A were alive on day 16 and only one was alive in group C. The remaining five mice in group B survived the observation period. _

719

*

CBA mice (data not shown). Shown in Table 3 are the results of an experiment in which both the cytotoxic and microbicidal effects of macrophages were mesured in CBA mice treated i.p. with MDP at various intervals before harvest. Regardless of the timing of treatment, macrophages were neither cytotoxic nor microbicidal. DISCUSSION Treatment of mice with MDP has been shown to stimulate nonspecific resistance to infection

TABLE 2. Cytotoxic capacity ofperitoneal macrophages from mice treated with MDP CytoTreatment com- Ro; Day toXICExpt S pound ity; Saline MDP (12 mg/kg) MDP (120 mg/kg) 2 SW Saline MDP (100 mg/kg) C. parvum 3 C57BL/6 Saline MDP (30 mg/kg) Saline MDP (30 mg/kg) MDP (30 mg/kg) MDP (30 mg/kg) C. parvum C. parvum 4 CBA Saline MDP (30 mg/kg) MDP (30 mg/kg) MDP (30 mg/kg) a 51Cr release (percent) from EL-4 triplicate cultures). 1

SW

S.C. S.C.

-1 -1 -1 -4 -4 -7 -1 -1 -1 -1 -7 -7 -7 -7 -1 -7 -3

-5.2 -5.6 s.c. -4.7 S.C. -7.5 s.c. -6.9 i.p. 9.2 i.p. -3.0 i.p. 2.5 i.v. 0.8 i.v. -2.6 i.p. 0.5 i.v. 2.8 17.9 i.p. i.v. 18.2 1.8 i.p. 0.8 i.p. -0.8 i.p. 0.8 i.p. -1 target celis (mean of

TABLE 3. Cytotoxic and microbicidal capacity of peritoneal macrophages from mice treated with MDP Microbicidal efTreatmenta

fectsc

Cytotoxicityb Oh 1.8 1.5 1.7 1.5

24h

5.9 1.5 Saline, day -1 4.3 0.4 MDP, day -7 4.7 1.1 MDP, day -3 5.6 -1.5 MDP, day -1 a MDP dose, 30 mg/kg, i.p. b 51Cr release (%) from EL-4 target cells (mean of triplicate cultures). 'No. of toxoplasma per infected cell.

720

KRAHENBUHL ET AL.

strain responsiveness to different in vivo and in vitro effects of this compound may vary. For example, whereas DBA/2, CBA, BALB/c, and AKR mice are strong responders to the in vitro blastogenic and in vivo adjuvant effects of MDP, C57BL/6 mice were consistently weak responders, as were (C57BL/6 x AKR)F1 and (C57BL/ 6 x DBA/2)F1 mice (6). However, marked protection against infection with Klebsiella was induced by MDP treatment in (C57BL/6 x AKR)F1 mice (5), and in vitro treatment with MDP induced in peritoneal exudate macrophages from (C57BL/6 x DBA/2)F1 the capacity to inhibit [3H]thymidine incorporation by P815 tumor target cells (10). The results of the present study are of special interest since our demonstration of the ability of MDP treatment to protect CBA but not SW or C57BL/6 mice from death due to Toxoplasma infection is in apparent contrast with the findings of Kierszenbaum and Ferraresi (17) who were unable to demonstrate the induction of resistance to T. cruzi in CBA mice, but did show that SW mice could be protected. The mechanism(s) underlying the remarkable resistance which MDP treatment affords against different infectious agents, including Toxoplasma, is unclear. Treatment with MDP has been shown to markedly enhance the humoral immune response to a variety of antigens (3, 8, 20, 21) and, in other models, to bolster certain cell-mediated immune responses (20, 21). The effects of MDP treatment on the relative contributions of either or both of these components of host response to infection have not been clearly defined. In the present study, there were no detectable differences in the level of cytolytic anti-Toxoplasma antibodies in treated compared with control mice. However, it is possible that other types of antibodies produced in the treated mice might, for example, possess a greater avidity for the parasite or kill it by mechanisms which do not result in lysis. An additional important consideration is that although the Toxoplasma organisms are highly susceptible to lysis by specific antibody in the presence of complement (18), their predominantly intracellular habitat protects them from the effects of humoral antibody. Thus, it seems unlikely that the protection observed in treated mice in the present study was due to an enhancement of the humoral antibody response. The presence of activated macrophages with enhanced microbicidal capacity represents a major mechanism of resistance to Toxoplasma in other animal models (19, 32, 35). If MDP treatment could be shown to activate macrophages

INFECT. IMMUN.

and increase their microbicidal capacity, this potent effector mechanism could underlie the results observed in the present study. A number of studies have suggested that the macrophage is an important target cell of the biological effects of MDP. Macrophages were shown to have a potentiating effect in MDP-induced enhancement of anti-sheep erythrocyte responses in spleen cell cultures (8, 20, 21). Similarly, MDP treatment of guinea pig peritoneal exudate macrophages elevated their synthesis of collagenase (38), stimulated their incorporation of ['4C]glucosamine (36), and inhibited their migration from capillary tubes (28). Each of the latter studies involved the exposure of macrophages to MDP in vitro. Although certain parameters of macrophage function were enhanced, none of these functions is directly linked to the enhanced microbicidal or cytotoxic capacity of the activated macrophage (15). More pertinent to the effector functions of macrophages is the report of Hadden et al. (11) in which guinea pig peritoneal exudate macrophages incubated in vitro with MDP acquired an enhanced phagocytic and bactericidal capacity for Listeria monocytogenes. In a different experimental model, the phagocytic capacity of mouse mnacrophages was not enhanced by treatment in vitro with MDP (24). However, other studies showed that mouse peritoneal macrophages and macrophage cell lines treated in vitro with MDP acquired an enhanced cytostatic (14) and cytocidal (38) capacity for tumor target cells. In the recent report by Galelli et al. (10), in vitro treatment of thioglycolate-induced macrophages from BDF1 mice induced a cytostatic but not a cytocidal capacity for P815 mastocytoma cells. Other than the demonstration that treatment with MDP enhances the carbon clearance capacity of the reticuloendothelial system (37), there is little direct evidence to suggest that altered macrophage function from in vivo treatment with MDP underlies enhanced resistance to infection. In earlier studies, Juy and Chedid (14) and Leclerc et al. (20) found that macrophages from BDF1 mice, treated i.p. with MDP, were not cytostatic for P815 target cells. However, by employing i.p. pretreatment with relatively high doses of MDP, Matter (26) has recently shown that the cytostatic capacity of peritoneal macrophages from DBA/2 mice is enhanced for Meth A tumor cells in vitro. In the present report, pretreatment with MDP failed to enhance the cytocidal capacity of peritoneal macrophages for EL-4 lymphoma cells or to enhance their microbicidal capacity for Toxoplasma. Others have shown that generation of a population of macrophages with full in vitro

VOL. 31, 1981

EFFECTS OF MDP ON TOXOPLASMA INFECTION IN MICE

cytotoxic capabilities requires more than one activation signal occurring in a definite sequence (33, 40). It is possible that in the present experiments these conditions were met in the intact animal protected from Toxoplasma infection by MDP treatment but were lacking in our attempt to determine whether peritoneal macrophages from treated mice possessed enhanced microbicidal or cytotoxic capabilities when tested in vitro. However, previous reports by ourselves (13) and others (4, 9) showed that regardless of dose, route, or timing of administration, MDP treatment did not protect mice against L. monocytogenes. Since resistance to this facultative intracellular bacterium is mediated solely by the enhanced microbicidal capacity of activated macrophages (25), these data, in addition to those of the present report, suggest that MDP treatment does not activate macrophages. Obvious differences in methodology, strain of mouse, and preparation of MDP could underlie many of the contrasting results cited above. Moreover, the knowledge that the cytotoxic functions of macrophages can be either cytostatic or cytocidal or both (16, 18a) and that neither function necessarily correlates with the microbicidal capacity of activated macrophages (19) forestalls any conclusions regarding the role of macrophages in MDP-induced resistance to infection. Thus, it appears that although MDP treatment has been shown to enhance resistance in mice to a variety of pathogens, including the obligate intracellular protozoan T. gondii, definition of the mechanism(s) of resistance will require further unravelling of the biological effects of MDP. ACKNOWLEDGMENTIS This work was supported by Public Health Service research grants AI07801 and AI04717 from the National Institute of Allergy and Infectious Diseases and grant SF0804100 from the U. S. Public Health Service Central Clinical Investigation Committee.

LUERATURE CITED 1. Azuma, I. K., Sugimura, T. Taniyama, M. Yamawaki, Y. Yamamura, S. Kusumoto, S. Okada, and T. Shiba. 1976. Adjuvant activity of mycobacterial functions: adjuvant activity of synthetic N-acetylmuramyldipeptide and the related compounds. Infect. Immun. 14:18-27. 2. Cerottini, J. C., and K. T. Brunner. 1971. In vitro assay of target cell lysis by sensitized lymphocytes, p. 369. In B. R. Bloom and P. R. Glade (ed.), In vitro methods in cell-mediated immunity. Academic Press, Inc., New York. 3. Chedid, L., F. Audibert, P. Lefrancier, J. Choay, and E. Lederer. 1976. Modulation of the immune response by a synthetic adjuvant and analogs. Proc. Natl. Acad. Sci. U.S.A. 73:2472-2475. 4. Chedid, L, M. Parant, F. Parant, F. Audibert, P. Lefrancier, J. Choay and M. Sela. 1979. Enhance-

721

ment of certain biological activities of muramyl dipeptide derivatives after conjugation to a multi-poly (Dlalanine) -- poly (L-lysine) carrier. Proc. Natl. Acad. Sci. U.S.A. 76:6557-6561. 5. Chedid, L., M. Parant, F. Parant, P. Lefrancier, J. Choay, and E. Lederer. 1977. Enhancement of nonspecific immunity to Kkbsiella pneumonia infection by a synthetic immunoadjuvant (N-acetylmuramyl-Lalanyl-D-isoglutamine) and several analogs. Proc. Natl. Acad. Sci. U.S.A. 74:2089-2093. 6. Damais, C., M. Parant, L. Chedid, P. Lefrancier, and J. Choay. 1978. In vitro spleen cell responsiveness to various analogs of MDP (N-acetyl-muramyl-L-alanylD-isoglutamine), a synthetic immunoadjuvant, in MDP high-responder mice. Cell. Immunol. 35:173-179. 7. Ellouz, F., A. Adam, R. Ciorbaru, and E. Lederer. 1974. Minimal requirements for adjuvant activity of bacterial peptidoglycan derivatives. Biochem. Biophys. Res. Commun. 59:1317-1325. 8. Fevrier, M., J. L Birrien, C. Leclerc, L. Chedid, and P. Liacopoulos. 1978. The macrophage, target cell of the synthetic adjuvant muramyl-dipeptide (MDP). Eur. J. Immunol. 8:558-562. 9. Finger, H., and C. H. Wirsing Von Konig. 1980. Failure of synthetic muramyl dipeptide to increase antibacterial resistance. Infect. Immun. 27:288-291. 10. Galelli, A., Y. LeGarrec, L. Chedid, P. Lefrancier, M. Derrien, and M. Level. 1980. Macrophage stimulation in vitro by an inactive muramyl dipeptide derivative after conjugation to a multi-poly (DL-alanyl)-poly-(Llysine) carrier. Infect. Immun. 28:1-5. 11. Hadden, J. W., A. Englard, J. R. Sadlik, and E. M. Hadden. 1979. The comparative effects of isoprinosine, levamisole, muramyl dipeptide and SM1213 on lymphocyte and macrophage proliferation and activation in vitro. Int. J. Immunopharmacol. 1:17-27. 12. Handman, E., and J. S. Remington. 1980. Serological and immunochemical characterization of monoclonal antibodies to Toxoplasma gondii. Immunology 40:579588. 13. Humphres, R. C., P. R. Henika, R. W. Ferraresi, and J. L Krahenbuhl. 1980. Effects of treatment with muramyl dipeptide and certain of its analogs on resistance to Listeria monocytogenes in mice. Infect. Immun. 29:462-466. 14. Juy, D., and L. Chedid. 1975. Comparison between macrophage activation and enhancement of nonspecific resistance to tumors by mycobacterial immunostimulants. Proc. Natl. Acad. Sci. U.S.A. 72:4105-4109. 15. Karnovsky, M. L, and J. K. Lazdins. 1978. Biochemical criteria for activated macrophages. J. Immunol. 121: 809-813. 16. Keller, R. 1976. Susceptibility of normal and transformed cell lines to cytostatic and cytocidal effects exerted by macrophages. J. Natl. Cancer Inst. 56:369-374. 17. Kierszenbaum, K., and R. W. Ferraresi. 1979. Enhancement of host resistance against Trypanosoma cruzi infection by the immunoregulatory agent muramyl dipeptide. Infect. Immun. 25:273-278. 18. Klainer, A. S., J. L. Krahenbuhl, and J. S. Remington. 1973. Scanning electron microscopy of Toxoplasma gondii. J. Gen. Microbiol. 75:111-118. 18a.Krahenbuhl, J. L., and J. S. Remington. 1980. Cytostatic effects of activated macrophages on tumor target cells: inhibition of cytotoxic action of ARA-C. J. Immunopharmacol. 2:325-348. 19. Krahenbuhl, J. L., J. S. Remington, and R. McLeod. 1980. Cytotoxic and microbicidal properties of macrophages, p. 1,631-1,653. In R. Van Furth (ed.), Mononuclear phagocytes: functional aspects. Martinus Nijhoff Publishing, The Hague, The Netherlands. 20. Leclerc, C., D. Juy, E. Bourgeois, and L. Chedid. 1979.

722

INFECT. IMMUN.

KRAHENBUHL ET AL.

In vivo regulation of humoral and cellular immune responses of mice by a synthetic adjuvant, N-acetylmuramyl-L-alanyl-D-isoglutamine, muramyl dipeptide for MDP. Cell. Immunol. 45:199-206. 21. Leclere, C., I. Ldwy, and L. Chedid. 1978. Influence of MDP and of some analogous synthetic glycopeptides on the in vitro mouse spleen cell viability and immune response to sheep erythrocytes. Cell. Immunol. 38:286293. 23. Lefrancier, P., and J. Choay. 1976. Distinctive adjuvanticity of synthetic analogs of mycobacterial water-soluble components. Cell. Immunol. 21:243-249. 24. Ldwy, I., C. Bona, and L. Chedid. 1977. Target cells for the activity of a synthetic adjuvant: muramyl dipeptide. Cell. Immunol. 29:195-199. 25. Mackaness, G. B. 1969. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129:973-992. 26. Matter, A. 1979. The effects of muramyl dipeptide (MDP) in cell mediated immunity: a comparison between in vitro and in vivo system. Cancer Immunol. Immunother. 6:201-210. 27. Matthews, T. R., and E. B. Fraser-Smith. 1980. Protective effect of muramyl dipeptide and analogs against Pseudomonas aeruginosa and Candida albicans infections in mice, p. 1734-1735. In J. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious disease. American Society for Microbiology, Washington, D.C. 27a.McLeod, R., K. G. Bensch, S. M. Smith, and J. S. Remington. 1980. Effects of human peripheral blood monocytes, monocyte-derived macrophages and spleen mononuclear phagocytes on Toxoplasma gondii. Cell. Immunol. 54:330-350. 28. Merser, C., P. Sinay, and A. Adam. 1975. Total synthesis and adjuvant activity of bacterial peptidoglycan derivatives. Biochem. Biophys. Res. Commun. 66:13161322. 29. Nagao, S., A. Tanaka, Y. Yamamoto, T. Koza, K. Onoue, T. Shiba, K. Kusumoto, and S. Kotani. 1979. Inhibition of macrophage migration by muramyl peptides. Infect. Immun. 241:308-312. 30. Parant, M. A., F. M. Audibert, L. A. Chedid, M. R. Level, P. L. Lefrancier, J. P. Choay, and E. Lederer. 1980. Immunostimulant activities of a lipophilic

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

muramyl dipeptide derivative and of desmuramyl peptidolipid analogs. Infect. Immun. 27:826-831. Parant, M., F. Parant, and L. Chedid. 1978. Enhancement of the neonate's nonspecific immunity to Klebsiella infection by muramyl dipeptide, a synthetic immunoadjuvant. Proc. Natl. Acad. Sci. U.S.A. 75:33953399. Remington, J. S., J. L. Krahenbuhl, and J. W. Mendenhall. 1972. A role for activated macrophages in resistance to infection with Toxoplasma. Infect. Immun. 6:829-834. Ruco, L. P., and M. S. Meltzer. 1978. Macrophage activation for tumor cytotoxicity: Development of macrophage cytotoxic activity requires completion of a sequence of short-lived intermediary reactions. J. Immunol. 121:2035-2042. Specter, S., H. Friedman, and L Chedid. 1977. Dissociation between the adjuvant vs mitogenic activity of a synthetic muramyl dipeptide for murine splenocytes (39804). Proc. Soc. Exp. Bio. Med. 155:349-352. Swartzberg, J. E., J. L. Krahenbuhl, and J. S. Remington. 1975. Dichotomy between macrophage activation and degree of protection against Listeria monocytogenes and Toxoplasma gondii in mice stimulated with Corynebacterium parvum. Infect. Immun. 12: 1037-1043. Takada, H., M. Tsujimot, K. Kato, S. Kotani, S. Kusumoto, M. Inage, T. Shiba, I. Yano, S. Kawata, and K. Yokogawa. 1979. Macrophage activation by bacterial cell walls and related synthetic compounds. Infect. Immun. 25:48-53. Tanaka, A., S. Nagao, R. Nagao, S. Kotani, T. Shiba, and S. Kusumoto. 1979. Stimulation of the reticuloendothelial system of mice by muramyl dipeptide. Infect. Immun. 24:302-308. Taniyama, T., and H. Holden. 1979. Direct augmentation of cytolytic activity of tumor-derived macrophages and macrophage cell lines by muramyl dipeptide. Cell Immunol. 48:369-374. Wahl, S. M., L M. Wahl, J. B. McCarthy, L Chedid, and S. E. Mergenhagen. 1979. Macrophage activation by mycobacterial water soluble compounds and synthetic muramyl dipeptide. J. Immunol. 122:2226-2231. Weinberg, J. B., H. A. Chapman, and J. B. Hibbs. 1978. Characterization of the effects of endotoxin on macrophage tumor cell killing. J. Immunol. 121:72-80.