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We have previously shown that prophylactic administration of the liposome-encapsulated immunomodulating agents muramyl tripeptide phosphatidylethanolamine (MTPPE) and gamma interferon (IFN-gamma) results in strongly increased survival of mice from a normally lethal septicemia with Klebsiella pneumoniae. It was anticipated that the treatment acts on macrophages and nonspecifically augments host resistance to various infections. In the present study, we provide evidence for a key role for T cells in host defense potentiation by the liposomal immunomodulators toward K. pneumoniae septicemia. It is shown that both CD4 and CD8 cells are important in immunomodulation, most likely due to production of IFN-gamma. Depletion of circulating IFN-gamma resulted in strong reduction of the antimicrobial host defense activation. Administration of interleukin-10 resulted in decreased antimicrobial host defense activation by liposomal immunomodulators. Moreover, administration of liposomal immunomodulators was shown to induce predominantly T-helper type 1 (Th1) cell populations in the spleen. These findings indicate that immunomodulation with liposomal MTPPE and IFN-gamma favors Th1 and NK cell activation.

Administration of free muramyl tripeptide phosphatidylethanolamide (MTPPE) or liposome-encapsulated MTPPE (LE-MTPPE) in a twofold-lower dose at 24 h before bacterial inoculation resulted in clearance of intravenously inoculated Klebsiella pneumoniae by tissue macrophages, whereas in control mice, bacteria were not effectively cleared from the blood. In addition, MTPPE and LE-MTPPE led to increased numbers of leukocytes in the blood, which could compensate for the leukopenia in mice resulting from infection with K. pneumoniae. In an attempt to elucidate the relative contributions of the activation of tissue macrophages and the recruitment of leukocytes to the antibacterial resistance induced by MTPPE and LE-MTPPE, mice were infected intraperitoneally with K. pneumoniae. In these MTPPE- and LE-MTPPE treated mice, intraperitoneal influx of leukocytes and the phagocytic capacity of leukocytes were not higher than in untreated control mice. However, MTPPE- and LE-MTPPE-treated mice survived much longer; eventually 33% of the LE-MTPPE-treated mice survived, whereas all untreated control mice died as a result of bacterial septicemia. This prevention of early death appeared to be the result of an increased clearance of bacteria from the blood by activated tissue macrophages. It was observed that depletion of these tissue macrophages in liver and spleen abrogates the effect of LE-MTPPE treatment, indicating that tissue macrophages are of major importance in the LE-MTPPE-induced resistance against K. pneumoniae infection.

Injection of type 6b pneumococci into pigs results in a sharp rise in levels of interleukin-6 (IL-6) in blood, with an average peak value of 7,700 U/ml at 4 h and mortality in five of seven of these animals. Pretreatment at -24 h with the monocyte activator MTP-PE prevents this strong increase in IL-6 (peak value, 1,000 U/ml) and reduces mortality to zero of six animals. This suggests that MTP-PE, in addition to activating monocytes, protects animals by preventing the harmful increase of cytokines such as IL-6.

The effect of free and liposome-encapsulated muramyl tripeptide phosphatidylethanolamide (MTPPE) on resistance to Klebsiella pneumoniae infection in mice was investigated. It was shown that administration of MTPPE, at 24 h before bacterial inoculation, led to a dose-dependent antibacterial resistance in terms of increased clearance of bacteria from the blood and bacterial killing in various organs. The lowest effective dose of MTPPE was 50 micrograms per mouse. Administration of liposome-encapsulated MTPPE was also effective at a dose of 25 micrograms per mouse. The time of administration of both free and liposome-encapsulated MTPPE, with respect to the appearance of bacteria in the blood, was very important and indicated that repeated administration is necessary to obtain protection for a prolonged period. In view of the toxicity of MTPPE, it was an important observation that repeated administration of MTPPE in the liposome-encapsulated form also produced antibacterial resistance. Administration of free and liposome-encapsulated MTPPE resulted in increased numbers of granulocytes, monocytes, and lymphocytes in the blood of uninfected mice and prevented leukopenia in infected mice.

Although it is clear that muramyl peptides are involved in sleep associated with bacterial infection, their role in normal physiological sleep is less certain. It has been speculated that "natural" muramyl peptides, derived from degraded gut flora, may pass into the bloodstream, where they play a role in normal sleep (M. Karnovsky, Fed. Proc. 45:2556-2560, 1986). Muramic acid serves as a chemical marker for muramyl peptides, since it is not synthesized by mammals. After injection of synthetic muramyl dipeptide in rabbits, muramic acid was readily detected (after release by acid hydrolysis) in the circulation; however, levels rapidly decreased. This was an important positive control in assessing circulating levels of natural muramyl peptides. Muramic acid was not found in normal serum (detection limit, approximately 500 pmol/ml), demonstrating the absence of appreciable amounts of circulating natural muramyl peptides. At this time we are unable to provide supportive evidence for Karnovsky's hypothesis.

In a porcine model of pneumococcal septicemia, animals were pretreated with 1 mg of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (MTP-PE) or with liposomes alone. After 24 h each animal received an injection of either 10(9) or 10(10) pneumococcal serotype 6B cells. MTP-PE pretreatment resulted in less pronounced leukocytopenia, with a nadir of 6,700 (versus 4,100) leukocytes per mm3 after injection of 10(9) bacteria and a nadir of 4,400 (versus 3,800) leukocytes per mm3 after injection of 10(10) bacteria. At the same time bacterial clearance was substantially improved by MTP-PE pretreatment. Finally, pretreatment with MTP-PE dramatically reduced mortality; the average death rates for both series of animals used were 55% for liposome-pretreated animals and 3% for animals pretreated with MTP-PE-containing liposomes. These results in a preclinical model suggest that treatment with MTP-PE-containing liposomes might be beneficial in controlling septicemia in patients at risk.

By subcutaneous treatment with an aqueous solution of 6-O-stearoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine [6-O-CH3-(CH2)16-CO-MurNAc-L-Ala-D-isoGln] [referred to here as L18-MDP(Ala)], an augmentation of the resistance of mice to Escherichia coli, Pseudomonas aeruginosa. Staphylococcus aureus, and Candida albicans infections was observed, but not to infections with Klebsiella pneumoniae and Listeria monocytogenes. Against E. coli infections, L18-MDP(Ala) was highly protective, irrespective of the administration route. Bacteremia occurring at an early phase of such infections was almost completely prevented by subcutaneous treatment 1 day before infection. Single or multiple doses were also effective against C. albicans infection. The phagocytosis of E. coli by mouse peritoneal polymorphonuclear cells was enhanced by treatment with the adjuvant, and the phagocytosis of K. pneumoniae was also enhanced, but only when the mice were treated either with rabbit normal serum or with a specific immune serum. The growth of the fungus in the kidneys was significantly inhibited, and growth was eliminated from the kidneys by treatment with the adjuvant once a day for 4 consecutive days, starting 1 day before infection. However, no growth suppression of L. monocytogenes in the livers or spleens of infected mice was observed when they were treated with a single dose of the adjuvant. This difference may be ascribed to the differences in the effector mechanisms of defense and to the different degree of augmentation of each defense mechanism by L18-MDP(Ala).

A synthetic muramyl dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine, dissolved in saline only and applied subcutaneously to rats of the Lewis inbred strain, produced arthritis, clinically manifest by hind feet paresis but without apparent paw swelling in most cases. Histologically, the disease was characterized by edema and hyperemia of connective tissues, joint synovias, and tendon sheaths, with massive accumulation of inflammatory cell infiltrates composed mainly of lymphoplasmocytes and partly of neutrophil leukocytes. Fibrin exudation and fibrinoid necrosis in connective tissues were observed in the most severe cases. Synovial layers of the talocrural joint, especially on their villi, exhibited marked swelling or cell desquamation of the inner zone. Clinical symptoms of the disease disappeared spontaneously within 5 days after cessation of the treatment; also, histological examinations showed that the effects were reversible. Our results prove that (i) muramyl dipeptide is the principal substance involved in the production of arthritis, (ii) there is no necessity for the presence of additional mycobacterial cell wall components, and (iii) the involvement of the oil moiety is not requisite for the production of arthritis.

Intravenous injection of muramyl dipeptide (MDP) and Salmonella lipopolysaccharides (LPS) enhanced lethal toxicity of the LPS in C57BL/6 mice. This was true for S (smooth)- and R (rough)-form LPS and free lipid A. Enhancement of toxicity was maximum when the LPS was administered 4 h after MDP, at which time the lethal doses for 50% of mice of S- and R-form LPS and free lipid A were between 1 and 10 micrograms, compared with more than 100 micrograms in normal animals. This sensitization was absent in endotoxin-resistant C3H/HeJ mice. Lethality usually commenced 15 h after LPS injection and was complete after 72 h. Higher doses of some S-form LPS (100 micrograms or more) administered 4 h after MDP led to a strong anaphylactoid reaction within 10 to 20 min of injection, with lethal outcomes in less than 1 h after LPS administration. This early anaphylactoid reaction was observed for various mouse strains, including LPS-resistant C3H/HeJ mice, but it was very weak or completely absent with R-form LPS or free lipid A even in concentrations of up to 1,000 micrograms. A strong anaphylactoid reaction comparable to that seen with S-form LPS was also obtained, after MDP treatment, with an LPS of low toxicity prepared from Bacteroides gingivalis. It is noteworthy that oral administration of MDP also contributed to the anaphylactoid reaction and enhanced the late-phase lethality of LPS. The present findings strongly suggest that the early- and late-phase reactions induced by MDP and LPS are caused by different mechanisms.

Encapsulation of N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine in multilamellar vesicles composed of phosphatidylcholine, cholesterol, and phosphatidylserine (7:6.7:3) or phosphatidylcholine and phosphatidylserine (7:3) reduced the amount of drug needed to protect against a Candida albicans intravenous infection. The 50% effective doses for encapsulated and free drug were 5.5 and greater than 80 mg/kg, respectively. The optimum treatment was twice (at days 4 and 2 preinfection) by the intravenous route. Intraperitoneal, subcutaneous, and oral routes of administration were ineffective. The same potentiation of anti-Candida activity was observed whether the lower dose of drug was encapsulated in multilamellar vesicles, mixed with multilamellar vesicles, or given either 1 h before or 1 h after multilamellar vesicles. It was postulated that the mechanism of action involved the retention of the liposomes by organs of the reticuloendothelial system, resulting in an enhanced response of the macrophages to the immunostimulating activity of the N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine given in conjunction with the vesicles.

A regimen of immunostimulation with 6-0-stearoyl-N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine, a lipophilic analog of muramyl dipeptide, combined with antimonial drug therapy was evaluated in the treatment of visceral leishmaniasis of mice and hamsters. The combined treatment was found to be more effective in the elimination of Leishmania donovani amastigotes from infected tissue macrophages than was either of the two treatments applied individually. In mice, it was found that immunostimulation of animals prophylactically, therapeutically, or both enhanced the effects of the antimonial drug (Glucantime) administered more than 1 week after a challenge of BALB/c and C57BL/6 mice. The superiority of the combined treatment of the parasite infection was demonstrable in both short-term (14 days) and long-term (40 to 45 days) infections of the two inbred strains of mice. The combined therapy was also effective in preventing the lethal course of leishmaniasis in hamsters which succumb to disseminated disease in the absence of therapeutic intervention. The efficacy of this dual approach to the therapy of disseminated leishmaniasis of experimental animals holds promise for similar application in the treatment of similarly afflicted human populations.

A nonpyrogenic desmuramyl peptidolipid, 1-O-(L-alanyl-D-isoglutaminyl-L-alanyl-glycerol-mycolate), had previously been shown to be inactive as adjuvant in guinea pigs, but to be very active in stimulating nonspecific resistance. We now show that 1-O-(L-alanyl-D-isoglutaminyl-L-alanyl-glycerol-mycolate) is capable of enhancing or suppressing the immune responses in mice when injected with or before an antigen. In vivo suppression of the immune response to sheep erythrocytes was also observed with high doses of murabutide, a nonpyrogenic adjuvant-active N-acetylmuramyl-L-alanyl-D-isoglutamine analog. Chemiluminescence measurements with mouse spleen cells show a very strong activity of 1-O-(L-alanyl-D-isoglutaminyl-L-alanyl-glycerol-mycolate) by far superior to the effect obtained with the corresponding muramyl peptide, N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanyl-glycerol-myco late.

Significant resistance against influenza virus and Mycobacterium tuberculosis infections was induced when trehalose dimycolate from M. tuberculosis or M. bovis but not M. avium was combined with muramyl dipeptide. Trehalose dimycolate from M. tuberculosis, in contrast to that from M. avium, could confer resistance against Toxoplasma gondii infections.

N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide [MDP]) enhanced resistance against Trypanosoma cruzi infection in mice. This effect was evidenced by significant reductions in both parasitemias and mortality rates and increased survival time in MDP-treated animals compared with untreated infected mice. MDP effectively augmented host resistance when administered in any one of the following ways: (i) continuous subcutaneous release from an osmotic minipump for a 7-day period starting 2 days before infection; (ii) as a single dose of 0.5 mg injected intraperitoneally 48 h before infection; or (iii) injected intraperitoneally at 48-h intervals during the first 16 days after infection. CBA/J mice, which exhibit very low, insignificant augmentation of reticuloendothelial activity by MDP but are susceptible to its adjuvant effect, failed to manifest enhancement of resistance to T. cruzi infection when treated with MDP under regimens that cause increased resistance in other mouse strains. These results suggest that MDP enhances resistance against T. cruzi infection by stimulating the activity of the phagocytic cells of the host. Adjuvant effect appears to play either a less significant role or no relevant role, except when MDP is administered repeatedly after infection.