Stimulation of mouse resistance to bacterial infection with muramyl dipeptide glycoside

We studied the capacity of 9 new muramyl dipeptide glycosides to stimulate mouse resistance to experimental sepsis induced by intraperitoneal injection of salmonella typhimurium culture. Preventive intraperitoneal injections of muramyl dipeptide -glycosides better improved survival of infected animals compared to the original (unmodified) muramyl dipeptide and muramyl dipeptide -glycosides. The most effective drug muramyl dipeptide -heptylglycoside injected during sepsis development also reduced animal mortality, decreased bacterial contamination of the viscera, and increased phagocytic activity of peritoneal macrophages in infected animals.     

胞壁酰二肽激活大鼠巨噬细胞抗肿瘤免疫效应的研究

目的:探讨胞壁酰二肽(MDP)激活大鼠巨噬细胞抗肿瘤效应的途径和机制。方法:以SD大鼠为骨肉瘤动物模型进行肿瘤抑制试验;采用中性红吞饮实验、间接MTT法及硝酸还原酶法,测定大鼠腹腔巨噬细胞的功能。结果:MDP(50μg～100μg/鼠)皮下注射可明显抑制UMR106骨肉瘤细胞在SD大鼠体内的生长,并可显著提高巨噬细胞的吞噬功能、杀伤活性及TNF和NO的分泌水平,且呈现一定的量效关系。结论:MDP激活的巨噬细胞可能是机体非特异性免疫抗瘤效应的主要基础。     

Increased synthesis of fibrinolytic inhibitor induced by muramyl dipeptide derivatives in human cultured endothelial cells

A decreased fibrinolytic activity induced by bacterial products and some muramyl peptides has been previously demonstrated in macrophages preparations. Since vascular endothelial cells are important for the fibrinolytic balance, we have studied the effects of MDP derivatives on cultured endothelial cells. The supernatant of MDP and murabutide treated cell cultures exhibited an increased fibrinolytic inhibitory activity when tested with urokinase. The MDP(D-D)-treatment had no effect. This increased inhibitory activity was detectable in the supernatant after a 6 h treatment and was suppressed by the addition of puromycin to the cell cultures. Furthermore, the endothelial cell culture supernatant also reduced the lytic activity of the human plasma plasminogen activator induced by venostasis. This was enhanced by MDP treatment of the cultures. These in vitro results suggested that adjuvant-active muramyl peptides may regulate the fibrinolytic balance at the vessel wall level. This could be of possible significance in the transendothelial cell migration where the role of plasminogen activator(s) has been involved.     

Effect of new muramyl dipeptide derivatives on the major components of immunity

Immunomodulatory activity of five new synthetic muramyl dipeptide (MDP) derivatives (β-heptylglycoside-MDP, β-hexadecylglycoside-MDP, polyacrylamide-MDP, polyacrylamide-MDP-phosphatidylethanolamine, and dexal-MDP) is studiedin vitro in different test systems. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 5, pp. 510–513, May, 1994 Presented by N. K. Permyakov, Member of the Russian Academy of Medical Sciences     

Degradation of muramyl dipeptide by mammalian serum.

Muramyl dipeptide, N-acetylmuramyl-L-alanine-D-isoglutamine (MDP), is the minimal biologically active subunit of bacterial peptidoglycan and elicits an acute inflammation in vivo. We now report that MDP is degraded by normal rat serum into its constituents, N-acetylmuramic acid and L-alanine-D- isoglutamine. The dipeptide is further degraded into its components L-alanine and D-isoglutamine. These results may help to explain how inflammation elicited by MDP is terminated in vivo.     

Pharmacokinetics and metabolism of muramyl dipeptide and nor-muramyl dipeptide [3H-labelled] in the mouse

The fates of ³H-muramyl dipeptide (MDP) and ³H-nor-MDP have been investigated after intravenous (i.v.), intraperitoneal, and subcutaneous injection of a range of doses in the mouse. After i.v. injection both compounds were cleared rapidly from the circulation, distributed initially to the tissues, and finally excreted largely intact in the urine. Most of the tissues contained intact material at 2 min after injection, but the much lower levels of radioactivity persisting at 1 h had undergone considerable metabolism (except in intestine, where some intact material persisted for as long as 24 h). Some accumulation of radioactivity was observed in liver and kidney and there were quantitative and qualitative differences between the two compounds. Characterisation of some of the metabolites in these tissues was undertaken, and the deamidated muramyl dipeptide was tentatively identified which is known to have some biological activity. The mechanism of the biological effects, which may be expressed over a relatively long time period, remains to be explained in view of the rapid excretion of most of the dose.     

Lipophilic derivative of muramyl dipeptide is more active than muramyl dipeptide in priming macrophages to release superoxide anion.

Mouse peritoneal macrophages, when treated with a lipophilic derivative of muramyl dipeptide either in vitro or in vivo by intraperitoneal injection, showed a more than fivefold increase in their ability to generate superoxide anion after stimulation of the macrophages with phorbol myristate acetate. This response was more than twice that observed with the parent molecule, muramyl dipeptide (MDP). Unlike MDP, which has a systemic effect, the lipophilic derivative, [B30]-MDP, did not alter the response of peritoneal macrophages when given subcutaneously in the flank, suggesting that [B30]-MDP remains localized at the site of injection. The enhanced effect of [B30]-MDP over MDP appeared to be due to the inherent lipophilicity of the molecule, and was probably not due to either stimulation of T lymphocytes or activation of the alternative pathway of complement.     

Effect of free or liposome-encapsulated muramyl dipeptide on uptake and intracellular survival ofListeria monocytogenes in mouse peritoneal macrophages in vitro

The effect of free versus liposome-encapsulated muramyl dipeptide (MDP) on the uptake and intracellular survival ofListeria monocytogenes in mouse peritoneal macrophages in vitro was investigated. Macrophages in monolayer culture were exposed to free MDP at various concentrations during different time periods before incubation withListeria monocytogenes. An increase in bacterial uptake dependent on the concentration of MDP and the length of exposure was observed. Exposure of macrophages to 200µg of free MDP per milliliter for 15 h led to a threefold increase in bacterial uptake, resulting in an average of 15 bacteria per macrophage after 30 min of incubation. In addition, intracellular bacteria were killed in the MDP-exposed macrophages, in contrast to the intracellular multiplication ofListeria monocytogenes in macrophages not exposed to MDP. Encapsulation of MDP within liposomes resulted in a significant enhancement of its activity: liposomal encapsulation led to a 1,000-fold reduction in the amount of MDP required to obtain these effects on bacterial uptake and intracellular killing, whereas empty liposomes had no effect at all. Liposomal encapsulation may be an appropriate means of increasing delivery of the muramyl peptides to the macrophages.     

Distinctive immunomodulating properties and interactivity with model membranes and cells of two homologous muramyl dipeptide derivatives differing by their lipophilicity

We have studied and compared the immunomodulating activities of two muramyl dipeptide (MDP) derivatives: β-heptylglycoside-MDP (C₇H₁₅MDP) and β-hexadecylglycoside-MDP (C₁₆H₃₃MDP). The amphiphilic derivative C₇H₁₅MDP has been found to be a more effective stimulator of T lymphocyte proliferation and allospecific cytotoxic T cell generation in a mixed lymphocyte culture, and a more effective activator of interleukin-1 (IL-1) and tumour necrosis factor (TNF) production by murine peritoneal macrophages, in comparison with MDP and C₁₆H₃₃MDP used in equimolar concentrations. C₇H₁₅MDP also stimulated cytotoxicity of natural killer (NK) cells. On the contrary, its lipophilic homologue C₁₆H₃₃MDP did not show such activities in vitro except for its influence on IL-1 and TNF production. We have found significant differences in the interaction of these two ₁₄C-labelled MDP derivatives with model membranes and in the uptake of these preparations by erythroleukemia K562 cells. We consider the hydrolipophilic balance of the above preparations to be the main cause of their different interactions with membranes and their uptake by cells and, as a result, their opposite immunomodulating activities in vitro.     

Stimulation of macrophage protease secretion via liposomal delivery of muramyl dipeptide derivatives to intracellular sites.

We have observed that murine macrophages can be activated for enhanced neutral protease secretion by exposing the cells to muramyl dipeptides (MDPs). A lipophilic derivative of nor-MDP is more efficacious than the parent hydrophilic nor-MDP. The efficacy and potency of the lipophilic and more prominently the hydrophilic drugs can be increased (10-10(3) fold) by encapsulating them in lipid vesicles (liposomes); however the encapsulation of drug causes a delay in the onset of activation. The enhanced effectiveness of liposomal MDPs seems in part, to be due to increased uptake, slow release and thus potentiated action of the drug at intracellular sites as emphasized by studies with [3H]-MDP. Appropriate distribution of the drug to intracellular compartments of the cell also seems to be an important factor in the activation process. The internalization of a relatively large amounts (greater than 5 ng/10(6) cells) of nor-MDP results in 'down regulation', that is reduced protease secretion, as compared to effects produced by internalization of lesser amounts of the drug. The macrophage activating effects of liposomal MDPs do not seem to require the processing of liposomes in the lysosomal compartment; thus lysosome-blocking agents, such as chloroquine and dextran sulphate, do not affect the induction of protease secretion.     

Activation of mouse peritoneal cells to kill Listeria monocytogenes by T-lymphocyte products.

An in vitro system has been used to demonstrate that glass-adherent mouse peritoneal cells can be activated to kill intracellular Listeria monocytogenes by antigen-stimulated T-lymphocytes derived from immunized mice. The soluble products of such stimulated lymphocyte cultures could only be shown to similarly activate peritoneal cells if the antigen used in both the immunization and lymphocyte stimulation was also present on the target intracellular organism.     

Treatment of experimental myasthenia with autologous idiotypes linked to muramyl dipeptide.

In order to develop a new treatment of experimental autoimmune myasthenia gravis (EAMG), rabbits were injected with purified acetylcholine receptor (AChR) from Torpedo californica. Polyclonal affinity-purified anti-AChR antibodies (idiotypes, Ids) were coupled covalently to muramyl dipeptide and injected back into the same (i.e. autologous) rabbits from which the Ids were obtained. Treated animals developed anti-Ids that bound to the F(ab')2 fragments of the Ids as demonstrated by ELISA and that also blocked binding of Ids to AChR in a radioimmunoassay. Treated animals showed a protective effect compared to control animals when challenged with a second injection of AChR. Anti-AChR titres in surviving animals achieved a steady-state equilibrium. No apparent toxicity from the treatment was noted.     

Control of the mitogenicity of muramyl dipeptide

Muramyl dipeptide (MDP) has been shown to be the smallest subunit of bacterial peptidoglycan which exhibits adjuvanticity in vivo. Among its numerous in vitro activities is the ability to enhance the rate of DNA synthesis of murine splenocytes. In the present study, the dividing splenocytes were shown to be B cells which required prolonged incubation with MDP before becoming committed to division. T cells contributed minimally, if at all, to the DNA synthesis response observed nor did they function as helpers. Macrophage depleted lymphocytes responded poorly but could be reconstituted with fresh macrophages or 2-mercaptoethanol. X-ray irradiation of the macrophages reduced their ability to serve as accessory cells.Because of the biological similarity between bacterial endotoxin (LPS) and MDP, the possibility was tested that the gene(s) controlling the mitogenic response to MDP were either linked to or identical with the genes controlling the response to LPS. The results obtained from the comparison of selected mouse strains and from backcross analysis indicated that the two responses are not controlled by closely linked genes.These same strains of mice were compared with respect to their ability to respond to MDP as an adjuvant for the secondary antibody response to bovine albumin in vivo. It was found that the intensity of the in vivo adjuvant response was not correlated with the intensity of the in vitro mitogen response. We conclude from these observations that the ability of MDP to act as a mitogen makes little or no contribution to its adjuvanticity in vivo.     

Failure of synthetic muramyl dipeptide to increase antibacterial resistance.

Synthetic muranyl dipeptide, which potentiates antibody production and cellular immune responses at a dosage of 100 to 500 micrograms, did not enhance resistance to intravenous infection with a sublethal dose of 2 X 10(3) to 4 X 10(3) viable Listeria monocytogenes cells in mice when intraperitoneally injected either 20 min or 5 days before infection. Similarly, blockade of the mononuclear phagocyte system by dextran sulfate 500 could not be overcome by pretreatment with muramyl dipeptide. In contrast, dextran sulfate 500-induced loss of antibacterial resistance was found to be completely abolished by intraperitoneal injection of 3 X 10(9) killed Bordetella pertussis organisms when given 4 days before injection of dextran sulfate 500, i.e., 5 days before infection. B. pertussis were also effective in enhancing antibacterial resistance when administered 5 days before infection. The different behavior of the two adjuvants tested is assumed to be due to their different nonspecific proliferative capacities. Thus, B. pertussis are assumed to act by direct stimulation of the mononuclear phagocyte system whereas muramyl dipeptide does not.     

Persistent enhancement of cell-mediated and antibody immune responses after administration of muramyl dipeptide derivatives with antigen in metabolizable oil.

Circulating antibody titers can be increased when the antigen is administered in an aqueous medium with N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide). Results reported here show that cell-mediated immunity can be demonstrated when this synthetic adjuvant or an active analog is injected with an antigen (ovalbumin) in metabolizable squalane emulsion. Under these conditions a lipophilic derivative of muramyl dipeptide was shown to be even more active and to enhance long-lasting immune responses.     

Arthritogenic activity of a synthetic immunoadjuvant, muramyl dipeptide.

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.     

Stimulation of chemiluminescence by synthetic muramyl dipeptide and analogs.

The effect on respiratory burst of murine splenic cells after in vitro exposure to synthetic muramyl dipeptide (MDP) and 6-O-acyl and quinonyl derivatives was studied at an early phase of interaction by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. The MDP molecule enhanced CL, but the degree of CL response varied with the kinds of fatty acids introduced in the chemical structure of synthetic glycopeptide analogs. A 6-O-acyl derivative possessing an alpha-branched fatty acid chain, B30-MDP, stimulated maximum levels of CL activity. High CL responses were obtained with L8-MDP having a short chain of linear fatty acids and with QS-10-MDP-66 containing a ubiquinone compound. CL was also stimulated by MDP and its analogs in the spleen cells of nude mice lacking mature T lymphocytes, but the extent of stimulation was decreased compared with that of normal spleen cells.