Modulation of natural killer activity by muramyl peptides: relationship with adjuvant and anti-infectious properties

Natural killer (NK) activity of spleen cells was studied in DBA/2 mice, 24 and 72 h after intravenous injection of various muramyl peptides: muramyl dipeptide (MDP) and derivatives which are both adjuvant-active and able to increase resistance against Klebsiella pneumoniae; derivatives which are adjuvant-active but devoid of anti-infectious properties; derivatives which are anti-infectious but devoid of adjuvant activity, and derivatives which are devoid of both activities such as the stereoisomer MDP[D-Ala]1. An early increase in NK activity was observed 24 h after injection of all nonadjuvant derivatives, whatever their effect on infection. A stimulation of natural cytotoxicity was always induced 72 h after injection of MDP and derivatives able to protect mice against Klebsiella pneumoniae infection. So, even if the reverse was not true, there seems to exist some correlation between the anti-infectious effect of muramyl peptides and the late increase in NK activity. The modulation of NK activity by muramyl peptides appeared to be independent of interferon production. Moreover, inhibition of the stimulatory effect by a cell cycle-specific drug, hydroxyurea, observed 72 h after MDP suggests a requirement for proliferation.     

The in vitro effect of new muramyl peptide derivatives on cytotoxic activity of NK (Natural Killer) cells from hamsters bearing AB Bomirski Melanoma

The modulation of NK activity by muramyl dipeptides derivatives against Ab (amelanotic) Bomirski melanoma and human erythroleukemia K562 cells was studied in vitro. The stimulatory effect was observed for 3 of 7 muramyl dipeptides: MDP(L-Ala)C921, MDPC857 and L18-MDP(Ala) in relation to cytotoxic activity of NK cells obtained from peripheral blood and spleen of healthy and Ab Bomirski melanoma bearing hamsters. An increased of cytotoxic activity NK cells isolated from animals before and during the transplantable phase of the tumor against K562 was found. A similar stimulation was received for NK cells obtained from animals against their own melanoma cells. The most significant influence of examined MDP derivatives on the cytotoxic activity of NK cells were obtained from animals between 10 to 12 days of tumor growth. The extent of the modulation of cytotoxic activity of NK cells was dependent on its initial value both in healthy control and Ab Bomirski melanoma bearing hamsters. If natural cytotoxic activity was high the stimulatory effect of the examined MDP derivatives was only slightly expressed.     

Synthesis, mass spectral characterization and immunoadjuvant activity of some novel lipophilic derivatives of muramyl dipeptides

Some novel lipophilic derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) have been prepared and rigorously evaluated by spectroscopic means. Fast atom bombardment and field desorption mass spectrometry provided information about both molecular weight and structural detail. The new MDP derivatives have been tested in guinea pigs for immunoadjuvant activity using egg albumin as the model antigen. Amongst these derivatives, 6-O-[3-(5-cholesten-3 beta-yloxycarbonyl) propionyl]-N-acetylmuramyl-L-alanyl-D-isoglutamine (CSMDP), 6-O-[3-1,2-dipalmitoyl-sn-glycero-3-carbonyl) propionyl]-N-acetylmuramyl-L-alanyl-D-isoglutamine (GSMDP) and N-palmitoyl muramyl-L-alanyl-D-isoglutamine (PMDP) possessed significantly better activity than MDP, as judged by the antigen-specific antibody and delayed hypersensitivity responses in the immunized animals. In addition, CSMDP was found to induce strong delayed hypersensitivity response even in saline. These three active compounds were also tested for their pyrogenic response in rabbits, and were found to be lesser pyrogenic than MDP. Some of these MDP derivatives hold promise as adjuvants in immunization.     

Modulation of natural killer cytotoxicity by muramyl dipeptide and trehalose dimycolate incorporated in squalane droplets

Summary The effect on natural killer (NK) cytotoxicity of splenic cells from BALB/c mice pretreated i. v. with squalane-in-water preparations of muramyl dipeptide (MDP), trehalose dimycolate (TDM), or the combination of MDP-plus-TDM was investigated. MDP or TDM augmented the NK cytotoxicity which peaked 48 h after the pretreatment whereas the combination of MDP and TDM induced an inhibition of the NK activity. Infection with influenza virus, a potent stimulator of NK cells, after the pretreatment with biological response modifiers resulted in a markedly enhanced NK activity on day 2 in MDP and control groups. Mice pretreated with TDM or the combination of MDP and TDM showed only moderate NK activity which peaked on day 3 after influenza infection. The NK activity was susceptible to asialo GM₁ and complement treatment. The cytotoxicity of MDP-plus-TDM cells could be significantly enhanced after treatment with anti-macrophage monoclonal antibody and complement. NK activity induced by MDP or TDM was reduced by mixing MDP-plus-TDM cells. Addition of adherent cell-depleted MDP-plus-TDM suspension to MDP or TDM cells had a NK restorative effect. Splenic cells from mice pretreated 2 days earlier with MDP or TDM, but not MDP-plus-TDM, generated enhanced levels of luminol-dependent chemiluminescence.     

Effect of terrilytin on the activity of nonspecific resistance factors during immunization with staphylococcal anatoxin and staphylococcal infection

Terrilytin and immobilized terrilytin enhance the activity and intensity of phagocytosis and increase the concentration of lysozyme in nonimmunized animals. Both preparations increase the production of antibodies to staphylococcal alpha-hemolysin, the titers of beta-lysins, the activity and intensity of the phagocytosis of bacterial cells by peripheral blood leukocytes in animals immunized with staphylococcal toxoid and challenged with live staphylococcal culture. In healthy animals terrilytin and immobilized terrilytin induce an increase in total proteolytic activity and in the activity of alpha-1-antitrypsin and alpha-2-macroglobulin, decreased as the result of staphylococcal infection.     

Stimulation of Nonspecific Host Resistance to Infection Induced by Muramyldipeptides

The effect of muramyldipeptide (MDP), N-acetylmuramyl-l-alanyl-d-isoglutamine [MDP(Ala)], and its analogs on bacterial infection was studied using the experimental model of sepsis infection in mice. Injection of MDP(Ala) gave mice definitive protection against E. coli infection, but only partial protection against P. aeruginosa or K. pneumoniae infection. Several factors influencing the protective activity of MDP (Ala) on E. coli infection were studied, and it was demonstrated that the activity was induced by various routes of administration of MDP(Ala), including the oral route, and was markedly influenced by the bacterial inoculum size. It was also shown that the effective dose of MDP(Ala) was 100 μg per mouse for intraperitoneal, intravenous or subcutaneous injections and 1,000 μg per mouse when administered orally. Furthermore, the optimal interval between MDP-treatment and infection was 24 hr when the treatment was carried out before infection. Clearance of bacterial cells in blood was observed after E. coli infection in mice treated with MDP(Ala). The efficacy of MDP(Ala) and two analogs, N-acetylmuramyl-l-valyl-d-isoglutamine [MDP(Val)] and N-acetylmuramyl-l-seryl-d-isoglutamine [MDP (Ser)], was evaluated for the E. coli infection; MDP(Val) was proven to be slightly less active than MDP(Ala), and MDP(Ser) to be the least effective, although MDP(Val) or MDP(Ser) was reported to have higher adjuvanticity than MDP (Ala) for the development of delayed-type hypersensitivity.     

Muramyl Peptides as Possible Endogenous Immunopharmacological Mediators

Synthetic N-acetylmuramyl-L -alanyl-d-isoglutamine, also called MDP for muramyl dipeptide, is a copy of a fragment of bacterial peptidoglycan. Soon after the recognition of MDP as being the minimal subunit responsible for the activity of Freund's complete adjuvant, a great number of derivatives were synthesized. Because of their very low molecular weight it was hoped that they could retain selectively certain of the numerous effects produced by complex bacterial agents. Evidence was gathered showing MDP's direct effect on lymphocytes and on macrophages. The ensuing studies reviewed that MDP and several of its derivatives have marked immunopharmacological and neuropharmacological activities. Thus, besides being adjuvants, they are capable of producing hyperthermia by acting directly on thermoregulation centers or by inducing in vivo and in vitro endogenous pyrogens (EP). More recently, Krueger et al have shown that slow-wave sleep (SWS) factor was a muramyl peptide of a molecular weight close to 1,000 daltons. They have also shown that MDP and several of its synthetic analogs had a somnogenic activity. It has previously been hypothesized that several of the immunological activities of the muramyl peptides could be due to biological mimicry with endogenous products. Recent observations argue in favor of the presence of an MDP bacterial structure in mammalian mediators which increase slow-wave sleep and/or produce fever. The implications of these findings will be discussed.     

Induction, in vivo and in vitro, of macrophage membrane interleukin-1 by adjuvant-active synthetic muramyl peptides

Recent evidence has shown that a membrane form of interleukin-1 (IL-1) serves as a necessary signal for antigen presentation, leading to T-cell activation. The synthetic immunostimulant muramyl dipeptide (MDP) is known to induce secretion of IL-1 and its adjuvant effect was found to be mediated through enhancement of T-helper cells. We have investigated the ability of MDP and 19 other adjuvant-active or -inactive MDP analogs and derivatives to induce membrane IL-1 in mouse peritoneal macrophages. Enhancement in vitro of membrane expression and secretion of IL-1 in fresh or aged cultures of macrophages was observed after stimulation with MDP or with adjuvant-active but not with adjuvant-inactive muramyl peptides. Administration in vivo of adjuvant-active doses of MDP or of any of 12 other active analogs induced high levels of macrophage membrane IL-1 detected by the lymphocyte-activating factor assay. This effect was not observed when 7 other adjuvant-inactive derivatives were used. Moreover, under conditions where MDP did not exert an adjuvant effect, this immunomodulator was found to be incapable of inducing the expression of macrophage membrane IL-1. These results demonstrate a very high correlation between the ability to induce membrane IL-1 and the adjuvant activity of muramyl peptides. The correlation was observed irrespective of other biological effects of the synthetic adjuvants such as pyrogenicity and/or anti-infectious activity.     

The immunomodulating activity of new muramyl dipeptide derivatives in vitro.]

The action of some new MDP derivatives on functional activity of murine T-lymphocytes and macrophages was studied. The following tests have been used: proliferation of spleen cells in one-way allo-MLC; IL-1 and TNF production by peritoneal macrophages treated with the preparations. The most expressed enhancement of lymphocyte proliferative response in MLC has been exerted by beta C7H15 MDP and beta C16H33 MDP (stimulation indexes 31-69%). beta C7H15 MDP, beta C16H33 MDP and polyacrylamide-MDP (P-MDP) alone or in combination with LPS caused elevated secretion of IL-1 by macrophages. While beta C7H15 MDP was as active as MDP, beta C16H33 MDP and P-MDP manifested increased ability to stimulate IL-1 production in comparison with MDP. beta C7H15 MDP, beta C16H33 MDP, P-MDP and MDP induced similar level of TNF production by murine macrophages. However, simultaneous treatment of macrophages with beta C16H33 MDP and LPS resulted in more significant enhancement of TNF production than combination LPS + MDP.     

Dissociation of immunostimulant activities of muramyl dipeptide (MDP) by linking amino-acids or peptides to the glutaminyl residue

N-acetyl-muramyl-L-alanyl-D-isoglutamine (muramyl dipeptide or MDP) is the minimal structure required for substituting mycobacteria in Freund's complete adjuvant. It is an adjuvant when injected in saline, protects mice non-specifically against infection, and enhances thymidine incorporation of lymphocytes. In this report it is shown that the linking of L-Lys, L-Ala-D-Ala, L-Lys-D-Ala or L-Lys-L-Ala to MDP permits the dissociation of anti-infectious activity from adjuvant activity. The optical configuration of the added residues plays the major role in this dissociation. It can be noted that the muramyl tetrapeptide MurNAc-L-Ala-D-isoGln-L-Lys-D-Ala mimicking the natural structure is devoid of anti-infectious activity.     

Structural specificity of synthetic peptide adjuvant for induction of experimental allergic encephalomyelitis

The peptide N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), which has adjuvant activities, and 17 of its derivatives and analogs were synthesized and assayed to elucidate the structure necessary for adjuvant activity in induction of experimental allergic encephalomyelitis (EAE) in guinea pigs. The results revealed the importance of the d configuration and the α-carboxamide group of the isoglutaminyl residue of MDP for adjuvant activity. Replacement of the l-alanyl residue of MDP by d-alanine, but not by l-serine or glycine, resulted in a marked decrease in the activity. The β-methyl glycoside of MDP was found to be more active than the α-methyl derivative. 6-O-Stearoyl-N-acetylmuramyl-l-alanyl-d-isoglutamme showed activity.     

Efficacy of human beta-casein fragment (54-59) and its synthetic analogue compound 89/215 against Leishmania donovani in hamsters

The characteristic feature of visceral leishmaniasis (VL) is the profound impairment of immune system of the infected host, which contributes significantly to the partial success of antileishmanial chemotherapy. Since in VL, cure is the combinatorial effect of drug and immune status of the host, the rationale approach towards antileishmanial chemotherapy would be to potentiate the immune functioning of the host to extract desired results. Towards this direction several rationally designed analogues of human beta-casein fragment (54-59) were evaluated for their ability to stimulate the non-specific resistance in hamsters against Leishmania donovani infection. By virtue of being derived from the food protein casein derivatives may be devoid of unwanted side effects associated with the substances of microbial origin, e.g. muramyl dipeptide (MDP). Out of this one peptide Val-Glu-Gly-Ile-Pro-Tyr (compound 89/215) had been reported to have such activity. In this communication, the prophylactic and therapeutic efficacy of the peptide along with its natural sequence has been evaluated in detail against experimental VL in hamsters. Their use as an adjunct to chemotherapy was also explored. Human beta-casein fragment, compound 89/215 and MDP were tested in vivo at various dose levels wherein compound 89/215 showed superiority over MDP at 3 mg/kg x 2 given intraperitoneally (i.p.). Compound 89/215 sensitized peritoneal macrophages acquired considerable resistance and only 24% of the cells were found infected in comparison to control peritoneal macrophages where 76.4% of the cells were found infected. Similarly, the efficacy of sodium antimony gluconate (SAG) in hamsters pretreated with compound 89/215 enhanced significantly (P < 0.001). This peptide also exhibited considerably good therapeutic efficacy when evaluated either alone or in combination with SAG in established infection of L. donovani.     

Augmentation of mouse natural killer cell activity by muramyl dipeptide and its analogs

The ability of muramyl dipeptide (MDP) and its structural analogs (des-MDP, abu-MDP, and des-abu-MDP) to influence mouse natural killer (NK) cells in two different strains of mice was examined. In CBA/J mice, administration of MDP by both intraperitoneal (ip) and intravenous (iv) routes enhanced splenic NK cell activity. Maximum augmentation of NK cell activity was observed 3 days after MDP treatment. NK cell activity was also stimulated upon in vitro culture of CBA/J mouse spleen cells with MDP. Only iv inoculation of MDP to C57BL/6 mice 7 days previously enhanced NK cell activity of spleen cells. Peritoneal NK cell activity was not affected in either strain of mice, regardless of the route of inoculation of MDP. Two structural analogs of MDP, abu-MDP and des-abu-MDP, enhanced peritoneal NK cell activity, whereas des-MDP had no effect when tested 3 days after ip treatment of CBA/J mice with these compounds. Peritoneal NK cell activity of C57BL/6 mice was not modulated by des-MDP, abu-MDP, or des-abu-MDP. A synergistic effect on peritoneal NK cell activity was observed in both CBA/J and C57BL/6 mice treated first with MDP and then with lipopolysaccharide (LPS) or Bacillus Calmette-Guerin (BCG).     

Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation

6-O-acyl-muramyldipeptides (MDP) with various lengths of fatty acid chains were examined for their dendritic cell (DC) maturation activity expressed through TLRs. Judging from anti-TLR mAb/inhibitor-blocking analysis, MDP derivatives with a single octanoyl or stearoyl fatty acid chain were found to activate TLR2 and TLR4 on human DCs, although intact and diacylated MDP expressed no ability to activate TLRs. Human DC activation profiles by the monoacylated MDP were essentially similar to those by Calmette-Guerin (BCG)-cell wall skeleton (CWS) and BCG-peptidoglycan (PGN) based on their ability to up-regulate costimulators, HLA-DR, beta(2)-microglobulin, and allostimulatory MLR. Monoacylated MDP induced cytokines with similar profiles to BCG-CWS or -PGN, although their potency for induction of TNF-alpha, IL-12p40, and IL-6 was less than that of BCG-CWS or -PGN. The MDP derivatives initiated similar activation in normal mouse macrophages, but exhibited no effect on TLR2/4-deficient or MyD88-deficient mouse macrophages. Mutation of d-isoGln to l-isoGln in monoacylated MDP did not result in loss of the DC maturation activity, suggesting marginal participation of nucleotide-binding oligomerization domain 2, if any, in monoacyl MDP-dependent DC maturation. These results define the adjuvant activity of 6-O-acyl MDP compounds at the molecular level. They target TLR2/TLR4 and act through the MyD88-dependent pathway in DCs and macrophages. Hence, the unusual combined activation of TLR2 and TLR4 observed with Mycobacterium tuberculosis is in part reflected in the functional properties of monoacylated MDP compounds. These findings infer that the essential minimal requirement for TLR2/4-mediated adjuvancy of BCG lies within a modified MDP.     

Effect of antibiotic and vaccine therapies on the succinate dehydrogenase and phosphatase activity of liver cells of mice infected with Staphylococcus

Changes in the activity of succinate dehydrogenase (SDH), total and acid phosphatase (TP and AP) were studied in treatment of laboratory animals with rifampicin, lincomycin and with inactivated staphylococcal vaccine used alone or in combinations. It was shown that immunization of the animals with inactivated staphylococcal vaccine under conditions of experimental staphylococcal infection promoted stimulation of the enzyme activity. Rifampicin and lincomycin used for the treatment of such animals lowered the activity of the enzymes. The suppressing effect of the antibiotics increased with an increase in the period of their use. It should be noted that the inhibitory effect of rifampicin on the activity of SDH, TP and AP was less pronounced than that of lincomycin. The combined use of the vaccine and antibiotics for the treatment of the animals promoted an increase in the enzyme activity as compared to the use of the antibiotics alone. Sometimes the activity of SDH, TP and AP reached the control levels in such animals or the levels observed in the animals treated with the vaccine alone. Stimulation of the enzyme activity was more pronounced when the vaccine was used in combination with rifampicin.     

Target cells for the activity of a synthetic adjuvant: muramyl dipeptide.

Muramyl dipeptide (MDP), a synthetic adjuvant, increased the primary response of CBA mice to sheep red blood cells (SRBC). In reconstituted irradiated recipients, cooperation between T and B lymphocytes was required for the expression of adjuvant activity and MDP increased the efficiency of SRBC-educated T cells. The role of T-derived lymphocytes in mediating the MDP adjuvant activity was also demonstrated in irradiated mice and in mice reconstituted with various splenic cellular types of donors which had received SRBC and MDP 24 hr earlier. In our experiments, the macrophage did not seem to be involved, since MDP did not increase the phagocytic capacity of peritoneal exudate cells and MDP- and SRBC-pretreated macrophages had no increased ability to induce an anti-SRBC immune response. These results demonstrate the importance of T lymphocytes as mediators of the adjuvant activity of MDP.     

Thiol-disulfide interchange between cystine and N-2-mercaptoethyl-1, 3-diaminopropane as a potential treatment for cystinuria

The radioprotective compound WR2721 is a thiophosphate, which, when administered orally, is activated at the acid pH of the stomach to its free thiol (MDP). The free thiol is a mucolytic compound which acts via the reduction of disulfide bonds of mucin molecules. An equimolar mixture of MDP and cysteine, in urine at pH 6.0 and 37 degrees C, when oxidized by molecular oxygen, preferentially forms the soluble mixed disulfide between MDP and cysteine. The disulfide cystine will undergo thiol-disulfide interchange with MDP; as a result, cystine crystals are effectively dissolved. Moreover, in the presence of catalytic amounts of free thiol, the disulfide of MDP will undergo thiol-disulfide interchange with cystine to dissolve cystine crystals. The mixed disulfide of MDP with cysteine is soluble in urine at pH 6.0 and 37 degrees C to at least 100 mg/ml. Chromatographic procedures which permit the analysis of MDP and its mixed disulfide derivatives as MDP-sulfonic acid are described. By these procedures, it was demonstrated that 20% of a single oral dose of WR2721 was excreted as MDP derivatives in the urine of normal volunteers. These procedures will permit the evaluation of WR2721 in the treatment of cystinuria.     

Production of anti-sporozoite antibodies in absence of response to carrier by coupling an MDP derivative to a malaria peptide-tetanus toxoid conjugate

A synthetic peptide (pep) representing a portion of the Plasmodium knowlesi circumsporozoite protein attached to a tetanus toxoid (TT) carrier, has been shown to be immunogenic when delivered in saline with derivatives of the synthetic adjuvant, muramyl dipeptide (MDP). The present study was designed to determine if the degree of substitution of pep and of MDP derivatives on the tetanus toxoid (TT) carrier, as well as the choice of MDP derivative used play a role in determining anti-pep and anti-TT antibody levels. One of the MDP derivatives used in the conjugates was epsilon-amino-caproic Murabutide, since Murabutide which is currently in clinical trials cannot be conjugated. The results show that low doses of this derivative coupled with pep on TT can be used to stimulate high levels of circulating anti-pep antibodies without augmenting the anti-carrier response. In addition, anti-pep antibodies elicited in response to one of the conjugates were biologically active since they produced shedding of the circumsporozoite coat of live parasites.     

Effect of antibiotic therapy and vaccine therapy on the phagocytic reaction in mice infected with Staphylococcus

The time course of changes in the activity, intensity and completeness of phagocytosis with leukocytes of the peritoneal exudate was studied on mice with experimental staphylococcal infection treated with rifampicin, lincomycin and inactivated staphylococcal vaccine used alone or in combination. It was shown that immunization of the animals with inactivated staphylococcal vaccine promoted stimulation of the phagocytic defense. Rifampicin and lincomycin applied therapeutically induced a decrease in the activity, intensity and completeness of phagocytosis. It should be noted that rifampicin had a less pronounced inhibitory effect than lincomycin. The combined use of vaccine and antibiotics with therapeutic purposes promoted an increase in phagocytosis as compared to the use of the antibiotics alone. The combined therapy sometimes resulted in completeness of phagocytosis making it reach the control values (the 10th and 15th days, rifampicin and vaccine). It should be noted that a more pronounced stimulation of the activity, intensity and completeness of the phagocytosis was observed with the use of the combination of rifampicin and the vaccine.     

Immunostimulating effects of muramyl dipeptide, glucosaminyl muramyl dipeptide and their synthetic derivatives in vitro

The effect of muramyldipeptide (MDP), glucosaminylmuramyldipeptide (GMDP) and their six synthetic derivatives on production of tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-2 (IL-2) by murine spleen cells in vitro was studied. MDP induced insignificant TNF production and did not stimulate production of IL-1 by the murine splenocytes within a 24-hour cultivation period whereas in combination with lipopolysaccharide (LPS) it induced significant production of both the cytokins. GMDP induced marked production of TNF (54 per cent cytotoxic index) and IL-1 (stimulation index 8). Addition of LPS in an amount of 10 ng/ml increased production of TNF by the murine splenocytes under the effect of GMDP but had no effect on production of IL-1. Neither MDP nor GMDP even in combination with LPS induced production of IL-2 by splenocytes of mice DVA/2 and C57B1/6 at activation for 24 hours. All the synthetic derivatives of MDP and GMDP except the MDP polymer activated TNF production by the murine spleen cells. GMDP lysine had the highest effect: 67 per cent cytotoxic index. In combination with LPS its cytotoxic index amounted to 87 per cent. The TNF activity was always higher when LPS in an amount of 10 ng/ml was added to the glycopeptides.