Muramyl Dipeptide Derivatives Research Articles

The Inhibitory Effects by Combined Doses of DDS and several Immunostimulants on the Growth of Leprosy Bacilli Inoculated into Footpads of hybrid Nude Mice, Jcl:AF-nu

The inhibitory effects by the combined doses of bacteriostatic DDS and several immunostimulants on the growth of leprosy bacilli (LB) inoculated into the footpads of nude mice were examined, using a strain of hybrid nude mice named Jcl:AF-nu, which is resistible to infections more than strain BALB/c-nu. The results found were: 1. LB could proliferate up to 10(9) level in the footpads of this strain of hybrid nude mice. 2. The combined dose of muramyl dipeptide (MDP) or a water-soluble lipoidal amine, CP-46665 and DDS mixed with chow in the content ratio of 0.005% completely inhibited the growth of LB. Whereas, the growth inhibition by DDS alone was only partial. 3. A derivative of MDP, muroctasin could partially inhibit the growth of LB without combined dose of DDS. 4. The combined dose of an antitumor beta-1, 3-glucan named ATSO could not enhance the partial growth inhibition due to the dose of DDS through 0.005%-DDS chow. Based on these results, the possibility of Jcl:AF-nu mice as a favorable animal model for examining the synergic inhibitory effect on the growth of LB due to the combined dose of an antileprous chemotherapeutic and an immunostimulant was discussed.

Interleukin-1-induced sleep and febrile responses differentially altered by a muramyl dipeptide derivative

Muramyl dipeptide ( N- acetylmuramyl- L - alanyl- D - isoglutamine; AcMur- L - Ala- D - iGln; MDP ) is a somnogenic, pyrogenic, and immunoadjuvant component of bacterial cell wall peptidoglycan. It enhances the production of the cytokine interleukin 1 (IL-1) in vivo and in vitro; IL-1 itself is somnogenic and pyrogenic. The pyrogenic effects of IL-1 may be separated from its somnogenicity by the use of antipyretics. A methyl ester derivative of MDP (murametide; AcMur- L-Ala- D-Gln-α-methyl ester) blocks IL-1-induced fever, but induces IL-1 production/release. Therefore, the effects of murametide on IL-1-induced sleep and fever responses were determined. Rabbits were pretreated with intravenous injections of murametide 0,30 or 60 min before intravenous injection of a pyrogenic/somnogenic dose of recombinant IL-1β (rIL-1β). Murametide alone produced no effects on sleep or brain temperatures. Murametide given simultaneously with rIL-1β potentiated increases of brain temperature, whereas when there was a 60 min delay between the injection of murametide and rIL-1β, responses were similar to those induced by rIL-1β alone. Murametide given 30 min before rIL-1β, blocked the expected IL-1-induced fevers but not the rIL-1β-mediated increased of slow-wave sleep and decreases of rapid-eye-movement sleep. These results reinforce the conclusion that, in part, independent brain mechanisms are involved in the somnogenic and pyrogenic actions of IL-1.

Synthesis of derivatives of N-acetylmuramyl-?-alanyl-d-isoglutamine containing activated disulfide bonds

N-Acetylmuramyl-l-alanyl-d-isoglutaminyl-l-S-(2-pyridylthio)cysteine and 1-(N-acetylmuramyl-l-alanyl-d-isoglutaminamido)-6-[3-(2-pyridylthio)-propionamido]hexane, derivatives of muramyldipeptide each with an activated disulfide bond, have been synthesized. Disulfide conjugates of muramyldipeptide with peptide fragment 136–147 of the heavy chain of the hemagglutinin of influenza virus A/Aichi/2/68 have been obtained.

Photoaffinity labelling of macrophages and B-lymphocytes using 125I-labelled aryl-azide derivatives of muramyldipeptide

Two 125I-labelled aryl-azide derivatives of MDP have been synthesized. Photoaffinity labelling experiments demonstrated cell surface binding sites for muramylpeptides on activated B-lymphocytes and intracellular muramylpeptides binding protein(s) of 40–45 kD in rat alveolar macrophages.

Synthesis of fluorescent muramyl dipeptide congeners. 2.

Muramyl dipeptide (MDP) analogues were prepared and utilized in the synthesis of new fluorescently labeled MDP derivatives for use as biologic probes. Thus, N alpha-(N-acetylmuramyl)-L-lysyl-D-isoglutamine (Lys-MDP, 4) and N alpha-(N-acetylmuramyl)-L-alanyl-D-isoglutaminyl)-L-lysine [MTP, 5] were synthesized and then reacted with 2-(fluoresceinylamino)-4,6-dichloro-s-triazine (DTAF, 2) to yield the fluorescent adducts, DTAF-Lys-MDP (6) and DTAF-MTP (7). The adjuvant activity of the fluorescent MDP derivatives was determined by the ability of the compounds to promote delayed skin test responses in guinea pigs immunized with ovalbumin (OA) and by evaluating the anti-OA activity of these guinea pigs.

Immunological activities of multiprenylacetyl derivatives of muramyldipeptides

The immunological properties of six kinds of multiprenylacetic acids and six kinds of multiprenylacetyl muramyldipeptide (MDP) derivatives were examined by using experimental models in mice and guinea-pigs. All the multiprenylacetyl MDP derivatives, particularly TMD-232, showed potent adjuvant activity on the circulating antibody formation against bacterial α-amylase in mice, and induction of delayed-type hypersensitivity to monoazobenzenearsonate N- acetyl- l-tyrosine in guinea-pigs. All multiprenylacetic acid preparations tested in this study, however, showed no adjuvant activity in these immune systems. Both TMD-17 and TMD-232 entrapped into multilamellar vesicles showed potent host stimulation activity against Sendai virus infection in mice.

Induction of tumoricidal macrophages and production of cytokines by synthetic muramyl dipeptide analogues

The ability of various synthetic muramyl dipeptide (MDP) derivatives to induce the production of interleukin-1 (IL-1) and colony stimulating factor (CSF) in vitro and in vivo and to induce cytotoxic macrophages was studied. 6-O-L18-MDP(Me) and MDP-Lys(L18), which were potent inducers of IL-1 and CSF production and of cytotoxic macrophages, had protective activity against Sendai virus infection in mice. In contrast, 1-O-L18-(6-O-P)-MDP(Me) and 2-N-L18-MDP exhibited weak or no ability to induce IL-1 and CSF production and no induction of tumoricidal macrophages, and did not protect against infection of Sendai virus. MDP derivatives, except 2-N-L18-MDP, efficiently rendered macrophages cytotoxic against target cells in the presence of murine recombinant interferon-gamma in vitro. The derivatives that induced cytokines and cytotoxic macrophages appeared to produce anti-viral activity.

Activation of macrophage in vitro to acquire antitumor activity by a muramyl dipeptide derivative encapsulated in microspheres composed of lactide copolymer

An immunopotentiator, lipophilic muramyi dipeptide derivative (MDP-B30), was encapsulated in microspheres composed of biodegradable polymers from l- lactic acid and glycolic acid copolymers. Tumor growth inhibitory activity of mouse peritoneal macrophages (PM) was investigated by the use of these microspheres. PM were activated by in vitro phagocytosis of the microspheres to suppress the growth of syngeneic, allogeneic, and xenogeneic tumor cells, but left normal (nontumorigenic) cells unharmed. When encapsulated in microspheres, MDP-B30 was far more efficient than free MDP-B30 in enhancing the tumor growth inhibitory activity of PM. The necessary minimum dose of MDP-B30 in the microspheres per unit volume of culture medium for PM activation was approximately one thousand times lower than that of free MDP-B30. A significant degree of activation was observed after incubation of PM with the microsphere-encapsulated MDP-B30 only for 3 h. The activated state of PM which phagocytosed microspheres maintained for a longer period than that of free MDP-B30. The growth inhibition of PM activated by the microspheres was found to increase with an enhancement in the phagocytosis which could be regulated by changing the microsphere size. Moreover, the maintenance of the in vitro activity could be readily controlled by changing the molecular weight, and/or monomer composition of polylactides and was in good accord with the release profiles of MDP-B30 from the microspheres. The growth inhibitory activity of PM was also observed when the microspheres were phagocytosed in situ. Mice bearing Meth-A tumor cells could survive when injected with the microsphereencapsulated MDP-B30, whereas injection with the free, unencapsulated MDP-B30 had little effect on the survival.

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.

Modulation of carrier-induced epitopic suppression by Bordetella pertussis components and muramyl peptide

Synthetic antigens employed in experimental synthetic vaccines are generally small haptenic peptides. Therefore, effective immunization with these antigens usually requires the use of an immunogenic carrier. Tetanus toxoid has been proposed for use as a carrier in future synthetic vaccines due to its high immunogenicity and acceptance for human use. Previous studies employing standard hapten/carrier systems such as DNP/KLH have demonstrated, however, that an epitope-specific suppression occurs when mice previously primed with carrier are subsequently immunized with an haptenic epitope conjugated to the same carrier. These same studies have shown that Bordetella pertussis vaccine administered at the time of carrier priming abrogates epitopic suppression. In the present investigation, epitopic suppression was studied in a synthetic vaccine model employing tetanus toxoid as a carrier. Results from these studies indicated that mice primed with tetanus toxoid 1 month before immunization with a peptide-tetanus toxoid conjugate exhibited enhanced secondary anti-tetanus toxin responses but decreased anti-peptide responses. Furthermore, injection of pertussis vaccine or purified B. pertussis toxin or endotoxin at the time of carrier priming could block the establishment of epitopic suppression. Administration of B. pertussis components enhanced antibody responses to both the carrier and the synthetic peptides as compared with responses of control animals. In addition, administration of an adjuvant-active nonpyrogenic derivative of muramyl dipeptide, Murabutide, with carrier priming reduced epitopic suppression of anti-peptide responses. B. pertussis toxin or endotoxin administered to mice previously suppressed by carrier priming with the first injection of carrier-peptide conjugate overcame epitopic suppression with resultant titers of anti-peptide antibody equal to or greater than nonsuppressed controls. These results suggest that the use of adjuvants with future synthetic vaccines may contribute the additional advantage of overcoming epitopic suppression, thus permitting the use of common, well-tolerated carrier systems such as tetanus toxoid in synthetic vaccine preparations.

Augmentation by priming with interferon-gamma of the binding of a muramyl dipeptide derivative to macrophages resulting in synergistic macrophage activation.

Macrophage (MA) activation by recombinant murine interferon-gamma (rMuIFN-gamma) and a synthetic muramyl dipeptide derivative, N alpha-(N-acetylmuramyl-L-alanyl-D-isoglutaminyl)-N epsilon-stearoyl-L-lysine [MDP-Lys(L18)], was examined. The cytostatic activity of MA that had been primed with rMuIFN-gamma against Lewis lung adenocarcinoma cells was augmented extensively by exposure to MDP-Lys(L18) for a minimum of 15 min, though such treatment in the reverse sequence interfered with the effects of rMuIFN-gamma on MA. The binding assays revealed that rMuIFN-gamma bound to MA with an apparent dissociation constant (Kd) of 0.93 X 10(-9) M (16,000 binding molecules/cell), while MDP-Lys(L18) bound nonspecifically and was endocytosed by MA at 37 degrees. The amount of MDP-Lys(L18) bound to the rMuIFN-gamma-primed MA was significantly greater than that bound to the MA without rMuIFN-gamma priming. A small increase in the amount of MDP-Lys(L18) associated with MA was also observed at 4 degrees, but the amount was one order of magnitude less than that at 37 degrees. The binding of rMuIFN-gamma to MA was significantly suppressed by priming with MDP-Lys(L18). These results indicate that the binding of rMuIFN-gamma in preference to MDP-Lys(L18) to MA is of great importance in the mechanisms of MA activation.

Modulation of pulmonary macrophage superoxide release and tumoricidal activity following activation by biological response modifiers

Following immunologic activation, pulmonary macrophages may prevent or cause regression of lung metastases by mechanisms which remain largely unknown. The studies described here were designed to determine if enhanced oxygen metabolite release was related to postactivation tumoricidal activity. We have shown that in vitro activation of Fischer 344 rat pulmonary macrophages by either free or liposome-encapsulated muramyl dipeptide leads to both enhanced release of superoxide anions and marked tumoricidal activity against syngenic (Fischer 13762), allogeneic (Schmidt-Ruppin RR 1022) and xenogeneic (Fibrosarcoma MCA-F) 125I-deoxy-uridine-labeled target cells. This immune modulator did not, however, metabolically activate pulmonary macrophages as effectively as liposome-encapsulated lipopolysaccharide. A 24-h in vitro incubation with either 150 U or 300 U of interferon-γ (3 × 106 U/mg) or 30 U, 150 U or 300 U of interferon-α (6 × 105 U/mg) caused a significant elevation in superoxide release above controls, whereas short-term exposure (2 or 4 h) had little or no effect. Free or encapsulated 6-O-stearoyl muramyl dipeptide, on the other hand, did increase superoxide levels at all 3 time periods. When either interferon-γ or free or encapsulated muramyl dipeptide derivative were administered to intact rats by either i.v. injection, intratracheal instillation or osmotic minipump infusion, pulmonary macrophage tumoricidal activity was observed 96 h after cell harvesting. Zymosan-stimulated superoxide release, however, was not consistently elevated above control or empty liposome treatment following this course of in vivo activation. The data collectively suggest that in vivo pulmonary macrophage activation to a tumoricidal state and metabolic activation resulting in enhanced superoxide may be separable events.

Synergistic induction of cytotoxicity in macrophages by murine interferon-gamma and biological response modifiers derived from microorganisms.

The ability of recombinant murine interferon-gamma (rMuIFN-gamma) to activate murine macrophages with or without several biological response modifiers (BRM), including synthetic muramyl dipeptide derivatives (MDPs), was investigated. Mouse peritoneal macrophages were activated by rMuIFN-gamma alone to the cytostatic state, but not the cytolytic state. Other BRM as well as bacterial lipopolysaccharide (LPS), including a lyophilized preparation of an attenuated strain of Streptococcus hemolyticus, a cell wall skeleton of bacillus Calmette-Guerin and synthetic MDPs, were highly active in generating the synergism with rMuIFN-gamma. Macrophages were endowed with the cytolytic activities by combinations of rMuIFN-gamma and MDP-Lys(L18); the combination of 100 U/ml of rMuIFN-gamma with 10 ng/ml of MDP-Lys(L18) was sufficient to induce cytolytic activities in macrophages. The synergism was observed when the macrophages primed with rMuIFN-gamma were treated with LPS or MDP-Lys(L18), but not when the sequence of treatment was reversed. The cytotoxicity of macrophages induced by rMuIFN-gamma with MDP-Lys(L18) was suppressed by priming with MDP-Lys(L18). The suppressive effect was also observed by priming with LPS in combinations of rMUIFN-gamma and LPS. The reason for the suppression of macrophage activation by priming with LPS and MDP-Lys(L18) is at present unknown.

Potentiation of direct antitumor cytotoxicity and production of tumor cytolytic factors in human blood monocytes by human recombinant interferon-gamma and muramyl dipeptide derivatives.

We investigated whether human peripheral blood monocytes isolated by centrifugal elutriation from healthy donors could be activated to become tumoricidal and release tumor cytolytic factor (TCF) subsequent to incubation with recombinant human interferon-gamma (r-IFN-gamma) or a derivative of muramyl dipeptide (nor-MDP), or both. Blood monocytes incubated in endotoxin-free medium containing up to 1000 U/ml of r-IFN-gamma or in medium containing less than 1 microgram/ml of nor MDP were not activated to lyse radiolabeled allogeneic human tumor cells. In contrast, the incubation of monocytes with various dose combinations of r-IFN-gamma and nor-MDP generated significant direct cytotoxic activity as well as production of TCF. Preincubation of the r-IFN-gamma and nor-MDP mixture with polymyxin B did not inhibit the synergism, thus ruling out the possibility that the process was due to endotoxin contamination. TCF harvested from monocyte culture supernatants was cytolytic against five allogeneic tumor targets, but not against a nontumorigenic cell line. Collectively, the data demonstrate that r-IFN-gamma can prime human blood monocytes to allow their activation by synthetic nor-MDP.

Type-specific opsonic antibodies evoked with a synthetic peptide of streptococcal M protein conjugated to polylysine without adjuvant.

A chemically synthesized copy (S-CB7) of a fragment (35 amino acid residues) of type 24 streptococcal M protein was covalently linked to polylysine with carbodiimide and injected subcutaneously into rabbits without adjuvant. Although the primary immune responses as measured by enzyme-linked immunosorbent assays at biweekly intervals were weak, the secondary responses as measured by both enzyme-linked immunosorbent assays and opsonophagocytic assays were as high as those obtained previously in rabbits immunized with the peptide conjugate emulsified in complete Freund adjuvant. Injection of murabutide, a synthetic muramyl dipeptide derivative of bacterial peptidoglycan, with the initial immunizing dose of peptide conjugate had no apparent effect on the secondary immune responses. These results indicate that protective immune responses may be raised against polylysine conjugates of chemically synthesized peptide copies of streptococcal M protein without adjuvant.

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives. I. Synthesis of a novel haptenic MDP derivative cross-reactive with Bacillus Calmette Guerin and its application to enhanced induction of tumor immunity.

A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.

Comparison of Murine B Cell Clonal Expansions by Synthetic Lipid A and Muramyl Dipeptide Analogs

Direct stimulations of murine B lymphocytes with synthetic lipid A analogs and synthetic muramyl dipeptide (MDP) derivatives were studied using a limiting dilution assay system. Synthetic lipid A analogs, GLA-27 and GLA-40, when conjugated with bovine serum albumin (BSA) had the ability to induce B cell clonal expansion of a single B cell from the spleen or bone-marrow. Their activities were almost the same as those of naturally obtained lipid A, but were lower than that of bacterial lipopolysaccharide (LPS). Addition of dextran sulfate (DXS) enhanced the effect of lipid A analogs. In contrast, synthetic MDP and its derivatives, although they had many biological and immunological activities in experimental animals, could not stimulate a single B cell to induce clonal expansion regardless of the presence or absence of DXS. These results suggested that lipid A analogs can directly cause the proliferation of B cells, but MDPs can not.

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 ε-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.

Biologically active antibodies elicited by a synthetic circumsporozoite peptide of Plasmodium knowlesi administered in saline with a muramyl dipeptide derivative.

A synthetic peptide whose sequence was derived from the circumsporozoite protein of Plasmodium knowlesi coupled to bovine gamma globulin has been shown to be immunogenic when administered with Freund complete adjuvant. The present experiments were designed to test the immunogenicity of the peptide when attached to a tetanus toxoid carrier and administered with alum or murabutide, both acceptable clinical adjuvants. In both cases, the use of an adjuvant increased the levels of circulating anti-peptide antibodies over those observed when no adjuvant was used. However, when the antisera were tested for reactivity with the native protein, animals of the group receiving the conjugate associated with murabutide always had titers greatly exceeding those observed in animals that received the conjugate with alum. Moreover, the sera of the murabutide-treated group were shown to be more active in eliciting shedding of the circumsporozoite protein than were sera of animals of the Freund complete adjuvant-treated group. The use of tetanus toxoid in secondary immunizations could be eliminated when the mice primed with peptide-tetanus toxoid and murabutide were boosted with a polymer of the peptide. The results indicate that the synthetic malarial peptide-tetanus toxoid conjugate is capable of stimulating high levels of biologically active antibodies only when administered with murabutide.

Protection of mice against fatal herpes simplex type 2 infection by liposomes containing muramyl tripeptide.

Intravenous administration of liposomes containing muramyl tripeptide phosphatidylethanolamine, a lipophilic derivative of muramyl dipeptide that activates macrophages to a cytolytic state in situ, significantly protected mice against lethal challenge with herpes simplex virus type 2. These findings suggest that the systemic activation of macrophages by liposomes containing an immunomodulator can lead to prophylaxis of severe infections caused by herpesviruses.