Combination Of Muramyl Dipeptide Research Articles

Strengthening peptide-based drug activity with novel glyconanoparticle.

The therapeutic application of peptide-based drugs is significantly limited by the rapid proteolytic degradation that occurs when in blood. Encapsulation of these peptide structures within a delivery system, such as liposomes, can greatly improve both stability and target delivery. As part of our work focused on novel ambiphilic mannosylated neoglycolipids as targeted drug delivery systems, we have developed a C14-alkyl-mannopyranoside that forms self-assembled monodisperse liposomes. Herein, these glycoliposomes are investigated as a potential method to improve the plasma stability of peptide-based drugs. Reversed phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (MS) methods were developed to assess the in vitro plasma stability of two structurally diverse peptides, including the kappa opioid receptor selective antagonist dynantin, and the NOD2 innate immune receptor ligand muramyl dipeptide (MDP). The RP-HPLC methods developed were able to resolve the peptides from background plasma contaminants and provided suitable response levels and linearity over an appropriate concentration range. Both compounds were found to be significantly degraded in rat plasma. Increasing degrees of both entrapment and stabilization were noted when dynantin was combined with the C14-alkyl-mannopyranoside in increasing peptide:glycoside ratios. The combination of MDP with the glycolipid also led to peptide entrapment, which greatly improved the plasma stability of the peptide. Overall, the results clearly indicate that the stability of peptide-based structures, which are subject to degradation in plasma, can be greatly improved via entrapment within C14-alkyl-mannopyranoside-bearing glycoliposomes.

Muramyl Dipeptide Synergizes with Staphylococcus aureus Lipoteichoic Acid To Recruit Neutrophils in the Mammary Gland and To Stimulate Mammary Epithelial Cells

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.

NOD2 receptors in adenopituitary folliculostellate cells: expression and function

Folliculostellate cells (FS cells) are non-endocrine cells from the pituitary gland that respond to bacterial endotoxins by producing cytokines. In immune cells, an important component of bacterial recognition are the toll-like receptors (TLRs). Previously, we showed that FS cells express TLR4. The TLR4 ligand lipopolysaccharide (LPS) stimulates interleukin-6 (IL6) production through nuclear factor kappaB (NFKB) induction. Binding of IL6 to gp130 receptor activates signal transducer and activator of transcription 3 (STAT3), an important mediator of inflammatory response. Another family involved in innate immune response following bacterial infection is the nucleotide-binding oligomerisation domain (NOD) intracellular receptor family. Herein, we describe for the first time the expression and function of NOD receptors in human pituitary and FS TtT/GF cell line. The NOD2 agonist muramyl dipeptide (MDP) increased Nf kappa b1-transcriptional activity, -protein expression and IL6 secretion in TtT/GF cells. Furthermore, these effects were potentiated by the combination of MDP and LPS. Silencing NOD2 abolished the action of LPS on NFKB transcriptional activity and IL6 production, indicating that, in TtT/GF cells, TLR4 transduces its signal through NOD2 receptor. We show here that in TtT/GF cells, Nod2 overexpression or stimulation by MDP increased STAT3 transcriptional activity. Furthermore, silencing STAT3 inhibited basal, LPS and MDP stimulated NFKB protein expression and overexpression of protein inhibitor of activated STAT3 (Pias3) markedly decreased basal NFKB activity. These data suggest that in TtT/GF cells, STAT3 acting upstream to NFKB mediates NOD2 receptor signalling pathway. In conclusion, the present study demonstrates that NOD molecules play a modulatory role in the pituitary by regulating the function and activation of FS cells in response to bacterial components.

Cooperation between NOD2 and Toll-like receptor 2 ligands in the up-regulation of mouse mFPR2, a G-protein-coupled Aβ42 peptide receptor, in microglial cells

Human G-protein-coupled formyl peptide receptor-like 1 and its mouse homologue formyl peptide receptor 2 (mFPR2) mediate the chemotactic activity of a variety of pathogen and host-derived peptides, including amyloid beta(42), a key causative factor in Alzheimer's disease. In mouse microglia, mFPR2 is up-regulated by pathogen-associated molecular patterns and proinflammatory cytokines, as shown, for instance, in our previous study using peptidoglycan (PGN) of Gram(+) bacteria. As PGN and its components have been reported to use TLR2 and an intracellular receptor nucleotide-binding oligomerization domain 2 (NOD2), we investigated the capacity of palmitoyl-cys[(RS)-2, 3-di(palmitoyloxy)-propyl]-Ala-Gly-OH (PamCAG), a specific TLR2 ligand, and muramyl dipeptide (MDP), a NOD2 ligand, to cooperatively regulate the expression and function of mFPR2 in microglia. We found that MDP and PamCAG as well as another TLR2-specific agonist palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4), when used alone, each increased the expression of functional mFPR2 in microglial cells, and the combination of MDP and PamCAG or Pam3CSK4 exhibited an additive effect. Mechanistic studies revealed that MDP increased the levels of TLR2 expression on the microglial cell surface and enhanced the levels of MAPKs p-38, ERK1/2, and NF-kappaB activated by PamCAG. Our results suggest that TLR2 and NOD2 cooperate to up-regulate the expression of mFPR2 and therefore, may actively participate in the pathogenic processes of brain inflammation and neurodegenerative diseases.

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Sex differences in the effects of muramyl dipeptide and lipopolysaccharide on locomotor activity and the development of behavioral tolerance in rats

Administration of bacterial agents, such as muramyl dipeptide (MDP) or lipopolysaccharide (LPS), induces a number of illness symptoms including decreased locomotor activity and weight loss. This study provides a detailed multivariate assessment of the effects of repeated exposures of various doses of MDP and LPS, alone and in combination, on various aspects of locomotion in male and female rats. Animals were given a single intraperitoneal injection of either MDP (0.8 or 1.6 mg/kg), LPS (100 or 200 μg/kg), a combination of MDP and LPS (0.8 mg/kg and 100 μg/kg, respectively), or vehicle on Days 1, 4, and 7. Two hours after each injection, locomotor activity was recorded for 30 min in an automated open-field. Both doses of LPS and the high dose of MDP produced significant decrements in locomotor activity in male and female rats, with tolerance becoming evident over repeated administrations, although LPS decreased activity more robustly than MDP. Sex differences were evident in the combined effects of MDP and LPS. Together, MDP and LPS reduced male activity levels in an additive manner but significantly potentiated both horizontal and vertical activity decrements in females. In addition, the rate of behavioral tolerance development to repeated bacterial injections was significantly higher in females than in males. These findings provide evidence for sex differences in the actions of MDP and LPS on various aspects of locomotor activity and in the development of behavioral tolerance to infection.

In vitro and in vivo production of interleukin-6 induced by muramyl peptides and lipopolysaccharide in normal dogs.

Interleukin-6 (IL-6) is a multifactorial cytokine produced by many cells including monocytes and macrophages in the immune-stimulated host. We measured IL-6 activity induced by muramyl dipeptide (MDP) and lipopolysaccharide (LPS) in vitro and by liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) in vivo in normal dogs. Adherent mononuclear cells were cultured with MDP, LPS, or MDP plus LPS for various time periods. After incubation, culture supernatants were collected and assayed for IL-6 activity. Sera from dogs following L-MTP-PE administration were also evaluated for IL-6 activity. IL-6 activity both in supernatants and sera was measured using a 7TD1 bioassay. Significantly elevated IL-6 activity could be measured as early as 2 hours after mononuclear cells were exposed to MDP, LPS, or MDP plus LPS. IL-6 activity induced by LPS was greater than that induced by MDP, and the combination of MDP and LPS induced the greatest increase in IL-6 activity. Serum IL-6 activity was elevated within 3 to 4 hours post L-MTP-PE administration and subsequently declined to pretreatment level at 24 hours post injection. Neutralization of supernatant and serum IL-6 activity was not achieved with goat or rabbit anti-recombinant human IL-6 polyclonal antibody. This study demonstrates that MDP and LPS, alone and in combination, can induce enhanced IL-6 activity of canine adherent mononuclear cells in vitro, and that intravenous injection of L-MTP-PE is capable of eliciting increased IL-6 activity in vivo in normal dogs. These findings suggest that IL-6 may play an important role in the biologic response observed in canine cancer patients treated with L-MTP-PE.

Maintenance of Tumoricidal Activity and Susceptibility to Reactivation of Subpopulations of Rat Liver Macrophages

The liver macrophage population was fractionated according to cell size into three subpopulations by means of elutriation centrifugation. The total liver macrophage population and the three subpopulations were cultured and exposed to the immunomodulators muramyl dipeptide (MDP), in a free or liposome-encapsulated form, and/or lipopolysaccharide (LPS). The tumor cytotoxic activity thus induced in the populations, the preservation of this activity and the response to a second stimulus were studied. The in vitro induced cytolytic activity was determined by a radioactivity release assay, using C26 colon adenocarcinoma cells, labeled with [methyl-3H]thymidine, as target cells. MDP or LPS readily activated the total macrophage population in maintenance culture to a tumor cytotoxic state during the first 2 days after isolation. Four days after isolation, the activation induced with both MDP and LPS was strongly reduced. The small to intermediate-size macrophages could be activated to tumor cytotoxic activity with MDP for up to 3 days and with LPS for up to 4 days in culture. The large-size macrophages could only be activated up to day 2 in culture with MDP or LPS or both. The combination of MDP and LPS, however, induced all cell populations in a synergistic way to become cytolytic for up to 4 days in culture. With free MDP as an activator, the activated state decayed within 1 day to almost zero levels, but less rapidly in the small cells than in the large cells. With liposome-encapsulated MDP, the activated state was preserved considerably longer, except in the largest cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative histology of muramyl dipeptide-potentiated induction of tumor necrosis by endotoxins.

Combinations of muramyl dipeptide (MDP) and toxic or detoxified endotoxin induced necrosis and subsequent disappearance of solid Meth A tumors in syngeneic mice. Toxic endotoxin alone was far less effective. MDP and detoxified endotoxin had negligible antitumor effects of their own. These observations were confirmed by histological examination. Neither MDP nor detoxified endotoxin induced significant changes in and around the tumor by 4, 24, and 48 h after intravenous administration when compared with saline treatment. MDP amplified various effects of toxic endotoxin such as the induction of hyperemia, mitotic arrest, mast cell depletion, non-hemorrhagic necrosis and reduction in lymphocyte infiltrates, but did not affect hemorrhagic necrosis or the influx of polymorphonuclear leukocytes. The combination of MDP and detoxified endotoxin lacked the latter two effects, but the other effects were similar to, although slightly less marked than those induced by the toxic combination. Because the degree of hyperemia was proportional to the degree of subsequent non-hemorrhagic necrosis, MDP seems to potentiate necrosis by enhancing mechanisms leading to hyperemia and mast cell mediators might be involved in the latter effect. Lymphocyte influx and the therapeutic outcome are likely to be related, since exclusively therapeutic treatments reduced the influx of these cells.

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

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.

Abrogation of species specificity for activation of tumoricidal properties in macrophages by recombinant mouse or human interferon-gamma encapsulated in liposomes.

Highly purified human blood monocytes, isolated by continuous Percoll density gradients under endotoxin-free conditions, and mouse peritoneal exudate macrophages (PEM) were activated in vitro by the combination of muramyl dipeptide (MDP) and recombinant interferon-gamma (r-IFN-gamma) to become tumoricidal against their respective tumorigenic target cells. The activation of human monocytes or mouse PEM by free unencapsulated r-IFN-gamma and MDP was species specific: human r-IFN-gamma activated human blood monocytes to lyse allogeneic melanoma cells, but did not activate mouse PEM. Mouse r-IFN-gamma activated mouse PEM to lyse syngeneic melanoma cells, but did not activate cytotoxic properties in human monocytes. The encapsulation of either mouse or human r-IFN-gamma with MDP within the same liposome preparation produced synergistic activation of cytotoxic properties in both PEM and monocytes without apparent species specificity. The activation of tumoricidal properties in macrophages by r-IFN-gamma and MDP occurred as a consequence of intracellular interaction. We base this conclusion on the data showing that whereas free r-IFN-gamma and MDP did not activate macrophages pretreated with pronase, liposome-encapsulated r-IFN-gamma and MDP did. Moreover, the i.v. injection of liposomes containing human or mouse r-IFN-gamma and MDP produced in vivo activation of mouse alveolar macrophages. These data suggest that in contrast to activation with free r-IFN-gamma, which requires binding to macrophage surface receptors, the intracellular interaction of r-IFN-gamma, which produces tumoricidal activity in macrophages, is not species specific.

Stimulation of chemiluminescence and resistance against aerogenic influenza virus infection by synthetic muramyl dipeptide combined with trehalose dimycolate.

The effect on respiratory burst of splenic cells from mice pretreated with oil-in-water emulsions of muramyl dipeptide (MDP), trehalose dimycolate (TDM), or the combination of MDP with TDM was studied by luminol-dependent chemiluminescence in response to stimulation by zymosan. Spleen cells from mice pretreated with TDM, but not those of mice treated with MDP, generated increased chemiluminescence. Spleen cells from animals pretreated with the combination of MDP and TDM exhibited markedly enhanced chemiluminescence activity. The effect of enhanced activity of preparations containing MDP combined with TDM was further examined in vivo by an aerosol infection of pretreated mice with a mouse-pathogenic influenza virus. Pretreatment with 6-O-acyl analogs and one ubiquinone derivative of MDP alone did not induce any resistance against influenza virus. Significant protection was conferred only when MDP and certain analogs were combined with TDM. The enhancement of nonspecific resistance to influenza virus infection was related to the chemical structure of the synthetic immunostimulant. A greater degree of protection was induced by the combination of TDM with the lipophilic derivatives like B 30-MDP and L-18 MDP.

Non-specific host defence stimulation and antibiotic-resistant infection.

The impact of a subinhibitory dose of clindamycin combined with stimulation of host defences by muramyl dipeptide (MDP) in an animal model of surgical infection with Escherichia coli was studied. Both the percentage of bacteria recovered from the implantation site and the degree of bacteraemia were quantitated at timed intervals following the bacterial challenge. Neither of these parameters was significantly different in animals treated with clindamycin alone compared to placebo animals. A significant reduction in bacteraemia was observed in animals treated with a combination of MDP and clindamycin when compared to animals receiving either agent alone. Local bacterial recovery was paradoxically greater in this combined treatment group. The presence of subinhibitory concentrations of clindamycin in vivo appears to render E. coli more susceptible to the systemic effects of host defence stimulation by MDP.