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Osteosarcoma (OS) is one of the most common primary bone sarcoma with high malignant degree and poor prognosis, for which there is an urgent need to develop novel therapeutic approaches. Recent research has revealed that mifamurtide significantly improved the outcome of OS patients when combined with adjuvant chemotherapy drugs including cisplatin (DDP). The present study aimed to construct a drug delivery system co-loading DDP and mifamurtide. Long-circulating targeted liposomes co-loading DDP and mifamurtide were constructed with Soy lecithin (SPC), cholesterol (Chol) and 1,2-distearoylglycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)] (DSPE-PEG), modified with MMP14 targeting peptide BCY-B in the surface of liposomes. In addition to characterization, the cellular uptake, endocytosis pathway and inhibition on cell viability, migration, invasion and cell apoptosis of MG-63 cells were explored. The constructed liposomal delivery possessed the basic characteristics of liposomes and showed high affinity to MG-63 cells, resulting in high uptake efficiency in MG-63 cells. The endocytosis might be involved in multiple pathways including caveolae-mediated endocytosis, clathrin-mediated endocytosis and macropinocytosis, dependently on energy. The constructed long-circulating targeted liposomes co-loading DDP and mifamurtide significantly inhibited the cell viability, migration, invasion and cell apoptosis of MG-63 cells, improving the antitumor effect of DDP and mifamurtide in vitro. The constructed liposomal delivery system is suitable for co-loading DDP and mifamurtide to achieve active tumor targeting, supplying a new strategy for the treatment of OS., Competing Interests: Declarations. Conflict of Interest: The authors declare no personal or financial conflicts of interest., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

In 3-month bone marrow transplants of CBA mice from bone marrow donors receiving single injections of TLR-4 ligand (LPS) or NOD-2 ligand (muramyl dipeptide, MDP) 24 h before transplantation, an increase in the total number of MSCs (by 2.6 and 1.9 times, respectively), as well as a slight increase in the number of nuclear cells and the mass of bone capsules (by 1.3 and 1.2 times) were observed. After combined administration of MDР and LPS to donors, the total content of MSCs in the grafts was higher by 1.6 times in comparison with the total result of their isolated administration (and by 7.2 times in comparison with the control). At the same time, the concentration of osteogenic MSCs in the grafts of all groups was almost the same and corresponded to the control level. The number of nuclear cells and the mass of bone capsules of the grafts after combined administration of LPS and MDP were close (~80%) to the sum of the results of their isolated administration. These findings suggest that activation of the stromal tissue and the success of bone marrow transplantation depend on the intensity of innate immune responses. These data can be useful for the development of optimal methods of tissue transplantation., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

GMDP (glucosoaminyl-muramyl-dipeptide), a synthetic analog of the peptidoglycan fragment of the bacterial cell wall, is an active component of the immunomodulatory drug Licopid. But the pharmacokinetic parameters of GMDP in humans after oral administration have not been investigated yet. The present study aimed at developing and validating a sensitive LC-MS/MS method for the analysis of GMDP in human plasma. The sample was prepared by solid-phase extraction using Strata-X 33 μm polymeric reversed-phase 60 mg/3 mL cartridges Phenomenex (Torrance, CA, USA). The analytes were separated using an Acquity UPLC BEN C18 column, 1.7 μm 2.1 × 50 mm Waters (Milford, USA). GMDP and internal standard growth hormone releasing peptide-2 (pralmorelin) were ionized in positive electrospray ionization mode and detected in multiple reaction monitoring mode. The developed method was validated within a linear range of 50-3000 pg/mL for GMDP. Accuracy for all analytes, given as the deviation between the nominal and measured concentration and assay variability , ranged from 1.61 to 3.02% and from 0.89 to 1.79%, respectively, for both within- and between-run variabilities. The developed and validated HPLC-MS/MS method was successfully used to obtain the plasma pharmacokinetic profiles of GMDP distribution in human plasma., (© 2020 John Wiley & Sons, Ltd.)

The relationship between chronic bacterial colonization in the brain and Alzheimer's disease is attracting extensive attention. Recent studies indicated that the components of bacterial biofilm drive the amyloid-β production. Muramyl dipeptide, the minimal bioactive peptidoglycan motif common to all bacteria, contributes to the development of many central inflammatory and neurodegenerative disorders. However, the involvement of Muramyl dipeptide in amyloid-β production is not completely defined. In our present study, wild type mice received an intracerebroventricular injection of normal saline or Muramyl dipeptide. Data showed that the production of Aβ1-42 oligomers was significantly increased after Muramyl dipeptide injection in the wild type mice or incubation of the SH-SY5Y cells with Muramyl dipeptide. Moreover, the action of Muramyl dipeptide was dose- and time-dependent. The above results suggested a possibility that the Muramyl dipeptide-induced Aβ1-42 oligomer production might be related to the NOD2/p-p38 MAPK/BACE1 pathway. To confirm this, the SH-SY5Y cells were transfected with siRNA NOD2. Data showed that the transfected SH-SY5Y cells exhibited decreased expression of Aβ1-42 oligomer, NOD2, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. Finally, SH-SY5Y cells were pretreated with SB203580, an inhibitor of the p-38-MAPK pathway. The results indicated that these pretreated SH-SY5Y cells exhibited decreased expression of Aβ1-42 oligomer, p-p38 MAPK, and BACE1 after treatment with Muramyl dipeptide. In conclusion, these results suggested that Muramyl dipeptide was the trigger factor for Aβ1-42 oligomer production, which probably acts via the NOD2/p-p38 MAPK/BACE1 signaling pathway., Competing Interests: All authors declare no conflicts of interest., (© 2020 Chen et al. Published by IMR press.)

Psoriasis is a multifactorial disease with a dysregulation in immune system. The aim of this study was to survey the clinical efficacy and safety of muramyl peptide-the ligand of the receptors of innate immunity (drug Licopid, AO Peptek, Moscow, Russia) in patients with psoriasis. The effect of muramyl peptide on 86 patients with different severity of plaque psoriasis was tested. The Psoriasis Area and Severity Index (PASI), cytokine status and production of nitric oxide in blood serum, and the subsequent course of psoriasis have been evaluated. Evaluation of significance of observed differences was presented by the Student's t -test. As a result of the treatment, clinical cure or improvement was detected in 98.2% of patients ( p < 0.05), while 24.4% had a complete cure. Subsequent observations during 4 years showed that patients who received muramyl peptide statistically significantly increased relapse-free period. Moreover, subsequent relapses of the disease after treatment with muramyl peptide were in much more milder form in the cases of mild psoriasis. The conducted studies showed that monotherapy with muramyl peptide stopped the clinical manifestations of psoriasis, normalized the processes of cytokine-dependent [interleukin (IL)-4, IL-10, IL-12, tumor necrosis factor alpha (TNF-α)] regulation of the immune response and nonspecific resistance, expressed in a decreasing amount of serum antigens sCD54 [soluble intercellular adhesion molecule-1 (sICAM-1)] to reference values ( p ≤ 0.01). Taken together, our research demonstrated the effectiveness of therapy with muramyl peptide and moreover, that elevated levels of sCD54 and MIF ( p ≤ 0.01) in the serum of patients with psoriasis considered as potential biomarkers of the severityof psoriasis and control over their dynamics have prognostic significance in determining the effectiveness of the therapy.

Emulsions play an important role in present-day subunit vaccine delivery. Squalane-based emulsion was formulated using surfactants viz., Pluronic F68, Span 85 along with Murabutide (MB) as immunepotentiator. Particle size and zetapotential of the final optimized emulsion was found to be 134 nm and -13 mV, respectively. The in vitro cellular uptake studies performed using fluorescein isothiocyanate (FITC)-labeled ovalbumin (OVA) clearly revealed the rapid uptake of antigen in the presence of emulsion. The in vivo subcutaneous studies involving measurement of OVA-specific IgG antibody titers, Th1/Th2 cytokines were performed and a marked up regulation in IL-2, IL-12 and IFN-γ cytokines indicate Th1 immune response. Results supported that the squalane-based delivery system enhanced the uptake of the antigen by immune cells and elicited humoral as well as cell-mediated immune response in mice. These results indicate the promising application of the new squalane based oil-in-water (O/W) emulsion as capable vaccine delivery system useful for vaccine development.

The aim of the present study was to evaluate the efficiency and side effects of mifamurtide in childhood osteosarcoma (OS). In total, 477 doses of 2 mg/m intravenous (IV) mifamurtide, along with paracetamol as a premedication, were given to 15 patients with primary nonmetastatic OS after complete surgical resection and to 3 patients with progressive OS. The most common side effects encountered in the patients were chills and fever (17/18). These reactions were observed in 4 patients during the administration of each dose, in a single patient during the last administration, and in the remaining 12 patients during the first or initial 2 administrations. Headache, myalgia, and arthralgia were observed in 2 patients during each infusion. Headache was observed in 1 patient with additional hearing loss during the first 2 infusions. One patient had back pain occuring within the first infusion. Of the 15 patients with primary nonmetastatic OS and treated with the addition of mifamurtide to chemotherapy, 13 showed a complete remission, and 2 patients were still under treatment with a complete remission. Of 3 patients with progressive disease, 2 died while the disease progressed further in the third case over a 51-month period. The 3-year overall survival and event-free survival distributions were 87.5% (mean follow-up time, 46.12; 95% confidence interval, 37.79-52.45 mo) and 75.6% (mean follow-up time, 31.30; 95% confidence interval, 26.54-36.06 mo), respectively. We consider that mifamurtide therapy is a safe and well-tolerated agent in childhood OS.

Drugs that are neither lipophilic nor suitable for encapsulation via remote loading procedures are generally characterized by low entrapment efficiencies and poor retention in liposomes. One approach to circumvent this problem consists in covalently linking a lipid to the drug molecule in order to permit its insertion into the vesicle membrane. The nature of the conjugated lipid and linker, as well as the composition of the liposomal bilayer were found to have a profound impact on the pharmacokinetic properties and biodistribution of the encapsulated drugs as well as on their biological activity. This contribution reviews the past and recent developments on liposomal lipid-drug conjugates, and discusses important issues related to their stability and in vivo performance. It also provides an overview of the data that were generated during the clinical assessment of these formulations. The marketing authorization of the immunomodulating compound mifamurtide in several countries as well as the promising results obtained with the lipid prodrug of mitomycin C suggest that carefully designed liposomal formulations of lipid-drug conjugates is a valid strategy to improve a drug's pharmacokinetic profile and with that its therapeutic index and/or efficacy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Subunit vaccines are often poorly immunogenic, and adjuvants and/or delivery vehicles, such as polymeric microparticles (MPs), can be used to enhance immune responses. MPs can also be used to understand cell activation kinetics and the significant impact antigen and adjuvant release has on adaptive immune responses. By controlling antigen and adjuvant release, we can determine if it is important to have precise temporal control over release of these elements to optimize the peak and duration of protective immunity and improve vaccine safety profiles. In order to study the effect of tunable adjuvant or antigen delivery on generation of adaptive immunity, we used acetalated dextran (Ace-DEX) MPs. Ace-DEX MPs were used because their tunable degradation can be controlled based on polymer cyclic acetal coverage (CAC). Ace-DEX MPs of varying degradation profiles were used to deliver murabutide or ovalbumin (OVA) as a model adjuvant or antigen, respectively. When murabutide was encapsulated within Ace-DEX MPs to test for controlled adjuvant delivery, fast-degrading MPs exhibited higher humoral and cellular responses in vivo at earlier time points, while slow-degrading MPs resulted in stronger responses at later time points. When OVA was encapsulated within Ace-DEX MPs to test for controlled antigen delivery, fast-degrading MPs induced greater antibody and cytokine production throughout the length of the experiment. This differential response suggests the need for distinct, flexible control over adjuvant or antigen delivery and its impact on immune response modulation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tuberculosis (TB) remains a main public health concern and 10.4 million new cases occurred in 2015 around the world. BCG is the only approved vaccine against TB, but has variable efficacy and new vaccines are needed. We developed two new mTB vaccine candidates based on the recombinant fusion proteins, rCMX and rECMX formulated with Advax4, a new combination adjuvant combining delta inulin, CpG oligonucleotide and murabutide. BALB/c mice were immunized three times intramuscularly with these vaccine formulations. Injection of Advax4 alone increased the percentage of lymphatic endothelial cells and activated macrophages (F480/CD11b + ) in the draining lymph nodes consistent with a chemotactic adjuvant effect. Advax4+CMX and Advax4+ECMX induced the highest levels of IgG1 and IgG2a antibodies against rCMX and rECMX, respectively. Immunized mice challenged with Mycobacterium tuberculosis (Mtb) had increased vaccine-specific Th1 responses in the lungs together with reduced Mtb - associated alveolar damage, although only the Advax4+ECMX vaccine demonstrated significant reduction of lung bacterial load. This study confirmed Advax4+ECMX as a potential TB vaccine candidate, with potential for further optimization and clinical development.

Ligands NLR2 (muramyldipeptide) and TLR (bacterial LPS, flagellin, CpG-dinucleotide, and Poly I:C) and S. typhimurium antigenic complex by 1.5-3-fold increase the efficiency of cloning and content of multipotent stromal cells (MSC) in the bone marrow of CBA mice as soon as 1 h postinjection. The counts of large colonies (150-500 cells) increased by 2.5-3.3 times in comparison with intact bone marrow cultures at the expense of a decrease in the number of smaller colonies, which attests to enhanced proliferation of stromal cells in the colonies. The efficiency of cloning and hence, MSC content in the femoral bone decreased by 1.2-1.9 times after 3 h and increased again after 24 h to the level 1.3-1.5 times higher than the level 1 h postinjection (LPS, Poly I:C, and S. typhimurium antigenic complex). The dynamics of bone marrow MSC cloning efficiency after 1-3 h corresponded to the dynamics of serum cytokine concentrations during the same period. However, the levels of serum cytokines after 24 h in general were similar to those in intact mice or were lower. The concentrations of osteogenic multipotent stromal cells in the bone marrow decreased 2-3-fold after 3 h and thus persisted by 24 h postinjection. Twofold (at 24-h interval) and a single injection of S. typhimurium antigenic complex to mice led to a significant increase of cloning efficiency, observed as early as just 1 h postinjection (1.9 and 1.5 times, respectively). The same picture was observed for serum cytokines. On the whole, injections of TLR and NLR ligands and of S. typhimurium antigenic complex led to stromal tissue activation within 1 h postinjection, this activation consisting in a significant increase of the efficiency of cloning and of MSC count in the bone marrow, and also in an increase in their proliferative activity and a decrease (after 3 h) of osteogenic MSC concentration.

Porphyromonas gingivalis (P.g.)-induced TNF-α can be affected by muramyl dipeptide (MDP) in a biphasic concentration-dependent manner. We found that in P.g.-exposed macrophages, treatment with 10 μg/mL of MDP (MDP-low) up-regulated TNF-α by 29%, while 100 μg/mL or higher (MDP-high) significantly decreased it (16% to 38%). MDP-high was found to affect the ubiquitin-editing enzyme A20 and activator protein 1 (AP1). An AP1 binding site was found in the promoter region of A20. A20 promoter activity was up-regulated after transfection of AP1 cDNA in cells. Four analogues of MDP (3-6) were prepared through a convergent strategy involving the synthesis of two unique carbohydrate fragments, 7a and 7b, using the peptide coupling reagents, EDCI and HOAt. Analogue 4 improved MDP function and P.g.-induced activities. We propose a new signaling pathway for TNF-α induction activated after exposing macrophages to both P.g. and MDP-high or analogue 4.

Background: NOD2 is the genetic cause of Blau syndrome, an autoinflammatory disease that manifests as coincident uveitis and arthritis. Since dysregulation of IL-1 signalling is considered a pathogenic mechanism in a number of related autoinflammatory conditions, we examined the extent to which unimpeded interleukin (IL)-1 signalling influences NOD2-dependent inflammation of the eye versus the joint., Methods: Mice deficient for IL-1R antagonist (IL-1Ra) were administered the NOD2 agonist muramyl dipeptide (MDP) by systemic (intraperitoneal) or local (intraocular and/or intra-articular) injections. NOD2-deficient mice received an intraocular injection of recombinant IL-1β. Uveitis was evaluated by intravital videomicroscopy and histopathology, and arthritis was assessed by near-infrared imaging and histopathology. Ocular levels of IL-1α, IL-1β and IL-1Ra were quantified by enzyme-linked immunosorbent assay., Results: IL-1Ra deficiency did not render mice more responsive to systemic exposure of MDP. Despite the increased production of IL-1R agonists IL-1α and IL-1β in response to intraocular injection of MDP, deficiency in IL-1Ra did not predispose mice to MDP-triggered uveitis, albeit intravascular cell rolling and adherence were exacerbated. NOD2 expression was dispensable for the potential of IL-1 to elicit uveitis. However, we find that IL-1Ra does play an important protective role in arthritis induced locally by MDP injection in the joint., Conclusions: Our findings highlight the complexity of NOD2 activation and IL-1 signalling effects that can be compounded by local environmental factors of the target organ. These observations may impact how we understand the molecular mechanisms by which NOD2 influences inflammation of the eye versus joint, and consequently, treatment options for uveitis versus arthritis., (© 2014 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: The aim of this work was to demonstrate an immunostimulatory and adjuvant effect of new apyrogenic lipophilic derivatives of norAbuMDP and norAbuGMDP formulated in nanoliposomes., Methods: Nanoliposomes and metallochelating nanoliposomes were prepared by lipid film hydration and extrusion methods. The structure of the liposomal formulation was studied by electron microscopy, AF microscopy, and dynamic light scattering. Sublethal and lethal γ-irradiation mice models were used to demonstrate stimulation of innate immune system. Recombinant Hsp90 antigen (Candida albicans) bound onto metallochelating nanoliposomes was used for immunisation of mice to demonstrate adjuvant activities of tested compounds., Results: Safety and stimulation of innate and adaptive immunity were demonstrated on rabbits and mice. The liposomal formulation of norAbuMDP/GMDP was apyrogenic in rabbit test and lacking any side effect in vivo. Recovery of bone marrow after sublethal γ-irradiation as well as increased survival of mice after lethal irradiation was demonstrated. Enhancement of specific immune response was demonstrated for some derivatives incorporated in metallochelating nanoliposomes with recombinant Hsp90 protein antigen., Conclusions: Liposomal formulations of new lipophilic derivatives of norAbuMDP/GMDP proved themselves as promising adjuvants for recombinant vaccines as well as immunomodulators for stimulation of innate immunity and bone-marrow recovery after chemo/radio therapy of cancer.

Introduction: Mepact(®) (mifamurtide) is the first drug approved for the treatment of high-grade resectable non-metastatic osteosarcoma in patients aged 2-30 in the last 20 years. It follows a randomized clinical trial showing a statistically-significant and clinically-relevant decrease in the risk of death without compromising safety., Aim: This study assessed the cost-effectiveness and budget impact of mifamurtide., Methods: The economic evaluation was done on a hypothetical cohort of young patients under the age of 30 with high-grade, non-metastatic, resectable osteosarcoma. Standard chemotherapy without mifamurtide was used as comparator. A Markov model was adapted using Spanish costs and the results of the INT-0133 Phase III study. The analysis has been carried out from the perspective of the Spanish National Health Service, with a time horizon of up to 60 years in the base analysis., Results: The observed greater efficacy of mifamurtide in the trial translates into a gain of 3.03 (undiscounted) and 1.33 (discounted) quality-adjusted life years at an additional cost of €102,000. The estimated budgetary impact of using mifamurtide in 10-100% of the potential population would cost €671,000 and €6.7 million respectively., Conclusion: The cost per quality-adjusted life years gained with mifamurtide in Spain is in the low band (<€100,000) of the iCERs obtained by other orphan drugs and would have a limited and affordable cost in Spain.

The role of muramyl dipeptide (MDP) and tuftsin in oral immune adjustment remains unclear, particularly in a Lactobacillus casei (L. casei) vaccine. To address this, we investigated the effects of different repetitive peptides expressed by L. casei, specifically the MDP and tuftsin fusion protein (MT) repeated 20 and 40 times (20MT and 40MT), in mice also expressing the D antigenic site of the spike (S) protein of transmissible gastroenteritis virus (TGEV) on intestinal and systemic immune responses and confirmed the immunoregulation of these peptides. Treatment of mice with a different vaccine consisting of L. casei expressing MDP and tuftsin stimulated humoral and cellular immune responses. Both 20MT and 40MT induced an increase in IgG and IgA levels against TGEV, as determined using enzyme-linked immunosorbent assay. Increased IgG and IgA resulted in the activation of TGEV-neutralising antibody activity in vitro. In addition, 20MT and 40MT stimulated the differentiation of innate immune cells, including T helper cell subclasses and regulatory T (Treg) cells, which induced robust T helper type 1 and T helper type 17 (Th17) responses and reduced Treg T cell immune responses in the 20MT and 40MT groups, respectively. Notably, treatment of mice with L. casei expressing 20MT and 40MT enhanced the anti-TGEV antibody immune responses of both the humoral and mucosal immune systems. These findings suggest that L. casei expressing MDP and tuftsin possesses substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration, and it may be useful in oral vaccines against TGEV challenge.

In the absence of long-term clinical trials that compare mifamurtide plus chemotherapy versus chemotherapy only for treatment of osteosarcoma, decision analysis is a useful tool that helps to determine the optimal treatment strategy. We analyzed the differences between mifamurtide plus chemotherapy versus chemotherapy only by using modeling to determine the treatment approach that results in longer life expectancy among children with osteosarcoma. We used the Markov model to compare the expected lifetime quality-adjusted life years (QALYs) between mifamurtide plus chemotherapy versus chemotherapy only. Our target cohort consisted of children with osteosarcoma. The starting age of the cohort was 12 years and cycle length was 3 months. The transition probabilities for each disease state and death were calculated using overall survival or progression free survival data from randomized controlled trials. Utility weights from scenario-based survey for 303 Korean general populations were applied to the model. Based on the base case analysis, the incremental benefit analysis indicated that mifamurtide plus chemotherapy resulted in an incremental QALY increase of 1.57 (a relative increase of 16.3 % in QALY expectancy) compared to chemotherapy only. Also, the incremental life years gained (LYG) from mifamurtide plus chemotherapy was 1.96 on comparison with chemotherapy only; this is a relative increase of 15.7 % in LYG expectancy. The decision analysis model indicated that mifamurtide plus chemotherapy was associated with a substantially longer survival than chemotherapy only among children with osteosarcoma during their lifetime.

Protein antigens encapsulated as vaccines in poly[(rac-lactide)-co-glycolide] (PLGA) microparticle carriers can induce immune responses. The intensity and directions of this response can be controlled by coloading the microparticles with immunomodulatory adjuvants, e.g., muramyl dipeptide (MDP) as adjuvant combined with ovalbumin (Ova) as protein antigen. In this study, methodologies for an individual quantification of both encapsulated substances should be reported, which comprise (i) a separation process to isolate and determine MDP as intact molecule and (ii) a simultaneous degradation of both analytes with subsequent specific quantification of Ova fragments. It was shown that coloading of both substances resulted in a substantially reduced encapsulation efficiency of MDP. This illustrates that correct conclusions on dose-response relationships in future vaccination studies can only be drawn, if a selective method for adjuvant and protein quantification will be applied., (Copyright © 2014. Published by Elsevier B.V.)

Aims: To evaluate the pharmacokinetics and pharmacodynamics following a single dose of liposomal mifamurtide (L-MTP-PE, MEPACT(®)) in adult subjects with mild (calculated creatinine clearance [CLcr ] of 50-80 ml min(-1)) or moderate (CLcr 30-50 ml min(-1)) renal impairment in comparison with age-, weight- and gender-matched healthy subjects with normal renal function (CLcr >80 ml min(-1))., Methods: Subjects received a 4 mg dose of liposomal mifamurtide via 1 h intravenous infusion. Blood samples were collected over 72 h for analysis of plasma pharmacokinetics of total and non-liposome-associated (free) mifamurtide and assessment of pharmacodynamics (changes in serum interleukin-6 [IL-6], tumour necrosis factor-α [TNF-α], C-reactive protein [CRP])., Results: Thirty-three subjects were enrolled: nine with mild renal impairment, eight with moderate renal impairment and 16 healthy subjects. Geometric mean (%CV) AUCinf for total mifamurtide was 89.5 (58.1), 94.8 (27.8), 85.1 (29.0), 95.4 (18.1) nM h in the mild renal impairment, mild-matched healthy subject, moderate renal impairment and moderate-matched healthy subject groups, respectively. Mifamurtide clearance was not correlated with CLcr, estimated glomerular filtration rate or iohexol clearance (all r(2) < 0.01). AUCinf of free mifamurtide was similar across the renal function groups. There were no readily apparent differences in serum pharmacodynamic effect parameters (baseline-adjusted AUEClast for IL-6 and TNF-α and Emax for CRP) between the renal function groups. No subjects reported grade ≥3 or serious adverse events., Conclusions: Mild or moderate renal impairment does not alter the clinical pharmacokinetics or pharmacodynamics of mifamurtide. No dose modifications appear necessary for these patients based on clinical pharmacologic considerations., (© 2013 The British Pharmacological Society.)

Subunit vaccines against anthrax based on recombinant protective antigen (PA) potentially offer more consistent and less reactogenic anthrax vaccines but require adjuvants to achieve optimal immunogenicity. This study sought to determine in a murine model of pulmonary anthrax infection whether the polysaccharide adjuvant Advax or the innate immune adjuvant murabutide alone or together could enhance PA immunogenicity by comparison to an alum adjuvant. A single immunization with PA plus Advax adjuvant afforded significantly greater protection against aerosolized Bacillus anthracis Sterne strain 7702 than three immunizations with PA alone. Murabutide had a weaker adjuvant effect than Advax when used alone, but when murabutide was formulated together with Advax, an additive effect on immunogenicity and protection was observed, with complete protection after just two doses. The combined adjuvant formulation stimulated a robust, long-lasting B-cell memory response that protected mice against an aerosol challenge 18 months postimmunization with acceleration of the kinetics of the anamnestic IgG response to B. anthracis as reflected by ∼4-fold-higher anti-PA IgG titers by day 2 postchallenge versus mice that received PA with Alhydrogel. In addition, the combination of Advax plus murabutide induced approximately 3-fold-less inflammation than Alhydrogel as measured by in vivo imaging of cathepsin cleavage resulting from injection of ProSense 750. Thus, the combination of Advax and murabutide provided enhanced protection against inhalational anthrax with reduced localized inflammation, making this a promising next-generation anthrax vaccine adjuvanting strategy.

Purpose: This non-randomized, patient-access protocol, assessed both safety and efficacy outcomes following liposomal muramyl-tripeptide-phosphatidylethanolamine (L-MTP-PE; mifamurtide) in patients with high-risk, recurrent and/or metastatic osteosarcoma., Methods: Patients received mifamurtide 2 mg/m(2) intravenously twice-weekly ×12 weeks, then weekly ×24 weeks with and without chemotherapy. Serum concentration-time profiles were collected. Adverse events within 24 hours of drug administration were classified as infusion-related adverse events (IRAE); other AEs and overall survival (OS) were assessed., Results: The study began therapy in January 2008; the last patient completed therapy in October 2012. Two hundred five patients were enrolled; median age was 16.0 years and 146/205 (71%) had active disease. Mifamurtide serum concentrations declined rapidly in the first 30 minutes post-infusion, then in a log-linear manner 2-6 hours post-dose; t1/2 was 2 hours. There were no readily apparent relationships between age and BSA-normalized clearance, half-life, or pharmacodynamic effects, supporting the dose of 2 mg/m(2) mifamurtide across the age range. Patients reported 3,679 IRAE after 7,482 mifamurtide infusions. These were very rarely grade 3 or 4 and most commonly included chills + fever or headache + fatigue symptom clusters. One- and 2-year OS was 71.7% and 45.9%. Patients with initial metastatic disease or progression approximated by within 9 months of diagnosis (N = 40) had similar 2-year OS (39.9%) as the entire cohort (45.9%), Conclusions: Mifamurtide had a manageable safety profile; PK/PD of mifamurtide in this patient access study was consistent with prior studies. Two-year OS was 45.9%. A randomized clinical trial would be required to definitively determine impact on patient outcomes., (© 2013 Wiley Periodicals, Inc.)

The purpose of this study was to elucidate the role of nucleotide binding oligomerization domain-containing protein 2 (NOD2) signaling in atherosclerosis and periodontal bone loss using an Apolipoprotein E(-/-) (ApoE(-/-)) mouse model based on the proposed role of NOD2 in inflammation. NOD2(-/-)ApoE(-/-) and ApoE(-/-) mice fed a standard chow diet were given an oral gavage of Porphyromonas gingivalis for 15 wk. NOD2(-/-)ApoE(-/-) mice exhibited significant increases in inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and the heart compared with ApoE(-/-) mice. In contrast, ApoE(-/-) mice injected i.p. with Muramyl DiPeptide (MDP) to stimulate NOD2 and given an oral gavage of P. gingivalis displayed a reduction of serum inflammatory cytokines, alveolar bone loss, cholesterol, and atherosclerotic lesions in the aorta and aortic sinus compared with ApoE(-/-) mice orally challenged but injected with saline. A reduction in body weight gain was observed in ApoE(-/-) mice fed a high-fat diet (HFD) and injected with MDP compared with ApoE(-/-) mice fed a high-fat diet but injected with saline. MDP treatment of bone marrow-derived macrophages incubated with P. gingivalis increased mRNA expressions of NOD2, Toll-like receptor 2, myeloid differentiation primary response gene 88, and receptor-interacting protein-2 but reduced the expressions of inhibitor of NF-κB kinase-β, NF-κB, c-Jun N-terminal kinase 3, and TNF-α protein levels compared with saline control, highlighting pathways involved in MDP antiinflammatory effects. MDP activation of NOD2 should be considered in the treatment of inflammatory processes affecting atherosclerosis, periodontal bone loss ,and possibly, diet-induced weight gain.

Objectives: Mifamurtide is an immune macrophage stimulant that when added to standard chemotherapy has demonstrated survival benefit for newly diagnosed osteosarcoma. The objectives of this study were to investigate the cost-effectiveness of adding mifamurtide to standard three- or four-agent chemotherapy for high-grade, resectable, nonmetastatic osteosarcoma following surgical resection and the issues of obtaining robust cost-effectiveness estimates for ultra-orphan drugs, given the shortage of data., Methods: An economic evaluation was conducted from the perspective of the UK's National Health Service as part of the manufacturer's submission to the National Institute for Health and Care Excellence. The disease process was simplified to a transition through a series of health states, modeled by using a Markov approach. Data to inform the model were derived from patient-level data of Study INT-0133, published literature, and expert opinion. The final efficacy measure was life-years gained (LYG), and utilities were used to obtain quality-adjusted life-years (QALYs)., Results: For a 60-year time frame and a discount rate of 3.5% for outcomes, patients receiving mifamurtide benefited from an average additional 1.57 years of life and 1.34 QALYs, compared with patients receiving chemotherapy alone, giving an incremental cost-effectiveness ratio (ICER) of £58,737 per LYG and £68,734 per QALY. Because treatment effects were both substantial in restoring health and sustained over a very long period, the National Institute for Health and Care Excellence changed its guidance to allow a discount of 1.5% for outcomes to be applied in these special circumstances. By using this discount factor, it was found that patients receiving mifamurtide had an average additional 2.58 years of life and 2.20 QALYs compared with patients receiving chemotherapy alone, resulting in an ICER of £35,765 per LYG and £41,933 per QALY., Conclusion: Mifamurtide's ICER is cost-effective compared with that of other orphan and ultra-orphan drugs, for which prices and corresponding cost-effectiveness estimates are high., (Copyright © 2013 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.)

The factors responsible for the conjunctivitis and iritis associated with acute ocular infection and post enteric inflammatory disease are not fully known. The pro-inflammatory activity of unilateral topical application of muramyl dipeptide (MDP; the smallest bio-active Gram-positive and Gram-negative bacterial cell wall component) was investigated in adult rabbits. The resultant bilateral conjunctivitis/iritis and pyogenic responses were characterized. Bilateral symptoms were graded by slit lamp examinations; tear fluid, Schirmer tests (tear production), blood and aqueous humor (AH) samples were obtained from MDP-treated and untreated rabbits. MDP concentration, gamma-glutamyltranspeptidase activity (GGT; key enzyme in glutathione recapture, xenobiotic detoxification, eicosanoid synthesis and neutrophil function), protein concentration, and tear cell density, cytology, and immunofluorescent antibody reactivity to GGT and calreticulin (CRT; MDP-binding protein) were determined. MDP was cleared from ipsilateral tears and serum by 6 h, but was undetected in mock-treated contralateral tears. Bilateral signs of acute transient pyogenic conjunctivitis, characterized by tearing, lid edema, conjunctival hyperemia, chemosis and leukocytic infiltrate with iritis (erythema and aqueous flare) were detected. Milder symptoms occurred in the mock-treated contralateral eyes. Bilateral symptoms, tear production, tear protein, GGT activity, and mucopurulent discharge (containing up to 2.5-5.0 × 10(6) cells/mL) were elevated 4-8 h post MDP and resolved to near pre-treatment levels by 24 h. Tear GGT activity and protein levels were higher in MDP-treated and mock-treated contralateral eyes than in eyes of untreated adult rabbits (p's < 0.001). Elevated tear GGT activity was associated with histopathology and increased vascular and epithelial permeability to serum protein, GGT-positive epithelia cells, macrophages and heterophils. Repeat MDP applications induced recurrent induction and resolution patterns of bilateral conjunctivitis/iritis and tear GGT activity, but ipsilateral GGT responses were lower. The results suggest unilateral topical MDP application to adult rabbit eyes induces a bilateral acute pyogenic conjunctivitis/iritis (PCI) characterized by increased vascular and epithelial permeability similar to acute bacterial conjunctivitis in man. The detection of CRT/GGT positive heterophils in tears suggests efferocytosis (phagocytosis of dead/dying cells). Tear GGT activity may be a useful means to quantify MDP-induced toxicity and extraocular inflammation., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Key intracytosolic pattern recognition receptors of innate immunity against bacterial infections are nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). We elucidated the NOD1 and NOD2-mediated activation of human eosinophils, the principal effector cells for allergic inflammation, upon interacting with human bronchial epithelial BEAS-2B cells in allergic asthma. Eosinophils constitutively expressed NOD1,2 but exhibited nonsignificant responses to release chemokines upon the stimulation by NOD1 ligand γ-D-glutamyl-meso-diaminopimelic acid (iE-DAP) and NOD2 ligand muramyl dipeptide (MDP). However, iE-DAP and MDP could significantly upregulate cell surface expression of CD18 and intercellular adhesion molecule (ICAM)-1 on eosinophils and ICAM-1 on BEAS-2B cells, as well as induce chemokines CCL2 and CXCL8 release in the coculture system (all P<0.05). Both eosinophils and BEAS-2B cells were the main source for CXCL8 and CCL2 release in the coculture system upon iE-DAP or MDP stimulation. Direct interaction between eosinophils and BEAS-2B cells is responsible for CCL2 release, and soluble mediators are implicated in CXCL8 release. ERK and NF-κB play regulatory roles for the expression of adhesion molecules and chemokines in coculture. Treatment with NOD1,2 ligand could induce the subepithelial fibrosis and significantly enhance the serum concentration of total IgE, chemokine CCL5 for eosinophils and T helper type 2 (Th2) cells and asthma Th2 cytokine IL-13 in bronchoalveolar lavage fluid of ovalbumin-sensitized allergic asthmatic mice (all P<0.05). This study provides further evidence of bacterial infection-mediated activation of NOD1,2 in triggering allergic asthma via the activation of eosinophils interacting with bronchial epithelial cells at inflammatory airway.

The aim of the present work was to compare in vitro the possibility of application of unmodified silica nanoparticles (UMNPs) and modified by aminopropyl groups silica nanoparticles (AMNPs) for topical delivery of immunomodulatory drug GMDP to the peritoneal macrophages of women with endometriosis. The absence of cytotoxic effect and high cellular uptake was demonstrated for both types of silica nanoparticles. The immobilization of GMDP on the UMNPs led to the suppression of the stimulatory effect of GMDP on the membrane expression of scavenger receptors SR-AI and SR-B, mRNAs expression of NOD2 and RAGE, and synthesis of proteolytic enzyme MMP-9 and its inhibitor TIMP-1. GMDP, immobilized onto AMNPs, enhanced the initially reduced membrane expression of SRs and increased NOD2, RAGE, and MMP-9 mRNAs expression by macrophages. Simultaneously high level of mRNAs expression of factors, preventing undesirable hyperactivation of peritoneal macrophages (SOCS1 and TIMP-1), was observed in macrophages incubated in the presence of GMDP, immobilized onto AMNPs. The effect of AMNPs immobilized GMDP in some cases exceeded the effect of free GMDP. Thus, among the studied types of silica nanoparticles, AMNPs are the most suitable nanoparticles for topical delivery of GMDP to the peritoneal macrophages.

Purpose: This study evaluated the pharmacokinetics (PK), pharmacodynamics (PD), safety/tolerability, and cardiac safety of liposomal muramyl tripeptide phosphatidyl-ethanolamine [mifamurtide (L-MTP-PE)] in healthy adults., Methods: L-MTP-PE 4 mg was administered intravenously over 30 min. Study participants were monitored from 24 h preinfusion until 72 h postinfusion. Blood samples were drawn over 0-72 h postdose to determine serum MTP-PE, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and C-reactive protein (CRP) concentrations. Electrocardiograpic (ECG) data were collected via continuous Holter monitoring beginning 24 h predose through 24 h postdose. Changes from time-matched pretreatment baseline QTc and associated two-sided 90 % confidence intervals were calculated., Results: Twenty-one participants received L-MTP-PE. Total serum MTP-PE declined rapidly with a terminal half-life of 2.05 ± 0.40 h. PK variability was low, with <30 % coefficient of variation in systemic exposure. Serum concentrations of IL-6, TNF-α, and CRP increased following L-MTP-PE infusion. Maximum observed increases in IL-6 and TNF-α occurred at 4 and 2 h, respectively, returning toward baseline by 8 h postdose. L-MTP-PE was generally well tolerated, with no adverse events greater than grade 3. Headache, chills, tachycardia, nausea, and pyrexia were the most frequent adverse events. L-MTP-PE infusion resulted in an increased heart rate without readily apparent QTc prolongation., Conclusions: MTP-PE PK following L-MTP-PE administration were characterized by a short serum half-life and low variability. Increases in IL-6, TNF-α, and CRP and the safety profile were consistent with the immunomodulatory mechanism of action. No clinically significant effect of L-MTP-PE on cardiovascular repolarization was observed based on analysis of ECG QTc intervals.

Yersinia pestis is the causative agent of plague. Cellular immunity seems to play an important role in defense against this disease. The subunit vaccine based on V (Lcr V) antigen has been proved to be immunogenic in animals and in humans. The multiple antigen peptide (MAP), incorporating all the relevant B and T cell epitopes is highly immunogenic in mice through intranasal route of immunization in PLGA particles containing CpG-ODN as an immunoadjuvant inducing humoral and mucosal immune response. In the present study, cell-mediated immune response using same MAP was studied in murine model. Primary and memory T cell responses were studied in outbred and inbred mice immunized intranasally with MAP in the presence of two immunoadjuvants (Murabutide and CpG-ODN). All the three compartments (Spleen, Lamina propria and Peyer's patches) of the lymphoid system showed increased lymphoproliferative response. Highest lymphoproliferative response was observed especially with CpG-ODN. Cytokine profile in the culture supernatant showed highest Th(1) and Th(17) levels. FACS analysis showed expansion of both CD4(+) and CD8(+) T cells producing gamma-interferon, perforin and granzyme-B with major contribution from CD4(+) T cells., (Copyright © 2012 Elsevier Inc. All rights reserved.)

N-trimethyl chitosan (TMC) nanoparticles have been shown to increase the immunogenicity of subunit antigens after nasal and intradermal administration. This work describes a second generation of TMC nanoparticles containing ovalbumin as a model antigen (TMC/OVA nanoparticles) and an immunopotentiator (TMC/OVA/immunopotentiator nanoparticles). The selection of immunopotentiators included Toll-like receptor (TLR) ligands lipopolysaccharide (LPS), PAM(3)CSK(4) (PAM), CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and the GM1 ganglioside receptor ligand, cholera toxin B subunit (CTB). The TMC/OVA/immunopotentiator nanoparticles were characterised physico-chemically and their immunogenicity was assessed by determining the serum IgG, IgG1, IgG2a titres and secretory IgA levels in nasal washes after intradermal and nasal vaccination in mice. After nasal vaccination, TMC/OVA nanoparticles containing LPS or MDP elicited higher IgG, IgG1 and sIgA levels than non-adjuvanted TMC/OVA particles, whereas nanoparticles containing CTB, PAM or CpG did not. After intradermal vaccination, the TMC/OVA/CpG and TMC/OVA/LPS nanoparticles provoked higher IgG titres than plain TMC/OVA particles. Altogether, our results show that co-encapsulation of an additional immunopotentiator with the antigen into TMC nanoparticles can further improve the immunogenicity of the vaccine. However, the strength and quality of the response depends on the immunopotentiator as well as the route of administration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Murabutide (MB) is a synthetic immunomodulator recognized by the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) receptor on mammalian cells. MB has previously been approved for testing in multiple human clinical trials to determine its value as an antiviral therapeutic, and as an adjuvant for injected vaccines. We have found a new use for this immunomodulator; it functions as a mucosal adjuvant that enhances immunogenicity of virus-like particles (VLP) administered intranasally. MB enhanced Norwalk virus (NV) VLP-specific IgG systemically and IgA production at distal mucosal sites following intranasal (IN) vaccination. A dose escalation study identified 100 µg as the optimal MB dosage in mice, based on the magnitude of VLP-specific IgG, IgG1, IgG2a and IgA production in serum and VLP-specific IgA production at distal mucosal sites. IN vaccination using VLP with MB was compared to IN delivery VLP with cholera toxin (CT) or gardiquimod (GARD) and to parenteral VLP delivery with alum; the MB groups were equivalent to CT and GARD and superior to alum in inducing mucosal immune responses and stimulated equivalent systemic VLP-specific antibodies. These data support the further testing of MB as a potent mucosal adjuvant for inducing robust and durable antibody responses to non-replicating subunit vaccines.

Microbial components stimulate innate immunity via Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and/or IL-1. We recently reported that in mice, Escherichia coli lipopolysaccharide (LPS, TLR4-ligand) promotes allergic responses to nickel (Ni) at both the sensitization and elicitation steps. Here, we examined in mice the effects of administering other microbial or inflammatory materials at the Ni-sensitization step. A mixture of 1mM NiCl(2) and a test solution was injected into BALB/c mice intraperitoneally (0.1 ml/10 g body weight), and 10 days later 5mM NiCl(2) was challenged intradermally into the ear pinnas of the mice (20 μl/ear). The following preparations or substances exhibited adjuvant activities: Prevotella intermedia LPS, Saccharomyces cerevisiae mannan, a synthetic muramyl dipeptide (NOD2-stimulating cell-wall component of bacteria), Pam(3)Cys-SKKKK (TLR2-stimulating synthetic peptide), poly I:C (TLR3-stimulating double-stranded RNA), concanavalin A (a typical T-cell mitogen and T-cell-mediated hepatitis-inducer), heat-killed Propionibacterium acnes (Gram-positive bacterium that causes pimples and induces macrophage-mediated experimental hepatitis), and nitrogen-containing bisphosphonates (chemicals stimulating IL-1 production). Unexpectedly, P. intermedia LPS, which displayed the most potent adjuvant activity among the tested preparations, was effective in TLR4-dysfunctional mutant mice, but not in TLR2-deficient mice, whereas the reverse was true for S. cerevisiae mannan. These results suggest that (i) for the establishment of Ni-allergy in mice, stimulation of innate immunity (including TLRs, NLRs, IL-1 production, and/or other factors) may be important at the sensitization step, and (ii) P. intermedia may produce a substance(s) that potently promotes Ni-allergy via stimulation of TLR2., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Introduction: The standard treatment for osteosarcoma requires both macroscopic surgical wide resection and postoperative multi-drug chemotherapy in neoadjuvant and adjuvant settings. However, the 5-year event-free survival has remained at a plateau of 60-70% of patients with nonmetastatic osteosarcoma for more than 30 years., Areas Covered: Mifamurtide (liposomal muramyl tripeptide phosphatidylethanolamine; L-MTP-PE) is a new agent. L-MTP-PE is a nonspecific immunomodulator, which is a synthetic analog of a component of bacterial cell walls. L-MTP-PE activates macrophages and monocytes as a potent activator of immune response in addition to standard chemotherapy. It also improves the overall survival from 70 to 78% and results in a one-third reduction in the risk of death from osteosarcoma. This review summarizes the most recent findings about L-MTP-PE and its therapeutic application for nonmetastatic osteosarcoma., Expert Opinion: Recently, L-MTP-PE has been approved in Europe for the treatment of nonmetastatic osteosarcoma with chemotherapy. L-MTP-PE in combination with traditional treatment is expected to go mainstream and to be beneficial for patients with osteosarcoma. Information about potential benefit regarding mifamurtide use in the neoadjuvant setting (i.e., before surgery) and/or usefulness of L-MTP-PE in metastatic in relapsed and metastatic osteosarcoma requires analysis of expanded access and/or future clinical trials of L-MTP-PE in high-burden and low-burden situations.

Intracellular nucleotide-binding oligomerization domain (NOD)-like receptors, NOD1 and NOD2, recognize the diaminopimelic acid (DAP)-containing peptide moiety and muramyldipeptide (MDP) moiety of bacterial peptidoglycan, respectively. Muramyldipeptide has been reported to exert analgesic activity to decrease the frequency of acetic acid-induced writhing movements in mice. In this study, we demonstrated the analgesic activities of NOD1 as well as NOD2 agonists. Intravenous injection of NOD2-agonistic MDP, 6-O-stearoyl-MDP (L18-MDP), and MDP-Lys (L18) exhibited analgesic activity at 10, 50, and 2.0 µg/head, respectively, in BALB/c mice. NOD1-Agonistic FK156 (D-lactyl-L-Ala-D-Glu-meso-DAP-L-Gly) and FK565 (heptanoyl-D-Glu-meso-DAP-D-Ala) were also analgesic at 50 µg/head and 1.0 µg/head, respectively. The analgesic effect of FK565 appeared from 30 min, reached maximum activity at 8 h, and continued until 24 h. The FK565 exhibited activity by various administration routes; intravenous, intraperitoneal, intramuscular, sublingual (1.0 µg/head each), subcutaneous, intragastric (oral), intragingival (10 µg/ head each) and intracerebroventricular (0.01 µg/head). The analgesic activity of FK565 was observed even in tumor necrosis factor (TNF)-α knockout, interleukin (IL)-1α/β double knockout, and their triple knockout mice. Naloxane, a non-selective antagonist for the opioid receptor, completely inhibited the analgesic effect of FK565. These findings suggest that NOD1 and NOD2 activation induces an analgesic effect via opioid receptors in a TNF-α and IL-1α/β independent manner.

Muramyldipeptide (MDP), the minimum essential structure responsible for the immuno-adjuvant activity of peptidoglycan, is recognized by intracellular nuclear-binding oligomerization domain 2 (NOD2). Muramyldipeptide enhances the activities of lipopolysaccharide (LPS), but the mechanism underlying this effect is unclear. Here, we obtained evidence that intravenously injected MDP augments LPS-induced hypothermia in wild-type mice, but not in mice deficient in interleukin (IL)-1α/β and/or tumor-necrosis factor (TNF)-α. Muramyldipeptide also: (i) increased pro-IL-1β in tissues, but did not increase IL-1β in serum (since caspase-1 was not activated by MDP); (ii) downregulated the expression of suppressor of cytokine signaling 1 (SOCS1; a negative-feedback regulator of LPS-induced signaling); and (iii) augmented the LPS-induced production of TNF-α, IL-12 p40, and interferon (IFN)-γ. Moreover, by performing in vivo and in vitro experiments, we obtained evidence that macrophages were involved in these effects of MDP. These results suggest that two different mechanisms may underlie the augmenting effect of MDP: namely, stimulation of pro-IL-1β production by, and down-regulation of SOCS1 in, macrophages. We consider that this work may help to elucidate the pathogenesis of mixed bacterial infections, including septic shock and multiple organ dysfunction syndrome (MODS).

Propionibacterium acnes was modified using biochemical extraction methods generating a suspension of microparticles (MIS416) comprising a minimal cell wall skeleton rich in immunostimulatory crosslinked muramyl dipeptide repeats and native bacterial DNA fragments, each which have known adjuvant activity. In vitro studies demonstrated that MIS416 was readily internalized by human myeloid and plasmacytoid DC inducing cytokine secretion and cell activation/maturation. Vaccination studies in mice using OVA as a model antigen demonstrated that MIS416 acts as a Th1 adjuvant, promoting cross-priming of cytotoxic CD8(+) T cell responses and enhanced anti-tumour immunity. Covalent attachment of OVA to MIS416 enabling simultaneous delivery of antigen and adjuvant to the antigen presentation system resulted in a dose-sparing vaccine formulation. Preclinical GLP toxicology studies demonstrated that MIS416 has a favorable safety profile in mouse and rabbit supporting its use in human vaccine formulations., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: Airway tolerance is critical for protecting the lung from inflammatory disease driven by allergens. However, factors that disrupt tolerance processes and then lead to susceptibility to developing allergic asthma remain elusive., Objective: To investigate whether recognition of bacterial microbial-associated molecular patterns in the lung may result in susceptibility to developing allergic reactions, and to understand the molecular mechanisms by which such triggers block natural tolerance., Methods: Ligands of intracellular microbial-associated molecular pattern recognition receptors-the nucleotide-binding oligomerization domain (Nod)-like receptors, Nod1 and Nod2-were given intranasally with antigen, and their ability to modulate airway tolerance was analyzed., Results: Intranasal Nod2 ligand rapidly induced lung expression of the innate cytokines thymic stromal lymphopoietin and IL-25, and thymic stromal lymphopoietin promoted expression of OX40 ligand, a T-cell-costimulatory ligand, on lung CD11c(+)CD11b(+) cells and B220(+) cells. Together these 3 molecules blocked the generation of antigen-specific CD4(+)forkhead box protein 3(+) adaptive regulatory T cells and concomitantly drove IL-4-producing CD4 T cells. By altering the regulatory T/T(H)2-cell balance, tolerance was blocked, and sensing of Nod2 ligand resulted in subsequent susceptibility to developing eosinophil-dominated airway inflammation., Conclusion: We show that a Nod-like receptor is a novel, previously unrecognized, pathway that adversely links innate and adaptive immunity and leads to allergic disease and asthmatic lung inflammation., (Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Vaccination with Echinococcus multilocularis 14-3-3 protein can protect mice against primary E. multilocularis infection. The present study investigated the efficacy and efficiency of the adjuvant muramyl dipeptide Gerbu, alone or together with recombinant 14-3-3 protein, to modulate the course of secondary E. multilocularis infection in C56BL/6 mice. The application of Gerbu alone already resulted in a parasite weight reduction when compared with infected control mice, while rec14-3-3 did not add to this effect. Immunological parameters were concurrently assessed with a mixed cell reaction including bone marrow-derived dendritic cells (BMDCs) together with lymph node cells from mice with or without immunisation and/or infection. While mice having received Gerbu adjuvant were found to highly proliferate in response to co-cultivation with 14-3-3-stimulated bone marrow dendritic cells, a sensitisation of BMDCs with vesicle fluid (VF) antigen lead to a striking decrease of the lymphoproliferative response in comparison to that of control mice, raising the hypothesis that immunosuppressive components may be part of this VF-antigen. Anti-14-3-3 antibody production was only found in those mice that had been previously 14-3-3-immunised, whereas all other only-infected mice failed to produce such antibodies. Conclusively, Gerbu adjuvant appears to directly generate a non-specific immune response that contributes to the control of the metacestode growth, putatively in association with a BMDC activity suppressed by components of the VF-antigen.

Structurally related peptidoglycan monomer (PGM) and muramyl dipeptide (MDP) differ in several aspects of biological activity but have in common immunostimulating properties. Comparative study of the effects of these adjuvants on humoral IgG immune response specific for protein antigen ovalbumin (OVA) was carried out in two inbred mouse strains, CBA and NIH/OlaHsd, and their ability to modulate the bias of immune response towards Th1/Th2 was evaluated. MDP had better adjuvant activity at some points than PGM, whereas both adjuvants stimulated Th2-biased immune response specific for OVA. In comparison to Complete Freund's adjuvant (CFA), as a golden standard of adjuvant action, both PGM and MDP exhibited considerably lower activity. Addition of PGM to Incomplete Freund's adjuvant (IFA) on humoral immune response was studied also, and the effect of such adjuvant formulation was compared to the effect of CFA. While CFA induced the switch towards Th1-biased immune response, the addition of PGM into IFA did have no impact on modulating the immune response towards more pronounced Th2-type of immune response, defined by IFA itself., (2010 Elsevier B.V. All rights reserved.)

We have shown that glucosaminyl muramyl dipeptide (GMDP) has been augmented the antitumor action of chemotherapy drug cisplatin and tumor necrosis factor-alpha (TNFalpha) on the Ehrlich ascites carcinoma and melanoma B-16 mouse tumor models. The doses of cisplatin, TNFalpha and GMDP and also the conditions of the drugs combination injection provided 100% survival of mice with Ehrlich ascites carcinoma were found. Furthermore, it was shown first that GMDP has been decreased toxicity of the cisplatin/TNFalpha combination and normalized the changes in the experimental mice hematological parameters which were produced by the CP/TNFalpha combination., ((c) 2009 Elsevier Masson SAS. All rights reserved.)

Intravenous injection of lipopolysaccharide (LPS, a component of the Gram-negative bacterial cell-surface) or mannan (Man, a component of the fungal cell-surface) into mice reportedly induces anaphylaxis-like shock (ALS) via complement-associated platelet degradation and platelet-activating factor (PAF), respectively. However, it is unclear whether PAF is involved in LPS-ALS or whether complements and/or platelets are involved in Man-ALS. Here, using preparations of Man from Saccharomyces cerevisiae and LPS from Klebsiella O3, we characterized and compared LPS-ALS and Man-ALS, with the following results. (1) ALS depended on mouse strain (ddY and BALB/c being highly responsive to Man and LPS, respectively), but not on Toll-like receptors 2 and 4. (2) In ddY mice, Man had little effect on platelets, K76 (C5a-inhibitor) did not prevent Man-ALS, and Man-ALS was augmented by prior platelet depletion. (3) CV-3988 (PAF antagonist) prevented Man-ALS, but not LPS-ALS. (4) LPS-ALS and Man-ALS were each augmented by prior injection of a muramyl dipeptide (MDP, a constituent abundant in the Gram-positive bacterial cell-surface), but prevented by prior macrophage depletion. (5) Co-administration of Man and LPS induced an augmented ALS in both ddY and BALB/c mice. These results indicate that (i) Man and LPS each induces ALS in mice in strain-dependent and macrophage-dependent (but not TLR-dependent) ways by stimulating a platelet-non-associated PAF pathway and a platelet-associated complement pathway, respectively, and (ii) these pathways are primed by MDP and exhibit mutually augmenting actions. Man-ALS and LPS-ALS may therefore serve as models for diseases involving augmentation by multiple or mixed infections.

Background: The addition of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) to chemotherapy has been shown to improve overall survival in patients with nonmetastatic osteosarcoma (OS). The authors report the results of addition of liposomal MTP-PE to chemotherapy for patients with metastatic OS., Methods: Intergroup-0133 was a prospective randomized phase 3 trial for the treatment of newly diagnosed patients with OS. The authors compared 3-drug chemotherapy with cisplatin, doxorubicin, and high-dose methotrexate (Regimen A) to the same 3 drugs with the addition of ifosfamide (Regimen B). The addition of liposomal MTP-PE to chemotherapy was evaluated., Results: Five-year event-free survival (EFS) for patients who received liposomal MTP-PE (n = 46) was 42% versus 26% for those who did not (n = 45) (relative risk for liposomal MTP-PE, 0.72; P = .23; 95% confidence interval [CI], 0.42-1.2). The 5-year overall survival for patients who received MTP-PE versus no MTP-PE was 53% and 40%, respectively (relative risk for liposomal MTP-PE, 0.72; P = 0.27; 95% CI, 0.40-1.3). The comparison of Regimen A with Regimen B did not suggest a difference for EFS (35% vs 34%, respectively; relative risk for Regimen B, 1.07; P = .79; 95% CI, 0.62-1.8) or overall survival (52% vs 43%, respectively; relative risk for Regimen B, 1.1, P = .75; 95% CI, 0.61-2.0)., Conclusions: When the metastatic cohort was considered in isolation, the addition of liposomal MTP-PE to chemotherapy did not achieve a statistically significant improvement in outcome. However, the pattern of outcome is similar to the pattern in nonmetastatic patients.

Tuberculosis (TB) infects one third of the world's population, and new infections occur at a rate of 1/s. Better vaccines are needed than the live mycobacterium Bacille Calmette-Guérin (BCG). Alveolar macrophages (AMPhis) play a central role in pulmonary manifestations of TB. Targeting immunomodulators to AMPhis, the first line of defense against Mycobacterium tuberculosis (Mtb), may initiate a potent cell-mediated immune response. Muramyl dipeptide (MDP) and trehalose dibehenate (TDB) have elicited strong immune response when delivered to the lungs as aerosols. AMPhis show toxicity in response to some immunomodulators. The objective of this work was to screen the immunomodulators MDP and/or TDB encapsulated in microparticles (MPs) and to evaluate certain indicators of toxicity in human AMPhi-like cells. Poly(lactide-co-glycolide) (PLGA) MPs containing MDP and/or TDB were prepared by spray-drying. The morphology, particle size distribution, and immunomodulator encapsulation efficiency of MPs were examined. THP-1 cells were exposed to these MPs for 24 h and characteristics of cell morphology, tumor necrosis factor-alpha (TNF-alpha) release, lactate dehydrogenase (LDH), N-acetyl-beta-D: -glucosaminidase (NAG) and alkaline phosphatase (ALP) activity in AMPhi culture supernatants were measured. MTT assay was used to assess the viability of cells. Spray-drying produced low-density MPs having volume median diameters between 4 and 6 microm as measured by laser diffraction and projected area diameter between 3 and 5 microm calculated by microscopy. More TNF-alpha was produced by THP-1 cells exposed to MPs composed of PLGA-MDP or PLGA alone than PLGA-TDB. LDH release following exposure to MPs of PLGA-MDP and PLGA alone was greater than controls. NAG release was higher following exposure to MPs of PLGA alone or PLGA-MDP 0.1% than PLGA-TDB (0.1% and 1.0%). Cells remained viable after exposure to MPs as per MTT assay. PLGA-MDP MPs demonstrated statistically elevated indicators of biochemical responses in cell culture compared to PLGA-TDB MPs, but the extent of their potential to elicit adverse effects in vivo must be studied independently.

We studied the capacity of anomeric pairs of alpha- and beta-dodecyl, alpha- and beta-(1-pentylhexyl), and alpha- and beta-cyclododecyl glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine (muramyl dipeptide) to stimulate the nonspecific resistance of mice to intraperitoneal infection of Staphylococcus aureus and Escherichia coli cultures. Intraperitoneal pretreatment with the test substances in a wide dose range increased survival of infected animals. No differences were found between the biological effects of alpha- and beta-dodecyl and alpha- and beta-(1-pentylhexyl) glycosides of muramyl dipeptide. An inverse relationship was found between stimulatory activity and dose of alpha- and beta-cyclododecyl glycosides of muramyl dipeptide during sepsis caused by Staphylococcus aureus.

Nucleotide oligomerization domain (NOD) proteins recognize peptidoglycan fragments, resulting in up-regulation of transcription factors, and may enhance the inflammatory response to infection. Specifically, NOD2 has been shown to sense muramyl dipeptide (MDP), which is released during bacterial cell growth and replication. Activation of NOD2 by MDP enhances the inflammatory response caused by LPS (endotoxin). Here, we investigated the effects of MDP on the organ injury/dysfunction caused by systemic administration of a low dose of LPS. Male Wistar rats were coadministered with either MDP (1 - 10 mg kg(-1), i.v.) or vehicle (0.5 mL kg(-1) saline, i.v.), and a low dose of LPS (1 mg kg(-1), i.v.) or vehicle (1 mL kg(-1), saline, i.v.). MAP and heart rate were continuously monitored for 6 h. Markers of organ dysfunction/injury, plasma cytokine levels, and lung myeloperoxidase activity were measured 6 h after MDP and LPS coadministration. In a separate study, MDP (3 or 10 mg kg(-1), i.v.) or vehicle (0.5 mL kg(-1) saline, i.v.) was administered 24 h before LPS infusion. When compared with animals receiving low-dose LPS alone, coadministration of MDP (10 mg kg(-1), i.v.) and LPS, or administration of MDP (10 mg kg(-1), i.v.) 24 h before LPS resulted in a significant increase in the degree of organ injury, cytokine release, and lung injury caused by LPS alone. Thus, our results demonstrate that the two bacterial wall components MDP and LPS work in concert to cause multiple organ injury and systemic inflammation. We hope that our results stimulate other studies designed to evaluate the effects of NOD ligands in animal models of inflammation.

The mechanisms underlying the susceptibility of individuals with caspase recruitment domain 15 (CARD15) mutations and corresponding abnormalities of nucleotide-binding oligomerization domain 2 (NOD2) protein to Crohn disease are still poorly understood. One possibility is based on previous studies showing that muramyl dipeptide (MDP) activation of NOD2 negatively regulates TLR2 responses and that absence of such regulation leads to heightened Th1 responses. We now report that administration of MDP protects mice from the development of experimental colitis by downregulating multiple TLR responses, not just TLR2. The basis of these in vivo findings was suggested by in vitro studies of DCs, in which we showed that prestimulation of cells with MDP reduces cytokine responses to multiple TLR ligands and this reduction is dependent on enhanced IFN regulatory factor 4 (IRF4) activity. Further studies of mouse models of colitis showed that this inhibitory role of IRF4 does in fact apply to MDP-mediated protection from colitis, since neither IRF4-deficient mice nor mice treated with siRNA specific for IRF4 were protected. These findings indicate that MDP activation of NOD2 regulates innate responses to intestinal microflora by downregulating multiple TLR responses and suggest that the absence of such regulation leads to increased susceptibility to Crohn disease.

Osteosarcoma is the most common form of primary malignant bone tumor. The use of chemotherapy drugs with many side effects, including high-dose methotrexate, doxorubicin, cisplatin and ifosfamide, has greatly improved osteosarcoma survival compared with surgery alone. However, for 20 years, overall survival remained at a plateau of 60-70% in nonmetastatic disease and 20-30% in metastatic osteosarcoma owing to lung metastases. Liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE) is a new agent that improves overall osteosarcoma survival (chemotherapy without L-MTP-PE 70% versus with L-MTP-PE 78%; p = 0.03). L-MTP-PE offers additional benefit for osteosarcoma treatment in combination with chemotherapy, particularly ifosfamide-containing regimens. Clinical experience indicates that side effects such as fever are temporary and controlled or prevented with ibuprofen and/or acetoaminophen premedication; severe side effects are rare. Although surgery will remain the main approach for osteosarcoma treatment of lung metastases, L-MTP-PE combined with other modalities, including chemotherapy, appears to be of benefit in these patients as well.

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose capped lipoarabinomannan) may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.

Liposomal muramyl tripeptide phosphatidyl ethanolamine (L-MTP-PE) is a synthetic biological investigational agent used for treating osteosarcoma. It has been used in both canine and human osteosarcoma to reduce pulmonary metastases, the most common pattern of treatment failure for sarcomas. L-MTP-PE has been well tolerated using the concept of biological cancer therapy during chemotherapy. The use of L-MTP-PE with ifosfamide is the best studied combination with single agent chemotherapy. This may represent a new treatment choice for osteosarcoma patients receiving ifosfamide. Such patients include those with a poor initial histological response to primary therapy and/or metastatic disease including pulmonary metastases. Reduction of side effects of L-MTP-PE, such as fever and/or flu-like symptoms, with ibuprofen has not reduced efficacy. Since improved symptom control is possible using drug combinations that are especially effective for delayed nausea, outpatient high-dose ifosfamide chemotherapy combined with L-MTP-PE may lead to a safe and effective therapy while maintaining the patients' quality of life.

Newborn piglets maintained germfree (GF) cannot respond to either thymus-dependent (TD) or type 2 thymus-independent Ags (TI-2) unless colonized with bacteria. We show here that pathogen-associated molecular patterns (PAMPs), including muramyl dipeptide (MDP), LPS, and a B-class CpG oligonucleotide (CpG-B), can substitute for gut flora in the induction of neonatal immunoresponsiveness. These PAMPs alone or in combination had little effect on serum IgG and IgA levels, but CpG-B and CpG-B + MDP elevated total IgM levels 3- to 7-fold above that seen in colonized controls after booster immunization. Although only CpG-B could alone stimulate immunoresponsiveness, co-administration of LPS or MDP resulted in a 5-fold increase in the IgG response to both immunogens. Co-administered MDP did not promote secondary IgG responses to either Ag but instead pronounced secondary IgM responses to the epitopes of both immunogens. LPS co-administered with CpG-B may promote class switch recombination or cause differentiation of previously switched cells that become responsive after exposure to CpG-B. Primary and secondary IgG responses equally recognized the epitopes of the TI-2 and TD immunogens, whereas IgM responses favored the TI-2 epitope. Because PAMPs alone can result in Abs to 2,4,6-triitrophenyl and FLU without immunization, it suggests they alone cause differentiation of B cells of the preimmune repertoire. The finding that both bacterial PAMPs and colonization are capable of stimulating Ab responses in both immunized and nonimmunized piglets suggests that PAMPs derived from host flora may play a major role in awakening adaptive immunity in neonates.

We have investigated the effects of continuous subcutaneous infusion of lipopolysaccharide (LPS), muramyldipeptide (MDP), or saline on abdominal temperature and voluntary activity in unrestrained rats. Both pyrogens were infused via osmotic pumps at a rate of approximately 2 microg.kg-1.min-1 for 7 d. LPS infusion evoked a 3-d and MDP a 1-d elevation in body temperature. Night-time activity was suppressed on days 1 and 2 during LPS infusion and on day 1 of MDP infusion. Body mass was significantly decreased on infusion day 4 in rats receiving either LPS or MDP; however, the rate of weight gain had been restored by day 8 (1 d after cessation of pyrogen infusion). We further tested the body temperature response of the same experimental animals to a single subcutaneous bolus injection (250 microg/kg) of the same pyrogen that had been infused for 7 d, 2 d after cessation of pyrogen infusion (day 9). The fever response in rats receiving a bolus injection of either LPS or MDP was significantly attenuated in rats that had previously been infused with the same pyrogen. These data suggest that tolerance developed to continuous infusion of both Gram-negative and Gram-positive pyrogens, and that mechanisms of tolerance development set in early during the 7-d infusion period of both pyrogens and persisted for at least 2 d after the cessation of pyrogen infusion. We propose that cytokine intermediates were involved or required in inducing these responses to continuous infusion of both LPS and MDP.