Association of the NOD2 genotype with bacterial translocation via altered cell-cell contacts in Crohn's disease patients.

BACKGROUND: Recent insights into the pathogenesis of Crohn's disease (CD) point to an important role of the mucosal barrier and intestinal microflora that may induce a chronic inflammation after crossing the intestinal barrier. The first detected susceptibility gene for CD, NOD2, is a pattern recognition receptor (PRR) for the recognition of the bacterial cell wall component muramyldipeptide (MDP). Binding of MDP to NOD2 is followed by activation of proinflammatory pathways mainly regulated by nuclear factor kappa B (NF-kappaB). In this study we investigated whether impaired recognition of MDP via NOD2 variants is associated with increased bacterial translocation across the epithelial barrier and whether this is followed by increased or decreased NF-kappaB activation. METHODS: NOD2 variants were analyzed in 36 CD patients and 30 controls. Endotoxin was stained by immunohistochemistry in 30 intestinal biopsies from patients carrying NOD2 variants (NOD2-mut) or being NOD2 wildtype (WT). Junctional proteins were visualized by immunofluorescence and quantified by Western blotting. NF-kappaB activation was analyzed by immunohistochemistry in specimens from NOD2-WT and NOD2-mut CD and control patients. RESULTS: We demonstrated the increased presence of endotoxin in the mucosal lamina propria of CD patients carrying NOD2 variants. This was associated with an altered composition of epithelial cell-cell contacts. Patients carrying NOD2 variants displayed increased NF-kappaB activation in the mucosa. CONCLUSIONS: This study for the first time demonstrates that translocation of luminal bacteria and/or bacterial products into the intestinal mucosa is increased in patients carrying NOD2 variants, leading to higher activation of proinflammatory signaling cascades.

Components of the peptidoglycan-recycling pathway modulate invasion and intracellular survival of Salmonella enterica serovar Typhimurium.

beta-Lactam resistance in enteric bacteria is frequently caused by mutations in ampD encoding a cytosolic N-acetylmuramyl- l-alanine amidase. Such mutants are blocked in murein (peptidoglycan) recycling and accumulate cytoplasmic muropeptides that interact with the transcriptional activator ampR, which de-represses beta-lactamase expression. Salmonella enterica serovar Typhimurium, an extensively studied enteric pathogen, was used to show that mutations in ampD decreased the ability of S. typhimurium to enter a macrophage derived cell line and made the bacteria more potent as inducers of inducible nitric oxide synthase (iNOS), as compared with the wild-type. ampG mutants, defective in the transport of recycled muropeptides across the cytoplasmic membrane, behaved essentially as the wild-type in invasion assays and in activation of iNOS. As ampD mutants also have reduced in vivo fitness in a murine model, we suggest that the cytoplasmic accumulation of muropeptides affects the virulence of the ampD mutants.

Factors limiting the oral bioavailability of N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) and enhancement of absorption in rats by delivery in a water-in-oil microemulsion.

The bioavailability (BA) of radio-labelled N-acetylglucosaminyl-N-acetylmuramyl dipeptide (GMDP) was low when administered by oral gavage as an aqueous solution to conscious male Sprague-Dawley rats (8.3+/-4.4% (mean+/-S.D., n=3)). To assess the likely factors contributing to the poor BA of GMDP, the stability of GMDP in the lumen of the gastrointestinal (GI) tract was examined in vitro, using ex vivo GI contents. GMDP was degraded by the contents of the small intestine, caecum and large intestine but was more stable in stomach contents. The permeability coefficient (p(app)) of GMDP in isolated sections of rabbit ileum was 1.67x10(-6) cm/s in the mucosal to serosal direction and was not significantly different in the serosal to mucosal direction, indicating that GMDP is poorly permeable and passively transported across the intestinal wall. First pass metabolism was considered to be unlikely to be the primary limitation to the oral bioavailability of GMDP and therefore, that the oral bioavailability of GMDP was likely limited by instability in the lumen of the gastrointestinal tract and low intestinal permeability. A water-in-oil (w/o) microemulsion formulation subsequently developed to address these problems was trialed in a preliminary bioavailability study in rats and enhanced the bioavailability of GMDP ten-fold when administered intraduodenally, indicating that w/o microemulsions may represent a viable mechanism for enhancing the bioavailability of poorly GI-stable and poorly permeable peptide-based molecules.

Enhanced antibody response to liposome-associated protein antigens: preferential stimulation of IgG2a/b production.

The effect of liposome encapsulation on the antibody response to bovine serum albumin (BSA), human carcinoembryonic antigen (CEA) and sheep IgG (sIgG) has been determined in the mouse. Dipalmitoylphosphatidylcholine/dimyristolylphosphatidylglycerol liposomes (10:1 molar ratio; 1 mumol) containing BSA, CEA or sIgG induced significant levels of IgG antibodies after one injection, and enhanced the proportion of IgG2a/2b to IgG1 on subsequent boost injection. The IgG antibody titre induced by liposomal antigen was 100-400-fold greater than immunization with antigen alone. Immunization with antigen and the water-soluble adjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP; 50 micrograms) resulted in antibody titres intermediate between free and liposomal antigen. MDP did not enhance the proportion of IgG2a/2b to IgG1. Incorporation of the lipid soluble MDP derivative MDP-glycerol dipalmitate (MDP-GDP; 10 micrograms) liposomes containing protein antigens resulted in higher titres and enhanced IgG2b isotype expression. Analysis of serum IgG antibody-isotype levels after immunization and boost with BSA/MDP showed that the half-life of IgG2a/2b and IgG3 was significantly less than that of IgG1. Liposomal encapsulation resulted in longer IgG2a/2b and IgG3 half-lives, especially when MDP-GDP was present in the liposome. These results demonstrate that, whereas MDP preferentially stimulates IgG1 antibodies, liposomes elicit high levels of IgG2a/2b isotypes with significantly longer serum half-lives.

Formulation of vaccine adjuvant muramyldipeptides (MDP). 1. Characterization of amorphous and crystalline forms of a muramyldipeptide analogue.

A relatively nonhygroscopic crystalline form of the glycopeptide, N-acetylmuramyl-L-alpha-aminobutyryl-D-isoglutamine (I), containing approximately one molecule of water was prepared from amorphous material. The crystalline material, consisting of a mixture of the alpha and beta anomers, exhibited better physical and chemical stability than the lyophilized amorphous material. The alpha/beta-anomer ratios of I in both the crystalline and the amorphous state were approximately equal but different from that in solution.

Formulation of vaccine adjuvant muramyldipeptides (MDP). 2. The thermal reactivity and pH of maximum stability of MDP compounds in aqueous solution.

The degradation of muramyldipeptides (MDPs) in aqueous solution obeys the rate law kobs = kH+aH+ + ko + kHO-aHO- and the Arrhenius equation. For example, the rate constants for degradation of N-acetylmuramyl-L-threonyl-D-isoglutamine, 3, at 25 degrees C are kH+ = 2.3 X 10(-6) M-1 sec-1, ko = 8.2 X 10(-10) sec-1, and kHO- = 0.19 M-1 sec-1. The degradation rates are dependent on the side-chain substituents; it is predicted that sterically hindered MDP compounds will show an extended shelf life in aqueous solution. Product studies in the weakly acid pH region (where the pH of maximum stability occurs) show that MDP compounds degrade largely by hydrolysis of the dipeptide side chain. These data show that MDP 3 exhibits a shelf life (t90) of greater than 2 years in aqueous solutions of pH 4-4.5, the pH of maximum stability.

Physico-chemical properties of muroctasin.

N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin) was studied to clarify its chemical structure and physico-chemical properties. The chemical structure of MDP-Lys(L18) was confirmed by UV, IR, NMR and MS analyses as well as by elemental analysis. The physico-chemical properties were investigated by thermal analysis, powder X-ray analysis, ionization constant measurement, high-performance liquid and thin-layer chromatography.

Specific absorption with monoclonal antibodies to muramyl dipeptide of the pyrogenic and somnogenic activities of rabbit monokine.

It is well established that muramyl dipeptide (MDP) can induce fever and enhance slow-wave sleep. Recently, crude or purified supernatants of activated macrophages containing endogenous pyrogen (EP) were also shown to enhance slow-wave sleep. These similarities and the recent finding that a mammalian factor that enhances slow-wave sleep is a muramyl peptide triggered us to study the possibility of the presence of this bacterial structure in the EP molecule. In the present study, EP was produced by stimulation of rabbit peritoneal cells with a nonpyrogenic, nonsomnogenic analog of MDP. The EP-containing supernatant lost its pyrogenicity and somnogenicity after passage over an immunoadsorbent column of monoclonal anti-MDP but not of another monoclonal antibody of different specificity. High percentage of the EP was recovered by elution of the anti-MDP columns with HCl/glycine buffer. Results suggest that bacterial muramyl peptides may be incorporated by mammalian cells into substances that act in picomole quantities to mediate immunological and physiological processes. In addition, the technique may be useful to extract interleukin 1 for structural studies.

[Demonstration of a bacterial structure in two human mediators: a sleep facilitating factor and a monokine]. / Mise en évidence d'une structure bactérienne dans deux médiateurs humains: un facteur favorisant le sommeil et une monokine.

A monoclonal anti-MDP antibody was found to bind to "Slow Wave Sleep" factor. This result confirms that this factor is a muramyl peptide and furthermore shows that it contains a structure characteristic of the synthetic adjuvant and of the bacterial cell wall, i.e. an acetylated muramic acid bound to L-alanine. This antibody was also shown to specifically inhibit a biological activity of a purified human monokine which induces fever. Because of these results and other recent observations we propose that a bacterial structure is present in certain mammalian mediators.