Activation of peritoneal macrophages from guinea pigs by various bacterial cell walls, M-1 endo-N-acetylmuramidase enzymatically digested bacterial cell walls and synthetic muramyl dipeptides was studied in terms of stimulation of ["4C] glucosamine incorporation. All test bacterial cell wall preparations significantly increased a [14C]glucosamine uptake by the macrophages. Some of the watersoluble M-1 enzyme digests also exerted stimulating effects on macrophages, although the activity of the digests was found to be weaker than those of original cell walls. Furthermore, an adjuvant-active synthetic MurNAc-L-Ala-D-isoGln (MDP) showed a weak but significant activity, whereas an adjuvant-inactive analog, MurNAc-L-Ala-L-isoGln, did not show a significant activity, at least with the dose of 100 ljg. Additional studies with 6-0-acyl derivatives of MDP revealed that 6-0-(2-tetradecylhexadecanoyl)-MDP and 6-0-(3-hydroxy-2-tetradecyloctadecanoyl)-MDP exhibit stronger macrophage-stimulating effects than MDP. It can be concluded from the above findings that MDP is the essential structure responsible for stimulating the activity of cell walls on guinea pig peritoneal macrophages, but it requires a particle state, which results from an additive character of lipophilicity, to exert the activity fully and effectively.Stimulation of radiolabeled glucosamine incorporation by macrophages as one expression of macrophage activation was first shown by Hammond and Dvorak (8, 9) on the reaction of peritoneal macrophages from antigen-primed guinea pigs with a specific antigen in vitro. This study preceded the finding that plant lectins, concanavalin A, and phytohemagglutinin stimulated macrophages from nonimmunized animals to increase glucosamine uptake (9) and that BCG cell walls had a similar activity (11).In this study, we investigated the stimulating effects of bacterial cell walls and related compounds, either enzymatically obtained or synthetic, upon peritoneal macrophages from guinea pigs in terms of elevation of glucosamine uptake. The study was undertaken to determine the chemical structure and physical state required for the macrophage activation as determined by the increase of glucosamine incorporation. MATERIALS AND METHODSCell wall preparations. Cells as a starting material for isolation of the walls were cultured under the 48 following conditions. Mycobacterium smegmatis ATCC 19420 was grown as a pellicle on 250 ml of Sauton medium in Roux flasks at 30'C for 14 days, Nocardia corynebacteriodes ATCC 14898 was grown in a medium (pH 7.2) containing 1% maltose, 1% meat extract, 1% polypeptone, 0.5% NaCl, and the deforming agents (0.2% silicone KM-70 and 0.01% Karaline 102). Streptomyces gardneri ATCC 23911 was grown with aeration in a medium (pH 7.2) consisting of 1% glucose, 0.5% polypeptone, 0.3% malt extract, and the above deforming agents. Cultivations of N. corynebacteriodes and S. gardneri were made in 20-liter Jar fermentors containing 10-liter portions of the above media by incubating each 100 ml of a seed culture ...
The experimental system utilized in investigating the correlation between the chemical structures of muramyl peptides and their protective activities in the sepsis type of systemic infections caused by Escherichia coli was applied in evaluating the enhancement of resistance to infection induced by 32 synthetic glycopeptide analogs, including 6-O-acyl derivatives and 1-alpha-O-benzyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine). In assessing the 6-O-acyl derivatives of muramyl dipeptide, we found that the degree of protective activity was attributable to the kinds of fatty acids introduced. Acylation of the 6-hydroxy group on the muramic acid moiety in muramyl dipeptide with natural mycolic acid or a synthetic fatty acid possessing either an alpha-branched or an alpha-branched, beta-hydroxylated group resulted in a decrease in or a disappearance of the protective activity of muramyl dipeptide. Acylation with a normal fatty acid or an iso fatty acid resulted in a retention or enhancement of muramyl dipeptide activity. The activity of acylated derivatives containing linear fatty acids was stimulated by increasing the chain length up to 18 carbon atoms. The highest degree of protective activity occurred with the derivatives acylated with straight-chain fatty acids, particularly with the derivatives acylated with palmitic acid and arachidic acid. Benzylation of the 1-hydroxy group of muramyl dipeptide resulted in a decrease in or a loss of protective activity.
α-Glycosyl phosphates and 4-phosphates of N-acylglucosamine derivatives were prepared in high yields via the reaction of corresponding O-lithium salts with dibenzyl or diphenyl phosphorochloridate at low temperature.
Stimulation of [3H]thymidine incorporation of thymocytes and splenocytes from guinea pigs by various bacterial cell walls and their peptidoglycans, by enzymatic digests, and by synthetic muramyl dipeptides was studied as an indication of mitogenic activity. Cell wall and peptidoglycan preparations, isolated from 19 strains belonging to 18 different species, definitely increased [3H]thymidine incorporation of thymocytes as well as splenocytes, regardless of mycolic acid contents as a non-peptidoglycan component. Both the cell walls from Nocardia corynebacteriodes (containing mycolic acids) and those from Streptomyces gardneri (lacking mycolic acids) showed far stronger mitogenic activities on splenocytes than other cell walls (stimulation index, 25 to 30). Furthermore, water-soluble enzymatic digests, notably the endopeptidase digests, which generally were greater in degree of polymerization of peptidoglycan subunits than the glycosidase digests obtained from representative cell walls, were found to have as distinct a stimulating activity on splenocytes as the original cell walls. In contrast, solubilization of the cell walls by enzymes, irrespective of endopeptidases or glycosidases, was accompanied by disappearance of the mitogenic activity on thymocytes. On the other hand, studies with synthetic 6-0-acyl-MurNAc-L-Ala-D-isoGln preparations (6-0-acyl-MDPs) revealed that 6-0-stearoyl-MDP and 6-0-(2-tetradecylhexadecanoyl)-MDP, unlike MDP, had distinct mitogenic activity on thymocytes, whereas their activity on splenocytes was rather weaker than MDP itself. The findings presented here suggest that MDP is the minimal structure for the mitogenic activities of bacterial cell walls on guinea pig splenocytes, but that MDP, though distinctively active by itself, requires a polymerized form to exert effectively its inherent stimulating activities on splenocytes. On the other hand, on thymocytes, MDP, unless it takes a particular form or has appropriate additive groups, cannot exert its mitogenic activities.Damais et al. reported that cell wall peptidoglycans obtained from Bacillus megaterium and Escherichia coli exerted mitogenic effects on spleen lymphocytes of nude mice and rabbit splenocytes, but those from Micrococcus lysodeikticus and the monomeric subunit of E. coli peptidoglycan lacked these activities (8). This study was followed by that of Ciorbaru et al., which demonstrated that a water-soluble polymer of peptidoglycan subunits prepared from Nocardia rubra cell walls by use of Streptomyces albus G (endo)peptidase exhibited mitogenic activity on murine B cells, but that a 645 peptidoglycan monomer obtained from the same cell walls digested by lysozyme could not (7). Then we revealed that adjuvant-active synthetic MurNAc-L-Ala-D-isoGln (MDP) (1,11,21) and MurNAc-L-Ala-D-Glu (21) had weak but significant mitogenic activity on splenocytes of guinea pigs and ICR mice, but none of the adjuvantinactive analogs, MurNAc-L-Ala-L-isoGln, MurNAc-L-Ala-D-Gln, MurNAc-L-Ala-L-Gln, MurNAc-L-Ala-L-Glu, and MurNAc-L-Ala-D-isoAsn,...
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