A synthetic Nod2 agonist, muramyldipeptide (MDP), and two Nod1 agonists, FK565 and FK156, mimic the bacterial peptidoglycan moiety and are powerful adjuvants that induce cell-mediated immunity, especially delayed-type hypersensitivity. In this study, we used human dendritic cell ( Freund's complete adjuvant (10), which contains killed mycobacterial cells, has been widely used as a powerful adjuvant to induce cell-mediated immunity, represented by delayed-type hypersensitivity, as well as to enhance humoral immunity against test antigens in laboratory animals. A series of studies on the mycobacterial component responsible for the unique adjuvant activity of Freund's complete adjuvant revealed that Wax D is the active entity, being composed of peptidoglycan (PGN), arabinogalactan, and mycolic acid, and thereafter the PGN moieties of various bacteria were revealed to also be active in this respect (37). In the mid-1970s, the minimal essential structure of PGN for adjuvant activity was demonstrated to be muramyldipeptide (MDP; N-acetylmuramyl-Lalanyl-D-isoglutamine) by use of a chemically synthesized compound (8,24). MDP reproduced various bioactivities of PGN (39,40), although the activities of MDP were generally weaker than those of PGN and MDP was scarcely active in some experiments. PGN activates macrophages via Toll-like receptor 2 (TLR2) (35,42,51), whereas MDP lacks TLR2-agonistic activity (41,46,49,51).Fleck et al. (9) reported that desmuramylpeptide (DMP)containing meso-diaminopimelic acid (meso-DAP) was also active as an adjuvant to induce cell-mediated immunity. meso-DAP-type PGN is found in most gram-negative bacteria and in some gram-positive bacteria, including mycobacteria, while most gram-positive bacteria such as Staphylococcus and Streptococcus strains possess L-lysine (Lys)-type PGN (34). In their report, Fleck et al. (9) suggested by mistake that Lys-type DMPs were similarly active to meso-DAP-type DMPs in this respect. Thereafter, French and Japanese investigators chemically synthesized adjuvant-active meso-DAP-type DMPs (1).In the course of the study, the Fujisawa Pharmaceutical Company generated a DMP, D-lactyl-L-alanyl-␥-D-glutamyl-meso-DAP-glycine, by chemically mimicking a counterpart purified from fermentation broths of Streptomyces strains, and this DMP was designated FK156; the company then synthesized various derivatives of FK156, among which the leading compound was FK565, or heptanoyl-D-glutamyl-meso-DAP-␥-Dalanine (13). Recently, an intracellular molecule carrying nucleotidebinding oligomerization domain 2 (Nod2) was revealed to be a receptor for MDP (12,17). Thereafter, another Nod family molecule, Nod1, was demonstrated to recognize a PGN motif containing meso-DAP (7, 11). We found that FK156 and FK565 were Nod1 agonists similar to ␥-D-glutamyl-meso-DAP (45), which is the minimal active structure of a Nod1 agonist (7,22).

Carbon nanotubes (CNTs) are single- or multi-cylindrical graphene structures that possess diameters of a few nanometers, while the length can be up to a few micrometers. These could have unusual toxicological properties, in that they share intermediate morphological characteristics of both fibers and nanoparticles. To date, no detailed study has been carried out to determine the effect of length on CNT cytotoxicity. In this paper, we investigated the activation of the human acute monocytic leukemia cell line THP-1 in vitro and the response in subcutaneous tissue in vivo to CNTs of different lengths. We used 220 nm and 825 nm-long CNT samples for testing, referred to as "220-CNTs" and "825-CNTs", respectively. 220-CNTs and 825-CNTs induced human monocytes in vitro, although the activity was significantly lower than that of microbial lipopeptide and lipopolysaccharide, and no activity appeared following variation in the length of CNTs. On the other hand, the degree of inflammatory response in subcutaneous tissue in rats around the 220-CNTs was slight in comparison with that around the 825-CNTs. These results indicated that the degree of inflammation around 825-CNTs was stronger than that around 220-CNTs since macrophages could envelop 220-CNTs more readily than 825-CNTs. However, no severe inflammatory response such as necrosis, degeneration or neutrophil infiltration in vivo was observed around both CNTs examined throughout the experimental period.

The cytokine‐inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll‐like receptors (TLRs). Tumor necrosis factor alpha (TNF‐α) production by monocytes was markedly induced in a dose‐dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 μg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF‐α production than respective preparations from the hyphal form. Only slight TNF‐α production was induced by the S. cerevisiae glucan. The TNF‐α production triggered by reference LPS and purified fungal mannans required the presence of LPS‐binding protein (LBP), and these responses were inhibited by anti‐CD14 and anti‐TLR4 antibodies, but not by anti‐TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan‐LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

The lipopeptide FSL-1 [S-(2,3-bispalmitoyloxypropyl)-Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe, Pam 2 CG DPKHPKSF] synthesized on the basis of the N-terminal structure of a Mycoplasma salivarium lipoprotein capable of activating normal human gingival fibroblasts to induce the cell surface expression of ICAM-1 revealed an activity to induce production of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-8. FSL-1 also activated macrophages to produce tumor necrosis factor alpha as the Mycoplasma fermentansderived lipopeptide MALP-2 (Pam 2 CGNNDESNISFKEK), a potent macrophage-activating lipopeptide, did. The level of the activity of FSL-1 was higher than that of MALP-2. This result suggests that the difference in the amino acid sequence of the peptide portion affects the activity because the framework structure other than the amino acid sequence of the former is the same as that of the latter. To determine minimal structural requirements for the activity of FSL-1, the diacylglyceryl Cys and the peptide portions were examined for this activity. Both portions did not reveal the activity. A single amino acid substitution from Phe to Arg and a fatty acid substitution from palmitic acid to stearic acid drastically reduced the activity. Similar results were obtained in measuring the NF-B reporter activity of FSL-1 to human embryonic kidney 293 cells transfected with Toll-like receptor 2 and 6, together with a NF-B-dependent luciferase reporter plasmid. These results suggest that both the diacylglyceryl and the peptide portions of FSL-1 are indispensable for the expression of biological activities and for the recognition by Toll-like receptors 2 and 6 and that the recognition of FSL-1 by Toll-like receptors 2 and 6 appears to be hydrophobic.Various bacterial cell wall components such as lipopolysaccharides (LPS), lipoteichoic acid, peptidoglycans, and lipoproteins (LP) have been shown to activate macrophages, fibroblasts, or lymphocytes to induce production of cytokines (16). Escherichia coli LP were first characterized and sequenced by Braun (9), and they have been demonstrated to be biologically active (5)(6)(7)(8)20). The part of LP responsible for biological activity is the N-terminal lipopeptide moiety, the structure of which is S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-SerSer-Asp-Ala-(Pam 3 CSNNA-) (7).Mycoplasmas, wall-less microorganisms, also possess LP capable of activating macrophages or fibroblasts (11,27,28,31,32). Mühlradt et al. (27,28) recently identified a 2-kDa lipopeptide, MALP-2, from Mycoplasma fermentans that is capable of activating monocytes/macrophages, and these authors determined the structure to be S-(2,3-bispalmitoyloxypropyl) Cys-Gly-Asn-Asn-Asp-Glu-Ser-Asn-Ile-Ser-Phe-Lys-Glu-Lys (Pam 2 CGNNDESNISFKEK). We have also found that Mycoplasma salivarium LP activate normal human gingival fibroblasts (HGF) to induce production of inflammatory cytokines and surface expression of ICAM-1 and have purified a 44-kDa LP (LP44) responsible for the activity (32). The structure of the N-ter...

S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) derived from Mycoplasma salivarium stimulated NF-κB reporter activity in human embryonic kidney 293 (HEK293) cells transfected with Toll-like receptor 2 (TLR2) or cotransfected with TLR2 and TLR6, but not in HEK293 cells transfected with TLR6, in a dose-dependent manner. The activity was significantly higher in HEK293 cells transfected with both TLR2 and TLR6 than in HEK293 cells transfected with only TLR2. The deletion mutant TLR2ΔS40-I64 (a TLR2 mutant with a deletion of the region of Ser40 to Ile64) failed to activate NF-κB in response to FSL-1. The deletion mutant TLR2ΔC30-S39 induced NF-κB reporter activity, but the level of activity was significantly reduced compared with that induced by wild-type TLR2. A TLR2 point mutant with a substitution of Glu178 to Ala (TLR2E178A), TLR2E180A, TLR2E190A, and TLR2L132E induced NF-κB activation when stimulated with FSL-1, M. salivarium lipoproteins, and Staphylococcus aureus peptidoglycans, but TLR2L107E, TLR2L112E (a TLR2 point mutant with a substitution of Leu112 to Glu), and TLR2L115E failed to induce NF-κB activation, suggesting that these residues are essential for their signaling. Flow cytometric analysis demonstrated that TLR2L115E, TLR2L112E, and TLR2ΔS40-I64 were expressed on the cell surface of the transfectants as wild-type TLR2 and TLR2E190A were. In addition, these mutants, except for TLR2E180A, functioned as dominant negative form of TLR2. This study strongly suggested that the extracellular region of Ser40-Ile64 and leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of TLR2 are involved in the recognition of mycoplasmal diacylated lipoproteins and lipopeptides and in the recognition of S. aureus peptidoglycans.