Masahito Hashimoto

Bacterial fimbriae activate human peripheral blood monocytes utilizing TLR2, CD14 and CD11a/CD18 as cellular receptors

Bacterial fimbriae are associated with a specific adherence factor, adhesin, in their microbial etiology. Porphyromonas gingivalis, as an anaerobic Gram‐negative periodontopathogenic organism, is known to possess fimbriae on its cell surfaces. In this study, we demonstrated that P.u2009gingivalis fimbriae and an active synthetic peptide composed of residues 69u2009–u200973 of thefimbrial subunit protein, ALTTE, induced IL‐6 mRNA expression and cytokine production, p38 mitogen‐activated protein (MAP) kinase phosphorylation, and NF‐κB activation in human peripheral blood monocytes. P.u2009gingivalis fimbriae and ALTTE also induced IL‐6 production via human Toll‐like receptor (TLR) 2, CD14, and CD11a/CD18 (LFA‐1) molecules on human monocytes. These results suggest that P.u2009gingivalis fimbriae and these degraded peptides may play an important role in the inflamed gingival and periodontal tissues seen in the development and progression of periodontal diseases.

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.

Chemical structure and immunobiological activity of lipid A from Prevotella intermedia ATCC 25611 lipopolysaccharide.

The novel chemical structure and immunobiological activities of Prevotella intermedia ATCC 25611 lipid A were investigated. A lipopolysaccharide (LPS) preparation of P. intermedia was extracted using a phenol–chloroform–petroleum ether method, after which its purified lipid A was prepared by weak acid hydrolysis followed by chromatographic separations. The lipid A structure was determined by mass spectrometry and nuclear magnetic resonance to be a diglucosamine backbone with a phosphate at the 4‐position of the non‐reducing side sugar, as well as five fatty acids containing branched long chains. It was similar to that of Bacteroides fragilis and Porphyromonas gingivalis, except for the phosphorylation site. P. intermedia lipid A induced weaker cytokine production and NF‐κB activation in murine cells via Toll‐like receptor (TLR) 4 as compared to Escherichia coli synthetic lipid A (compound 506). Our results indicate that P. intermedia lipid A activates cells through a TLR4‐dependent pathway similar to E. coli‐type lipid A, even though these have structural differences.

Cell activation by monosaccharide lipid A analogues utilizing Toll-like receptor 4

Lipid A is the bioactive centre of lipopolysaccharide (LPS), and its properties exhibit various endotoxic and biological effects toward host cells. We examined whether Toll‐like receptors (TLRs) are mediated by the signals from various synthetic acylated derivatives of d‐glucosamine monophosphate. All test synthetic monosaccharide lipid A analogues similar to acylated β‐(1‐6)‐d‐glucosamine disaccharide bisphosphates, such as Escherichia coli‐type lipid A (compound 506) and its precursor (compound 406), clearly induced nuclear factor (NF)‐κB activation in Ba/F3 cells expressing murine TLR4 and its accessory protein MD‐2 (Ba/mTLR4/mMD‐2), but no induction was found in those expressing murine TLR2 (Ba/mTLR2). Compound 411, the non‐reducing sugar moiety of compound 506, exhibited interleukin‐8 (IL‐8) and tumour necrosis factor‐α (TNF‐α)‐producing activities in human peripheral blood mononuclear cells (PBMC), whereas compound 401, the reducing moiety of compounds 506 and 406, and Gifu lipid A‐46 (GLA‐46), the non‐reducing moiety of compound 406, induced no production of IL‐8 and TNF‐α, which was similar to the findings for compound 406. Among the synthetic triacylated monosaccharide lipid A analogues, some compounds with three tetradecanoyl (C14) groups or that included a dodecanoyl (C12) group were more active toward murine and human cells than were other analogues with a decanoyl (C10) or hexadecanoyl (C16) group. Furthermore, IL‐8 production in PBMC stimulated with the active monosaccharide lipid A analogues as well as compound 506 was clearly inhibited by the monoclonal antibody to human TLR4. These findings suggest that monosaccharide lipid A analogues similar to disaccharide lipid As are capable of activating both murine and human cells through TLR4.

Treponemal glycoconjugate inhibits Toll-like receptor ligand-induced cell activation by blocking LPS-binding protein and CD14 functions

Spirochetes are well‐known as causative agents of various chronic infectious diseases. Glycolipids of small‐sized Treponema spirochetes have been shown to exhibit immunostimulating activities. In this study, we found that a glycoconjugate preparation obtained from Treponema medium (Tm‐Gp), an intermediate‐sized oral Treponema seen in subgingival plaque from patients with chronic periodontal diseases, diminished the LPS‐induced activation of a human monocytic cell line, while TNF‐α‐induced activation was unaffected. NF‐κB activation in Ba/F3 cells expressing murine Toll‐like receptor (TLR) 4 and MD‐2 was inhibited by Tm‐Gp in a CD14/LPS‐binding protein (LBP)‐dependent manner when stimulated with LPS or its active center lipid A but not whenstimulated with Taxol. Tm‐Gp blocked the binding of LPS to immobilized CD14 and LBP and inhibited nitric oxide (NO) production by a murine Mϕ cell line following stimulation with peptidoglycan and LPS in the presence of FBS, while NO production in response poly (I:C) RNA or CpG DNA remained unaffected. The inhibitory effects of Tm‐Gp seem to be attributable to the lipophilic portion of the glycoconjugate. These results indicate that T. medium contains a glycoconjugate possessing an inhibitory effect on TLR‐mediated cell activation through interaction with LBP and CD14.

A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: A fundamental structure of the hydrophilic part

Previously, we showed that quantitatively minor several glycolipids only less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possessed cytokine-inducing activity, whereas the major component (over 90%) did not [Suda et al. (1995) FEMS Immun Med Microbiol 12:97–112]. The major inactive component was shown to have the chemical structure as was proposed for the LTA by Fischer [Hashimoto et al. (1997) J Biochem 121:779–86], suggesting that so-called LTA is not a cytokine-inducing component in the Gram-positive bacteria. In the present paper, the structure of the hydrophilic part of one of the cytokine-inducing glycolipid tentatively named GL4 is elucidated. GL4 was first subjected to hydrolysis with aqueous HF to give a polysaccharide and a mixture of low molecular weight products. The polysaccharide was composed mainly of highly branching mannan as concluded from NMR and MS analyses of its acetolysis products. The low molecular weight products consisted of phosphate and glycerol, suggesting the presence of a poly(glycerophosphate) structure in the original GL4. From these observations, the hydrophilic part of GL4 was shown to consist of mannose-rich polysaccharide and poly(glycerophosphate), the latter being bound to the former by a phosphodiester linkage.

Treponema medium Glycoconjugate Inhibits Activation of Human Gingival Fibroblasts Stimulated with Phenol-Water Extracts of Periodontopathic Bacteria:

Oral treponemes are well-known as causative agents of periodontal diseases; however, the details have not been fully clarified. Here, we examined the effects of Treponema medium glycoconjugate on the activation of human gingival fibroblasts using phenol-water extracts from Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans. The phenol-water extracts activated human gingival fibroblasts to mediate IL-8 production, as well as IL-8 mRNA expression, phosphorylation of p38 mitogen-activated protein kinase, and expression of intercellular adhesion molecule-1. T. medium glycoconjugate exhibited no activation of human gingival fibroblasts, while phenol-water extract-induced activation of human gingival fibroblasts was clearly inhibited by T. medium glycoconjugate. Furthermore, binding of biotinylated phenol-water extracts to CD14 in the presence of LPS-binding protein was blocked with T. medium glycoconjugate. These results suggest that T. medium glycoconjugate has an inhibitory effect on host cell activation by periodontopathic bacteria caused by binding to CD14- and LPS-binding protein.

Exo-Vivo Cytokine Inducing Activity of Hydrophobized Poly(Maleic Acid-Alt-7, 12-Dioxaspiro-[5,6]-Dodec-9-Ene) [Poly(MA-CDA)]

In order to confirm the immuno-stimulating activity of fractionated and hydrophobized poly(maleic acid-alt-7,12-dioxaspiro-[5,61-dodec-9-ene) [poly(MA-CDA)], we evaluated exo-vivo cytokine inducing activity of the polymers using peripheral whole blood cells. The highest inducing activity of interleukin 6 (IL-6) and tumor necrosis factor-α(TNF-α) was found with the polymer with a molecular weight of ~5,000 and containing 10 mol% aniline moiety.