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J Dent Res 84(5):456-461, 2005
© 2005 International and American Associations for Dental Research

RESEARCH REPORT
Biological

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

Y. Asai, Y. Ohyama, Y. Taiji, Y. Makimura, R. Tamai, M. Hashimoto, and T. Ogawa*

Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi, Mizuho, Gifu 501-0296, Japan;

* corresponding author, tomo527{at}dent.asahi-u.ac.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.

KEY WORDS: Treponema medium • glycoconjugate • periodontopathic bacteria • human gingival fibroblasts


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Periodontitis is a chronic inflammatory disease characterized by destruction of gingival connective tissues and resorption of alveolar bone. It is widely recognized that more than 400 species of bacteria colonize the subgingival area, of which Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum subsp. nucleatum, and Actinobacillus actinomycetemcomitans are widely accepted as major pathogenic organisms, since they have been specifically isolated from the periodontal pockets of patients with various types of periodontal diseases (Moore, 1987; Slots et al., 1991; Socransky et al., 1998). In addition, several cell-surface components from Gram-negative periodontopathic bacteria—such as LPS, fimbriae, glycoprotein, and outer membrane protein—have been proposed to be inducers of the synthesis of inflammatory mediators by host cells, and are thought to play pivotal roles in bone resorption caused by periodontal lesions (Chiang et al., 1999; Sugawara et al., 2001; Ogawa et al., 2002b; Asakawa et al., 2003).

Spirochetes are Gram-negative, anaerobic, motile, and helical rods that are associated with various chronic infectious diseases. Oral spirochetes, generally belonging to the genus Treponema, have been found in subgingival plaque from patients with periodontal diseases (Riviere et al., 1991; Dewhirst et al., 2000). Among them, Treponema medium, an intermediate-sized treponeme, has been isolated from the subgingival plaque of patients with adult periodontitis (Umemoto et al., 1997; Nakazawa et al., 2003). Further, using a real-time PCR assay, we recently showed that the number of T. medium organisms was increased in subgingival plaque samples from deep periodontal pockets (Asai et al., 2002). However, the role of T. medium in the formation of periodontal lesions has not been fully examined. Recently, we demonstrated that a glycoconjugate from T. medium exhibited inhibitory actions against LPS- and peptide-glycan-mediated monocyte/macrophage activation, by blocking the functions of CD14- and LPS-binding protein in serum (Asai et al., 2003).

The purpose of this study was to evaluate the effect of T. medium glycoconjugate on the activation of human gingival fibroblasts by the use of phenol-water extracts from various Gram-negative periodontopathic bacteria. We also examined the effect of T. medium glycoconjugate on the interaction between phenol-water extracts and CD14/LPS-binding protein.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria and Preparation of Bacterial Phenol-Water Extracts
T. medium ATCC 700293T organisms were grown anaerobically in trypticase-yeast extract-gelatine-volatile fatty acids-rabbit serum broth containing 5% rabbit serum, and T. medium glycoconjugate was extracted as described previously (Hashimoto et al., 2003). P. gingivalis 381, P. intermedia ATCC 25611T, and F. nucleatum subsp. nucleatum ATCC 25586T were grown anaerobically at 37°C in Gifu anaerobic medium broth (Nissui, Tokyo, Japan), supplemented with 5 µg/mL of hemin and 0.5 µg/mL of vitamin K3. A. actinomycetemcomitans Y4 was also grown at 37°C in a 5% (v/v) CO2 atmosphere in Todd-Hewitt broth (Difco Laboratories, Detroit, MI, USA) supplemented with 1% yeast extract. The bacterial cells were harvested by centrifugation, washed 3 times with phosphate-buffered saline (PBS), and freeze-dried. Phenol-water extracts from P. gingivalis, P. intermedia, F. nucleatum subsp. nucleatum, and A. actinomycetemcomitans were prepared as previously described (Westphal and Jann, 1965), and then treated by enzymatic digestion with DNase and RNase, followed by proteinase K. To remove contaminated proteins, we subjected the digest again to phenol-water extraction. LPS from the non-oral bacterium Escherichia coli O55:B5 (List Biological Lab., Inc. Campbell, CA, USA) was used as a control stimulant.

Cells
Human gingival fibroblasts were prepared from clinically normal gingival tissues according to a method similar to that described previously (Ogawa et al., 2002a). The tissue sample was collected from subjects who provided written informed consent under a protocol approved by the Institutional Review Board of Asahi University. The cells were cultured in {alpha}-MEM (Sigma Chemical Co., St. Louis, MO, USA) containing 10% FBS (Sigma Chemical Co.), 50 µg/mL of gentamicin, and 50 ng/mL of amphotericin B (Sigma Chemical Co.) at 37°C in a 5% (v/v) CO2 atmosphere and were used for the assay at the 5th and 13th passages.

Cytokine Assay
Human gingival fibroblasts (2 x 104 cells) were plated in 96-well cell culture plates (BD Biosciences, San Jose, CA, USA). After an overnight incubation, the confluent monolayer was stimulated with the indicated doses of each phenol-water extract with or without 25 µg/mL of T. medium glycoconjugate in {alpha}-MEM containing 5% FBS, or 500 ng/mL of CD14 and 50 ng/mL of LPS-binding protein at 37°C for 24 hrs. IL-8 production was measured in the culture supernatants by means of a commercial ELISA kit system (GT, Cambridge, MA, USA). The assays were performed according to the manufacturer’s instructions, and the results were determined based on a standard curve prepared for each assay. We used a commercial kit (Roche, Mannheim, Germany) to determine cell viability by measuring the leakage of lactate dehydrogenase (LDH) from the cells into the culture supernatant.

RT-PCR
Human gingival fibroblasts (5 x 105 cells/mL) were plated in 60-mm cell culture dishes. After an overnight incubation, the confluent monolayer was stimulated with each phenol-water extract with or without 25 µg/mL of T. medium glycoconjugate in {alpha}-MEM containing 5% FBS for 4 hrs at 37°C. Total RNA was extracted by a single-step extraction method with TRIzol (Nippon Gene, Toyama, Japan). RT-PCR was performed, and total RNA was extracted by means of an RNA PCR kit (Takara Biomedicals, Shiga, Japan), with sense and antisense oligonucleotides specific for IL-8 or ß-actin used as primers. As a negative control, a PCR reaction was performed without an RT sample. PCR products were detected by electrophoresis on a 1% agarose gel.

Western Blotting
Human gingival fibroblasts (5 x 105 cells/mL) were plated in 60-mm cell culture dishes (BD Biosciences). After an overnight incubation, the confluent monolayer was stimulated with the indicated doses of each phenol-water extract with or without 25 µg/mL of T. medium glycoconjugate in {alpha}-MEM containing 5% FBS for 30 min at 37°C. After being washed 3 times with PBS, the cells underwent lysis in the sample buffer under reducing conditions, and were subjected to SDS-PAGE (10% acrylamide) and Western blotting. Phosphorylated p38 mitogen-activated protein kinase (MAPK) and p38 MAPK were detected by means of a PhosphoPlus® p38 MAP Kinase Antibody Kit (Cell Signaling Technology, Beverly, MA, USA) according to the manufacturer’s protocol.

Flow Cytometry
Human gingival fibroblasts (5 x 105 cells/mL) were plated in 60-mm cell culture dishes. After an overnight incubation, the confluent monolayer was incubated with fluorescein isothiocyanate (FITC)-conjugated anti-human CD14 (BD Biosciences) or mouse IgG1{kappa} (BD Biosciences) at 25°C for 15 min. For intercellular adhesion molecule-1 (ICAM-1) detection, the cells were stimulated with the indicated doses of each phenol-water extract with or without 25 µg/mL of T. medium glycoconjugate in {alpha}-MEM containing 5% FBS for 24 hrs at 37°C. The cells were then treated with trypsin/EDTA for 10 min, and detached cells were incubated with mouse anti-human ICAM-1 (eBioscience, San Diego, CA, USA) or mouse IgG1{kappa} (eBioscience) at 25°C for 15 min. After being washed with PBS containing 0.1% NaN3, the cells were incubated with FITC-conjugated goat anti-mouse IgG (Dako, Glostrup, Denmark) at 25°C for 15 min, then washed with PBS containing 0.1% NaN3 and fixed with 1% paraformaldehyde. Stained cells were analyzed by a FACS Calibur with Cell Quest software (BD Biosciences).

Binding Assay
Phenol-water extracts and E. coli LPS were biotinylated as previously described (Luk et al., 1995). Binding of 1 µg/mL of biotinylated phenol-water extracts and E. coli LPS to CD14 adsorbed onto the ELISA plates was detected as previously described (Asai et al., 2003). Binding was carried out for 30 min at 37°C in 0.1% BSA in PBS containing 50 ng/mL of LPS-binding protein, with or without 25 µg/mL of T. medium glycoconjugate. After the plate was washed with PBS containing 0.1% Tween 20, the bound biotinylated phenol-water extracts and E. coli LPS were detected with streptavidin-peroxidase and 3,3',5,5'-tetramethylbenzidine (TMB) substrate (KPL, Guildford, UK).

Statistical Analysis
Data were analyzed by a one-way analysis of variance (ANOVA) according to the Bonferroni or Dunn method, and the results are presented as the mean ± standard error of the mean (SEM). When an individual result is presented, it is representative of at least 3 independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the cell-activating capacities of the tested phenol-water extracts from Gram-negative periodontopathic bacteria, we first examined IL-8 production by human gingival fibroblasts, which exhibited no or little CD14 expression, stimulated with phenol-water extracts from P. gingivalis, P. intermedia, F. nucleatum subsp. nucleatum, or A. actinomycetemcomitans (Fig. 1AGo). The IL-8 production-inducing activity of phenol-water extracts from A. actinomycetemcomitans was detected at 1 ng/mL, which was comparable with that of E. coli LPS and significantly stronger than that of phenol-water extracts from P. gingivalis, P. intermedia, and F. nucleatum subsp. nucleatum (Fig. 1BGo). Those later 3 phenol-water extracts showed IL-8 production at 100 ng/mL, with the maximum observed in human gingival fibroblasts stimulated with phenol-water extracts from F. nucleatum subsp. nucleatum. In contrast, T. medium glycoconjugate showed no activation of or lethality toward human gingival fibroblasts at the highest concentration tested (100 µg/mL) (data not shown).



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  		Figure 1. CD14 expression and IL-8 production in human gingival fibroblasts. (A) Cell-surface expression of CD14 on human gingival fibroblasts was determined with a specific antibody (bold line) or its isotype control (thin line), as described in MATERIALS & METHODS. (B) IL-8 production in human gingival fibroblasts stimulated with various periodontopathic bacterial phenol-water extracts. The cells were stimulated with the indicated doses of the phenol-water extracts or with E. coli LPS in the presence of 5% FBS. The results are shown as the mean ± SEM. Representative results from 3 independent experiments are presented.

 
We also examined the effects of T. medium glycoconjugate on the activation of human gingival fibroblasts stimulated with the phenol-water extracts. Human gingival fibroblasts were stimulated with phenol-water extracts from P. gingivalis (10 µg/mL), P. intermedia (10 µg/mL), F. nucleatum subsp. nucleatum (10 µg/mL), A. actinomycetemcomitans (10 ng/mL), or E. coli LPS (10 ng/mL) in the presence or absence of T. medium glycoconjugate (25 µg/mL) at 37°C for 24 hrs, and the culture supernatants were measured for IL-8 production (Fig. 1BGo). IL-8 production induced by each of the phenol-water extracts was significantly inhibited by T. medium glycoconjugate (Fig. 2AGo). To evaluate whether T. medium glycoconjugate abolished the secreted IL-8 in the culture supernatants, we next examined IL-8 mRNA expression by, and p38 MAPK phosphorylation in, human gingival fibroblasts. T. medium glycoconjugate clearly inhibited all phenol-water extract-mediated IL-8 mRNA expressions (Fig. 2BGo). In contrast, A. actinomycetemcomitans phenol-water extract- and E. coli LPS-induced p38 MAPK phosphorylation was almost completely inhibited by T. medium glycoconjugate, but the inhibition was weaker in phenol-water extracts from P. gingivalis, P. intermedia, and F. nucleatum subsp. nucleatum. These differences seem to be due to the concentrations of phenol-water extracts and the stimulation times. Together, these results indicated that the T. medium glycoconjugate-mediated inhibition was due to a modulation upstream of the signaling cascade in human gingival fibroblasts initiated by phenol-water extracts. Local induction of cell adhesion molecules such as ICAM-1 is one of the mechanisms involved in initiating and potentiating inflammatory and immunological responses (Ebnet et al., 1997). Sonic extracts prepared from P. gingivalis and P. intermedia have been reported to up-regulate ICAM-1 expression on the surfaces of human gingival fibroblasts (Hayashi et al., 1994). Therefore, we examined ICAM-1 expression by human gingival fibroblasts stimulated with phenol-water extracts and found that they, as well as E. coli LPS, up-regulated ICAM-1 expression in human gingival fibroblasts, which was clearly suppressed by T. medium glycoconjugate (Fig. 2DGo).



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  		Figure 2. Inhibitory effects of T. medium glycoconjugate on phenol-water extract-induced activation of human gingival fibroblasts. Cells were stimulated with the phenol-water extracts or with E. coli LPS with (closed bar) or without (open bar) T. medium glycoconjugate in the presence of 5% FBS. (A) The results of IL-8 production are presented as the mean ± SEM. Data were analyzed by a one-way analysis of variance (ANOVA) with the Bonferroni or Dunn method, and asterisks indicate statistically significant (p < 0.01) inhibition by T. medium glycoconjugate. Representative results from 3 independent experiments are presented. (B) IL-8 mRNA expression. The ß-actin gene was assayed as a positive control, and PCR products of non-RT samples were used as a negative control. (C) p38 MAPK phosphorylation (pp38). (D) The cell-surface expression of ICAM-1 on human gingival fibroblasts was determined with a specific antibody (bold line) or its isotype control (thin line) as described in MATERIALS & METHODS. Representative results from 3 independent experiments are presented in B-D.

 
T. medium glycoconjugate-induced inhibitory action is dependent on CD14- and LPS-binding protein (Asai et al., 2003). Consequently, using an IL-8 production assay, we examined the CD14/LPS-binding protein dependency of each phenol-water extract. Human gingival fibroblasts were stimulated with the indicated doses of each phenol-water extract in culture medium with or without CD14- and LPS-binding protein at 37°C for 24 hrs, and the culture supernatants were measured for IL-8 production. The phenol-water extracts as well as E. coli LPS definitely induced IL-8 production in the presence of CD14/LPS-binding protein, but not in its absence (Fig. 3Go). In addition, IL-8 production was significantly inhibited by T. medium glycoconjugate. We also demonstrated that T. medium glycoconjugate blocked the binding of biotinylated phenol-water extracts, as well as biotinylated E. coli LPS to immobilized CD14 in the presence of LPS-binding protein (Fig. 4Go).



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  		Figure 3. Correlations between CD14/LPS-binding protein and activation of human gingival fibroblasts induced by phenol-water extracts. Cells were stimulated with the phenol-water extracts or with E. coli LPS with (closed bar) or without (open bar) T. medium glycoconjugate in the presence or absence of CD14- and LPS-binding protein. The results are presented as the mean ± SEM. Data were analyzed by a one-way analysis of variance (ANOVA) with the Bonferroni or Dunn method, and an asterisk indicates statistically significant inhibition by T. medium glycoconjugate (p < 0.01). Representative results from 3 independent experiments are presented.

 


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  		Figure 4. Inhibition of phenol-water extract binding to immobilized CD14 by T. medium glycoconjugate. Biotinylated phenol-water extract or E. coli LPS with or without T. medium glycoconjugate were added to CD14- or BSA-coated wells in the presence or absence of LPS-binding protein. The binding of biotinylated phenol-water extract or E. coli LPS was detected with the use of HRP-conjugated streptavidin. Representative results from 3 independent experiments are presented.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Tissue destruction that is associated with periodontal disease may occur as an outcome of the host response to exclude various bacteria from the gingival sulcus. Further, activation of host cells to secrete endogenous inflammatory mediators and cytokines may play important roles in local amplification of immune responses, as well as in the breakdown of periodontal tissues (Page, 1991; Ranney, 1991). IL-8, a CXC chemokine produced by human gingival fibroblasts, is considered to be an important factor that initiates inflammatory reactions in gingival tissues (Takashiba et al., 1992). It has been detected in the gingival tissues of patients with periodontal disease, and the level of IL-8 mRNA has also been demonstrated to correspond to disease severity (Tonetti et al., 1994). In the present study, we prepared phenol-water extracts from periodontopathic bacteria and examined their IL-8-producing effects with human gingival fibroblasts (Fig. 1Go). It has been generally regarded that a phenol-water extract from Gram-negative bacteria is an LPS; however, we recently showed that an unknown contaminant contained in a phenol-water extract of P. gingivalis, so-called P. gingivalis LPS, exhibited a powerful cell activation (Ogawa et al., 2002a), which indicated that the phenol-water extract from Gram-negative bacteria is not necessarily LPS alone. Therefore, we termed the phenol-water extracts from periodontopathic bacteria as ‘phenol-water extracts’ and not LPS in this study.

Several spirochetal cell-surface components have been reported to exert immunobiological effects toward host cells. Among them, lipoproteins from Treponema pallidum and LPS from Leptospira interrogans showed cell-activating capabilities (Lien et al., 1999; Werts et al., 2001), while glycolipids from the oral treponemes Treponema maltophilum and Treponema brennaborense also exhibited NF-{kappa}B activation and cytokine production in host cells (Opitz et al., 2001). However, we recently found that T. medium glycoconjugate blocked CD14/LPS-binding protein functions, resulting in inhibition of E. coli LPS-induced cell activation (Asai et al., 2003). It was previously demonstrated that human gingival fibroblasts possessed membrane CD14 at various levels, and human gingival fibroblasts, which expressed no or low membrane CD14, recognized LPS through soluble CD14-dependent mechanisms (Sugawara et al., 1998). Since we used no or little membrane CD14-expressing human gingival fibroblasts (Fig. 1AGo), the phenol-water extract-induced cell activation seems to be dependent on soluble CD14 rather than on membrane CD14. The present results demonstrated that the inhibitory effects of T. medium glycoconjugate were effective toward phenol-water extracts obtained from various periodontopathic bacteria (Fig. 2Go), and that phenol-water extract-induced IL-8 production was CD14/LPS-binding protein-dependent (Fig. 3Go). Furthermore, we showed that T. medium glycoconjugate blocked the binding of phenol-water extracts to CD14 in the presence of LPS-binding protein (Fig. 4Go). It was previously demonstrated that higher levels of CD14 in gingival crevicular fluid and a greater number of sites containing CD14 were associated with fewer deep periodontal pockets (Jin and Darveau, 2001). Those results indicated that CD14 has a protective role in bacterially induced periodontal destruction, while T. medium glycoconjugate, which has an ability to inhibit CD14/LPS-binding protein function, may be associated with the progression of periodontal diseases.

Taken together, the present and previous results demonstrate that T. medium glycoconjugate inhibits the activation of human gingival fibroblasts stimulated by the cell components of periodontopathic bacteria, indicating that T. medium glycoconjugate may act as a bacterial modulator against host immune responses in periodontal lesions.


   ACKNOWLEDGMENTS
 
This work was supported by a grant-in-aid for scientific research from the Japan Society for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology (No. 15791052) and by the Miyata Research Foundation (A) of Asahi University (No. 04019). We thank Mr. Mark Benton for his critical reading of the manuscript.

Received June 3, 2004; Last revision January 6, 2005; Accepted January 12, 2005


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Asai Y, Jinno T, Igarashi H, Ohyama Y, Ogawa T (2002). Detection and quantification of oral treponemes in subgingival plaque by real-time PCR. J Clin Microbiol 40:3334–3340.[Abstract/Free Full Text]

Asai Y, Hashimoto M, Ogawa T (2003). Treponemal glycoconjugate inhibits Toll-like receptor ligand-induced cell activation by blocking LPS-binding protein and CD14 functions. Eur J Immunol 33:3196–3204.[ISI][Medline]

Asakawa R, Komatsuzawa H, Kawai T, Yamada S, Gonçalves RB, Izumi S, et al. (2003). Outer membrane protein 100, a versatile virulence factor of Actinobacillus actinomycetemcomitans. Mol Microbiol 50:1125–1139.[ISI][Medline]

Chiang CY, Kyritsis G, Graves DT, Amar S (1999). Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide. Infect Immun 67:4231–4236.[Abstract/Free Full Text]

Dewhirst FE, Tamer MA, Ericson RE, Lau CN, Levanos VA, Boches SK, et al. (2000). The diversity of periodontal spirochetes by 16S rRNA analysis. Oral Microbiol Immunol 15:196–202.[ISI][Medline]

Ebnet K, Brown KD, Siebenlist UK, Simon MM, Shaw S (1997). Borrelia burgdorferi activates nuclear factor-kappa B and is a potent inducer of chemokine and adhesion molecule gene expression in endothelial cells and fibroblasts. J Immunol 158:3285–3292.[Abstract]

Hashimoto M, Asai Y, Jinno T, Adachi S, Kusumoto S, Ogawa T (2003). Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293. Eur J Biochem 270:2671–2679.[ISI][Medline]

Hayashi J, Saito I, Ishikawa I, Miyasaka N (1994). Effects of cytokines and periodontopathic bacteria on the leukocyte function-associated antigen 1/intercellular adhesion molecule 1 pathway in gingival fibroblasts in adult periodontitis. Infect Immun 62:5205–5212.[Abstract/Free Full Text]

Jin L, Darveau RP (2001). Soluble CD14 levels in gingival crevicular fluid of subjects with untreated adult periodontitis. J Periodontol 72:634–640.[ISI][Medline]

Lien E, Sellati TJ, Yoshimura A, Flo TH, Rawadi G, Finberg RW, et al. (1999). Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J Biol Chem 274:33419–33425.[Abstract/Free Full Text]

Luk JM, Kumar A, Tsang R, Staunton D (1995). Biotinylated lipopolysaccharide binds to endotoxin receptor in endothelial and monocytic cells. Anal Biochem 232:217–224.[ISI][Medline]

Moore WE (1987). Microbiology of periodontal disease. J Periodontal Res 22:335–341.[ISI][Medline]

Nakazawa F, Hoshino E, Fukunaga M, Jinno T, Asai Y, Yamamoto H, et al. (2003). Amended biochemical characteristics and phylogenetic position of Treponema medium. Oral Microbiol Immunol 18:127–130.[ISI][Medline]

Ogawa T, Asai Y, Hashimoto M, Takeuchi O, Kurita T, Yoshikai Y, et al. (2002a). Cell activation by Porphyromonas gingivalis lipid A molecule through Toll-like receptor 4- and myeloid differentiation factor 88-dependent signaling pathway. Int Immunol 14:1325–1332.[Abstract/Free Full Text]

Ogawa T, Asai Y, Hashimoto M, Uchida H (2002b). Bacterial fimbriae activate human peripheral blood monocytes utilizing TLR2, CD14 and CD11a/CD18 as cellular receptors. Eur J Immunol 32:2543–2550.[ISI][Medline]

Opitz B, Schröder NW, Spreitzer I, Michelsen KS, Kirschning CJ, Hallatschek W, et al. (2001). Toll-like receptor-2 mediates Treponema glycolipid and lipoteichoic acid-induced NF-kappaB translocation. J Biol Chem 276:22041–22047.[Abstract/Free Full Text]

Page RC (1991). The role of inflammatory mediators in the pathogenesis of periodontal disease. J Periodontal Res 26:230–242.[ISI][Medline]

Ranney RR (1991). Immunologic mechanisms of pathogenesis in periodontal diseases: an assessment. J Periodontal Res 26:243–254.[ISI][Medline]

Riviere GR, Weisz KS, Simonson LG, Lukehart SA (1991). Pathogen-related spirochetes identified within gingival tissue from patients with acute necrotizing ulcerative gingivitis. Infect Immun 59:2653–2657.[Abstract/Free Full Text]

Slots J, Rams TE, Feik D, Taveras HD, Gillespie GM (1991). Subgingival microflora of advanced periodontitis in the Dominican Republic. J Periodontol 62:543–547.[ISI][Medline]

Socransky SS, Haffajee AD, Cugini MA, Smith C, Kent RL Jr (1998). Microbial complexes in subgingival plaque. J Clin Periodontol 25:134–144.[ISI][Medline]

Sugawara S, Sugiyama A, Nemoto E, Rikiishi H, Takada H (1998). Heterogeneous expression and release of CD14 by human gingival fibroblasts: characterization and CD14-mediated interleukin-8 secretion in response to lipopolysaccharide. Infect Immun 66:3043–3049.[Abstract/Free Full Text]

Sugawara S, Yang S, Iki K, Hatakeyama J, Tamai R, Takeuchi O, et al. (2001). Monocytic cell activation by nonendotoxic glycoprotein from Prevotella intermedia ATCC 25611 is mediated by toll-like receptor 2. Infect Immun 69:4951–4957.[Abstract/Free Full Text]

Takashiba S, Takigawa M, Takahashi K, Myokai F, Nishimura F, Chihara T, et al. (1992). Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1beta or tumor necrosis factor alpha. Infect Immun 60:5253–5258.[Abstract/Free Full Text]

Tonetti MS, Imboden MA, Gerber L, Lang NP, Laissue J, Mueller C (1994). Localized expression of mRNA for phagocyte-specific chemotactic cytokines in human periodontal infections. Infect Immun 62:4005–4014.[Abstract/Free Full Text]

Umemoto T, Nakazawa F, Hoshino E, Okada K, Fukunaga M, Namikawa I (1997). Treponema medium sp. nov., isolated from human subgingival dental plaque. Int J Syst Bacteriol 47:67–72.[Abstract/Free Full Text]

Werts C, Tapping RI, Mathison JC, Chuang TH, Kravchenko V, Saint Girons I, et al. (2001). Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat Immunol 2:346–352.[ISI][Medline]

Westphal O, Jann K (1965). Bacterial lipopolysaccharides: extraction with phenol-water and further applications to the procedure. In: Methods in carbohydrate chemistry. Vol. 5. Whistler RL, editor. New York, NY: Academic Press, pp. 83–91.





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