Hiromasa Yoshie

Toll-Like Receptors Confer Responsiveness to Lipopolysaccharide from Porphyromonas gingivalis in Human Gingival Fibroblasts

ABSTRACT Gingival fibroblasts produce proinflammatory cytokines in response to lipopolysaccharide (LPS) from periodontopathic bacteria. Recently it has become evident that the human homologue of DrosophilaToll can transduce intracellular signaling by LPS stimulation. Toll-like receptors (TLRs) have been identified in myeloid cells; however, their role in nonmyeloid cells such as gingival fibroblasts has not been fully elucidated. Here, we report that human gingival fibroblasts constitutively express TLR2 and TLR4 and that their levels of expression are increased by stimulation with LPS fromPorphyromonas gingivalis. Upregulated expression of interleukin-6 gene and protein in fibroblasts stimulated with LPS is inhibited by anti-TLR4 antibody. These findings suggest that TLRs may confer responsiveness to LPS in gingival fibroblasts.

FGF-2 Stimulates Periodontal Regeneration Results of a Multi-center Randomized Clinical Trial

The efficacy of the local application of recombinant human fibroblast growth factor-2 (FGF-2) in periodontal regeneration has been investigated. In this study, a randomized, double-blind, placebo-controlled clinical trial was conducted in 253 adult patients with periodontitis. Modified Widman periodontal surgery was performed, during which 200 µL of the investigational formulation containing 0% (vehicle alone), 0.2%, 0.3%, or 0.4% FGF-2 was administered to 2- or 3-walled vertical bone defects. Each dose of FGF-2 showed significant superiority over vehicle alone (p < 0.01) for the percentage of bone fill at 36 wks after administration, and the percentage peaked in the 0.3% FGF-2 group. No significant differences among groups were observed in clinical attachment regained, scoring approximately 2 mm. No clinical safety problems, including an abnormal increase in alveolar bone or ankylosis, were identified. These results strongly suggest that topical application of FGF-2 can be efficacious in the regeneration of human periodontal tissue that has been destroyed by periodontitis.

Elevated humoral immune response to heat shock protein 60 (hsp60) family in periodontitis patients

The presence of antibodies to the 60‐kD human and Porphyromonas gingivalis GroEL hsp60 in the sera and inflamed gingival tissues of periodontitis patients was examined. In order to obtain the antigens, recombinant plasmids carrying human hsp60 and P. gingivalis GroEL genes were constructed and expressed as histidine‐tagged recombinant proteins. Immunoreactivities of these proteins were confirmed by MoAbs specific to mammalian hsp60 and cross‐reactive with both mammalian and bacterial hsp60. Western blot analysis clearly demonstrated that the number of periodontitis patients showing a positive response to P. gingivalis GroEL was higher than the number of periodontally healthy subjects. Furthermore, anti‐P. gingivalis GroEL antibody was detected in all samples of gingival tissue extracts. For human hsp60, a higher frequency of seropositivity was found in the periodontitis patients than in the healthy subjects. In addition, the periodontitis patients demonstrated stronger reactivity compared with the healthy subjects. Quantitative analysis of serum antibodies by ELISA also demonstrated that the levels of antibodies in the sera of patients were significantly higher than those of control subjects. In the gingival tissue extracts, seven out of 10 patients demonstrated a positive response to human hsp60 and tso of these demonstrated strong positivity. Affinity‐purified serum antibodies to human hsp60 and P. gingivalis GroEL from selected patients reacted with P. gingivalis GroEL and human hsp60, respectively, suggesting cross‐reactivity of antibodies. These results suggest that molecular mimicry between GroEL of the periodontopathic bacterium P. gingivalis and autologous human hsp60 may play some role in immune mechanisms in periodontitis.

Polymorphisms in the IL-6 receptor (IL-6R) gene: strong evidence that serum levels of soluble IL-6R are genetically influenced.

We have investigated the association of the recently identified IL6R polymorphisms with the serum levels of soluble IL-6 receptor (sIL-6R). sIL-6R is generated by shedding of the membrane-bound receptor (IL-6Rα) or alternative mRNA splicing. In total, 115 healthy volunteers were genotyped, with 70 of them analyzed for sIL-6R levels. Using the PCR/RFLP methods, two important polymorphic sites were selected for genotyping: the 48892A/C (D358A) in exon 9 and the −183G/A in the promoter region. In exon 9, C allele carriers had higher sIL-6R level (P<0.0001) showing that this sequence variation, which corresponds to the proteolytic cleavage site of IL-6Rα, strongly influences the serum sIL-6R levels. In the promoter region, G allele carriers had lower sIL-6R levels (P<0.0082) compared with the A allele carriers. This could be attributed to the linkage disequilibrium (D′=0.54, χ2=51.3, P<0.0001) between the −183G/A and the 48892A/C gene polymorphisms.

The effect of periodontal treatment on serum leptin, interleukin-6, and C-reactive protein.

BACKGROUND Previous studies suggest that periodontitis is closely related to obesity and metabolic syndrome. Leptin, a pleiotrophic hormone produced by adipose tissue, has been reported to be related to periodontitis. This study investigates the effects of periodontal treatment on the serum levels of leptin and other cytokines in patients with chronic periodontitis (CP). METHODS Serum samples were taken from 33 CP patients (22 non-smokers, 11 smokers) and 18 healthy subjects. The serum leptin, adiponectin, tumor necrosis factor-alpha, interleukin (IL)-6, and C-reactive protein (CRP) levels were measured before and after non-surgical periodontal treatment. RESULTS Significant differences between healthy and CP patients were found in serum leptin, IL-6, and CRP levels (P = 0.0018, P = 0.0064, and P = 0.0095, respectively). The serum leptin level was associated with mean probing depth, mean clinical attachment level, mean alveolar bone loss, and body mass index. There were significant associations between serum leptin levels and IL-6 and CRP levels. After non-surgical periodontal treatment, serum leptin, IL-6, and CRP levels were significantly decreased (mean +/- SD before and after, P value, respectively: leptin, 8.02 +/- 5.5, 7.10 +/- 4.4, P = 0.015; IL-6, 1.73 +/- 1.02, 1.36 +/- 0.73, P = 0.048; and CRP, 802.0 +/- 1065, 491.2 +/- 479.3, P = 0.047). CONCLUSIONS Periodontal treatment is effective in reducing serum leptin, IL-6, and CRP levels. The results suggest that leptin, IL-6, and CRP could be mediating factors that connect metabolic syndrome and periodontitis.

Accumulation of Human Heat Shock Protein 60-Reactive T Cells in the Gingival Tissues of Periodontitis Patients

ABSTRACT Heat shock protein 60s (hsp60) are remarkably immunogenic, and both T-cell and antibody responses to hsp60 have been reported in various inflammatory conditions. To clarify the role of hsp60 in T-cell responses in periodontitis, we examined the proliferative response of peripheral blood mononuclear cells (PBMC), as well as the cytokine profile and T-cell clonality, for periodontitis patients and controls following stimulation with recombinant human hsp60 and Porphyromonas gingivalis GroEL. To confirm the infiltration of hsp60-reactive T-cell clones into periodontitis lesions, nucleotide sequences within complementarity-determining region 3 of the T-cell receptor (TCR) β-chain were compared between hsp60-reactive peripheral blood T cells and periodontitis lesion-infiltrating T cells. Periodontitis patients demonstrated significantly higher proliferative responses of PBMC to human hsp60, but not to P. gingivalis GroEL, than control subjects. The response was inhibited by anti-major histocompatibility complex class II antibodies. Analysis of the nucleotide sequences of the TCR demonstrated that human hsp60-reactive T-cell clones and periodontitis lesion-infiltrating T cells have the same receptors, suggesting that hsp60-reactive T cells accumulate in periodontitis lesions. Analysis of the cytokine profile demonstrated that hsp60-reactive PBMC produced significant levels of gamma interferon (IFN-γ) in periodontitis patients, whereas P. gingivalis GroEL did not induce any skewing toward a type1 or type2 cytokine profile. In control subjects no significant expression of IFN-γ or interleukin 4 was induced. These results suggest that periodontitis patients have human hsp60-reactive T cells with a type 1 cytokine profile in their peripheral blood T-cell pools.

The Fcγ Receptor Genotype as a Risk Factor for Generalized Early-Onset Periodontitis in Japanese Patients

BACKGROUND Genetic polymorphisms of immunoglobulin G (IgG) Fc receptors (FcγR) were recently shown to be associated with recurrence rates of adult periodontitis (AP). The purpose of this study was to evaluate whether FcγR polymorphisms are also associated with generalized early-onset periodontitis (G-EOP) in Japanese patients. METHODS Thirty-eight Japanese patients with G-EOP and 83 Japanese patients with AP were identified according to established clinical criteria, including measurements of probing depth, clinical attachment level, and alveolar bone level. FcγR genotypes for 3 bi-allelic polymorphisms were determined in these G-EOP and AP patients and 104 race-matched healthy controls by means of allele-specific polymerase chain reactions. RESULTS There was a significant difference in the distribution of FcγRIIIb genotypes between G-EOP patients and healthy controls (P = 0.02). Additionally, a significant over-representation of FcγRIIIb-NA2 allele was observed in G-EOP patients as compared to AP patients and controls (P = 0.02, P = 0.009, respectively). Moreover, we found a strong association between GEOP and the composite genotype comprising FcγRIIIb-NA2 and FcγRIIIa-158F (G-EOP versus controls: odds ratio 2.4, 95% CI 1.0-6.0, X2 = 4.13, P = 0.04). CONCLUSIONS This study indicates that the FcγRIIIb-NA2 allele and possibly FcγRIIIa-158F could be associated with susceptibility to G-EOP in Japanese patients. J Periodontol 2000;71:1425-1432.

Self-heat shock protein 60 induces tumour necrosis factor-α in monocyte-derived macrophage: possible role in chronic inflammatory periodontal disease

Heat shock protein 60 (hsp60) has been increasingly recognized as an important molecule in infectious and autoimmune diseases. We have demonstrated previously that serum antibodies to both human hsp60 and Porphyromonas gingivalis GroEL were elevated in periodontitis patients compared with healthy subjects. In order to clarify the relative importance of hsp60 in the inflammatory response in periodontal disease, the stimulatory effect of human and bacterial hsp60 on the production of tumour necrosis factor‐α (TNF‐α) was examined in phorbol myristate acetate (PMA)‐stimulated THP‐1 cells. As bacterial hsp60s, recombinant P. gingivalis and Actinobacillus actinomycetemcomitans GroEL was used. Human hsp60 but not P. gingivalis or A. actinomycetemcomitans GroEL demonstrated stimulatory activity similar to lipopolysaccharide (LPS) derived from the bacteria. The activity of hsp60 was inhibited by anti‐CD14 and anti‐Toll‐like receptor 4 (TLR4) antibodies, suggesting that both CD14 and TLR4 mediate hsp60 signalling. Immunohistochemical analysis demonstrated that hsp60 is abundantly expressed in periodontitis lesions. Therefore, it is postulated that periodontopathic bacteria stimulate the cells in the periodontium to up‐regulate the expression of hsp60, which in turn may stimulate macrophage and possibly other cells to produce proinflammatory cytokines. These mechanisms may be involved in the chronicity and tissue destruction of periodontal disease.

A novel PCR-based method for direct Fcγ receptor IIIa (CD16) allotyping

Abstract Leukocyte IgG receptors (FcγR) are important immune-response modulating molecules. FcγRIIIa is expressed on macrophages, NK-cells and γδ-T cells and exhibits a genetically determined, functional polymorphism at nucleotide 559. This allelic difference predicts either a phenylalanine (F158) or valine (V158) at amino acid 158 in the membrane-proximal extracellular domain, and has been shown to be associated with autoimmune and infectious diseases. Published methods to determine FcγRIIIa genotypes are cumbersome. Therefore, we developed a novel, rapid and reliable PCR-based method to determine FcγRIIIa genotypes. Comparison of genotyping results with direct FcγRIIIa sequencing of 60 blood donors showed 100% accuracy of this new method. Since genotype frequencies of FcγR polymorphisms depend strongly on race and ethnicity, we compared FcγRIIIa genotype frequencies of 176 Caucasian Dutch and 104 Japanese blood donors. Interestingly, these frequencies were not significantly different (P>0.1), in contrast to the FcγRIIa and FcγRIIIb genotype frequencies (P

Inhibitory Effects of Lactoferrin on Growth and Biofilm Formation of Porphyromonas gingivalis and Prevotella intermedia

ABSTRACT Lactoferrin (LF) is an iron-binding antimicrobial protein present in saliva and gingival crevicular fluids, and it is possibly associated with host defense against oral pathogens, including periodontopathic bacteria. In the present study, we evaluated the in vitro effects of LF-related agents on the growth and biofilm formation of two periodontopathic bacteria, Porphyromonas gingivalis and Prevotella intermedia, which reside as biofilms in the subgingival plaque. The planktonic growth of P. gingivalis and P. intermedia was suppressed for up to 5 h by incubation with ≥130 μg/ml of human LF (hLF), iron-free and iron-saturated bovine LF (apo-bLF and holo-bLF, respectively), and ≥6 μg/ml of bLF-derived antimicrobial peptide lactoferricin B (LFcin B); but those effects were weak after 8 h. The biofilm formation of P. gingivalis and P. intermedia over 24 h was effectively inhibited by lower concentrations (≥8 μg/ml) of various iron-bound forms (the apo, native, and holo forms) of bLF and hLF but not LFcin B. A preformed biofilm of P. gingivalis and P. intermedia was also reduced by incubation with various iron-bound bLFs, hLF, and LFcin B for 5 h. In an examination of the effectiveness of native bLF when it was used in combination with four antibiotics, it was found that treatment with ciprofloxacin, clarithromycin, and minocycline in combination with native bLF for 24 h reduced the amount of a preformed biofilm of P. gingivalis compared with the level of reduction achieved with each agent alone. These results demonstrate the antibiofilm activity of LF with lower iron dependency against P. gingivalis and P. intermedia and the potential usefulness of LF for the prevention and treatment of periodontal diseases and as adjunct therapy for periodontal diseases.