Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Background: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. Methods: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real‐time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase‐1 (HO‐1) mRNA expression. iNOS and HO‐1 protein expression and phosphorylation of c‐Jun N‐terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate. Results: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS‐stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO‐1 expression in LPS‐activated cells. Inhibition of HO‐1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS‐induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS‐induced NO. Conclusions: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS‐stimulated RAW264.7 cells at the translational level via HO‐1‐mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.     

l‐arginine‐dependent nitric oxide production of a murine macrophage‐like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg‐LPS). RAW264.7 cells were incubated with i) various concentrations of Pg‐LPS or Salmonella typhosa LPS (St‐LPS), ii) Pg‐LPS with or without l ‐arginine and/or N^G‐monomethyl‐l ‐arginine (NMMA), an arginine analog or iii) Pg‐LPS and interferon‐γ (IFN‐γ) with or without anti‐IFN‐γ antibodies or interleukin‐10 (IL‐10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg‐LPS, but was observed after stimulation with St‐LPS. Exogenous l ‐arginine restored the ability of Pg‐LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg‐LPS with exogenous l ‐arginine was abolished by NMMA. IFN‐γ induced independent NO production by Pg‐LPS‐stimulated macrophages and this stimulatory effect of IFN‐γ could be completely suppressed by anti‐IFN‐γ antibodies and IL‐10. These results suggest that Pg‐LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an l ‐arginine‐dependent mechanism which is itself independent of the action of IFN‐γ.     

β2‐Glycoprotein I‐Dependent Anti‐Cardiolipin Antibodies Associated With Periodontitis in Patients With Systemic Lupus Erythematosus

Background : It was reported that patients with systemic lupus erythematosus (SLE) exhibited increased levels of anticardiolipin (anti‐CL) antibodies, a class of antiphospholipid antibodies associated with thrombosis. β2‐glycoprotein I (β2GPI) has been considered as the actual target antigen for anti‐CL antibodies. This study investigates the association of periodontal infection with anti‐CL antibodies in patients with SLE. Methods: Fifty‐three SLE female patients and 56 healthy female volunteers were recruited in this case‐control study. All participants received periodontal examinations. The presence of Porphyromonas gingivalis and Treponema denticola in saliva and plaque samples was detected by polymerase chain reaction. Levels of serum anti‐CL and anti‐β2GPI antibodies were examined using enzyme‐linked immunosorbent assay. Results: Patients with SLE exhibited more periodontal attachment loss and increased titers of serum anti‐CL and anti‐β2GPI antibodies compared with healthy controls. Patients with active SLE who harbored P. gingivalis or P. gingivalis together with T. denticola intraorally exhibited significantly higher anti‐CL and anti‐β2GPI antibodies than those without these bacteria. Anti‐CL and anti‐β2GPI antibody levels correlated positively with clinical attachment level. Furthermore, increased anti‐β2GPI antibody levels were significantly associated with C‐reactive protein and erythrocyte sedimentation rate. Conclusions: Elevated anti‐CL and anti‐β2GPI antibody levels were associated with periodontopathic bacteria and periodontal breakdown in patients with SLE. Periodontitis might be a modifiable risk factor for SLE.     

E‐selectin expression induced by Porphyromonas gingivalis in human endothelial cells via nucleotide‐binding oligomerization domain‐like receptors and Toll‐like receptors

Porphyromonas gingivalis, an important periodontal pathogen, has been proved to actively invade cells, induce endothelial cell activation, and promote development of atherosclerosis. Innate immune surveillance, which includes the activity of nucleotide‐binding oligomerization domain (NOD)‐like receptors (NLRs) and Toll‐like receptors (TLRs), are essential for the control of microbial infections; however, the roles of receptor families in P. gingivalis infections remain unclear. Here, we examined the roles of NLRs and TLRs in endothelial cell activation caused by P. gingivalis. Live P. gingivalis and whole cell sonicates were used to stimulate endothelial cells, and both showed upregulation of E‐selectin as well as NOD1, NOD2, and TLR2. In addition, silencing of these genes in endothelial cells infected with P. gingivalis led to a reduction in E‐selectin expression. Porphyromonas gingivalis also induced nuclear factor‐κB (NF‐κB) and P38 mitogen‐activated protein kinase (MAPK) activity in endothelial cells, whereas small interfering RNA targeting NOD1 significantly reduced these signals. Moreover, inhibition of either NOD2 or TLR2 inhibited NF‐κB significantly, but had only a weak inhibitory effect on P38 MAPK signaling. Direct inhibition of NF‐κB and P38 MAPK significantly attenuated E‐selectin expression induced by P. gingivalis in endothelial cells. Taken together, these findings suggest that NOD1, NOD2, and TLR2 play important, non‐redundant roles in endothelial cell activation following P. gingivalis infection.     

NO production in RAW264 cells stimulated with Porphyromonas gingivalis extracellular vesicles

Oral Diseases (2010) 17 , 83–89 Objective: This experiment was carried out in order to prove the inducible nitric oxide synthase (iNOS) expression and the nitric oxide (NO) production in mouse macrophage cells (RAW264) which were stimulated by vesicles released from Porphyromonas gingivalis, and discussed about the role of vesicles in advance periodontal diseases. Materials and Methods: Production of NO₂^? in RAW264 cells was investigated after 0, 1, 3, 6 and 12 h of stimulation with P. gingivalis vesicles. NO was analyzed by HPLC‐based flow reactor system with Griess reagent. The cells stained by the enzyme‐labeled antibody method, after being stimulated with vesicles for 12 h. The iNOS proteins, which were expressed in RAW264 cells after 12 h of stimulation with vesicles, were detected by western blot. Results: When stimulated with vesicles from W83 and from ATCC33277, the RAW264 cells produced NO, but cell proteins that came in contact with the vesicles were degraded by protease activities in vesicles. When stimulated with vesicles from gingipain‐deficient mutant strain KDP136, the RAW264 cells produced NO, but the quality was about 60%, compared with the vesicles from ATCC33277. Conclusion: The results suggest that vesicles are not only just a part of bacterial component, but also are a toxic complex of lipopolysaccharide and protease, and one of the putative virulence factor for periodontal diseases that continue inflammation and cause chronic conditions.     

Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis

Takahashi N, Honda T, Domon H, Nakajima T, Tabeta K, Yamazaki K. Interleukin‐1 receptor‐associated kinase‐M in gingival epithelial cells attenuates the inflammatory response elicited by Porphyromonas gingivalis. J Periodont Res 2010; 45: 512–519. © 2010 John Wiley & Sons A/S Background and Objective: Recent studies have revealed that negative regulatory molecules, including interleukin‐1 receptor‐associated kinase‐M (IRAK‐M), control the overactivation of Toll‐like receptor (TLR) signaling. The role of IRAK‐M in human gingival epithelial cells (HGECs), which express TLRs, remains unclear. The present study examined the role of IRAK‐M on interleukin‐8 and macrophage chemoattractant protein‐1 (MCP‐1) expression in HGECs stimulated with Porphyromonas gingivalis and TLR ligands. Material and Methods: Primary HGECs and an SV40 T‐antigen‐immortalized HGEC line (epi 4) were stimulated with live or heat‐killed P. gingivalis, P. gingivalis lipopolysaccharide or the synthetic lipopeptide PAM₃CSK₄, and subsequent expression of IRAK‐M, interleukin‐8 and MCP‐1 was evaluated at the mRNA and protein levels. The effects of IRAK‐M on interleukin‐8 and MCP‐1 expressions were evaluated by IRAK‐M‐specific RNA interference (RNAi)‐based loss‐of‐function assay. Results: All tested stimulants up‐regulated the expression of IRAK‐M in HGECs. The P. gingivalis lipopolysaccharide or PAM₃CSK₄ increased MCP‐1 expression, whereas live P. gingivalis down‐regulated the MCP‐1 expression in HGECs. However, IRAK‐M RNAi increased the expression of MCP‐1 irrespective of up‐ or down‐regulation mediated by the respective stimulants. Interleukin‐8 gene expression, up‐regulated by all tested stimulants, was further enhanced by IRAK‐M RNAi. In contrast, IRAK‐M RNAi had no effect on the interleukin‐8 protein levels, irrespective of the stimulant, indicating that post‐translational modification, not IRAK‐M, controls interleukin‐8 protein expression. Conclusion: Interleukin‐1 receptor‐associated kinase‐M appeared to have distinct regulatory roles on the interleukin‐8 and MCP‐1 produced by HGECs, further suggesting an important role for interleukin‐8 in the immune reponse to periodontopathic bacteria.     

Activation of toll‐like receptors 2 and 4 by gram‐negative periodontal bacteria

Background/aims: Periodontitis is a chronic infectious disease associated with a gram‐negative subgingival microflora. Bacterial components stimulate, among other receptors, Toll‐like receptor (TLR) 2 and/or TLR4. Accumulating evidence indicates that both qualitatively and quantitatively distinct immune responses result from the triggering of TLR2 as compared to TLR4 triggering. The aim was to study the interaction of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Tannerella forsythensis, Prevotella intermedia, Prevotella nigrescens, Fusobacterium nucleatum and Veillonella parvula with TLR2 and TLR4. We investigated all known serotypes (K⁻, K1–K6) of P. gingivalis and A. actinomycetemcomitans serotype a–e strains for their potency to stimulate cytokine production. Methods: Human embryonic kidney (HEK) cells, stably transfected with CD14, CD14‐TLR2, or CD14‐TLR4 and whole blood were stimulated with bacterial sonicates. Cytokine production (interleukin‐6, ‐8, ‐10 and ‐12) was measured in the supernatant by enzyme‐linked immunosorbent assay. Results: All test bacteria stimulated HEK‐CD14‐TLR2, but only A. actinomycetemcomitans and V. parvula stimulated HEK‐CD14‐TLR4. No differences were found in the activation of HEK‐CD14‐TLR2/4, or cytokine production in whole blood between serotypes of P. gingivalis and A. actinomycetemcomitans. Conclusion: Gram‐negative periodontal bacteria predominantly stimulated TLR2, which may be of importance for the Th1/Th2 cell orientation of the immune response in periodontitis.     

The role of Toll‐like receptors 2 and 4 on reactive oxygen species and nitric oxide production by macrophage cells stimulated with root canal pathogens

Introduction: Periapical lesions arise as a result of the activation and interaction of the host immune responses against root canal infection. Recently identified Toll‐like receptors (TLR) seem to be involved in the recognition and development of immune responses against a myriad of microorganisms. However, very little information is available on the role of TLR in the induction of periapical lesions. Method: The role of TLR‐2 and TLR‐4 in the activation of murine macrophages stimulated using Fusobacterium nucleatum and Peptostreptococcus anaerobius was investigated. The production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed. Results: The results demonstrate that TLR‐2 and TLR‐4 are involved in the production of ROS by activated macrophages. The microorganisms induced similar levels of NO production by TLR‐2‐competent and TLR‐2‐deficient macrophages, regardless of the addition of interferon‐γ (IFN‐γ), ruling out a role for TLR‐2 in the NO production induced by these bacteria. Only P. anaerobius induced NO production by TLR‐4‐competent macrophages without the addition of IFN‐γ. However, after IFN‐γ addition, F. nucleatum induced macrophage NO production. Therefore, NO production stimulated by IFN‐γ and these microorganisms seems to be TLR‐4‐independent. Conclusion: TLR‐2 seems to be involved in the induction of ROS production by macrophages in response to prevalent root canal bacteria, while only F. nucleatum induced ROS production by TLR‐4‐competent macrophages. Both microorganisms significantly induced large amounts of NO independent of TLR‐2 and TLR‐4. We conclude that microorganisms may participate in the induction and progression of periapical lesions through NO and ROS production by activated macrophages.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Effects of Resolvin D1 on Cell Survival and Cytokine Expression of Human Gingival Fibroblasts

Background: Tissue breakdown in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis, and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils to initiate resolution. The effects of resolvins on human gingival fibroblasts (HGFs) are unknown. This study examines the effects of resolvin D1 on HGF survival and cytokine expression when treated with or without P. gingivalis supernatant. Methods: Cytotoxicity of resolvin D1 on HGFs with or without a toxic level of P. gingivalis supernatant was measured with lactate dehydrogenase assays. Cytokine arrays were performed on HGF‐conditioned media treated with or without resolvin D1 and with or without P. gingivalis supernatant. Results: Resolvin D1 had no cytotoxic effects on HGFs at concentrations between 1 and 1,000 nM (all P > 0.05). Resolvin D1 (1,000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant on HGFs (P = 0.002). Resolvin D1 significantly reduced the expression of interleukin (IL)‐6 (P = 0.010) and monocyte chemoattractant protein (MCP)‐1 (P = 0.04) in untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the expression levels of granulocyte‐macrophage colony‐stimulating factor (CSF), granulocyte CSF, growth‐regulated oncogene (GRO), IL‐5, IL‐6, IL‐7, IL‐8, IL‐10, MCP‐1, MCP‐2, MCP‐3, and monokine induced by γ‐interferon. Resolvin D1 significantly reduced the expression of GRO (P = 0.04), marginally reduced the levels of MCP‐1 (P = 0.10), and marginally increased the levels of transforming growth factor (TGF)‐β1 (P = 0.07) from HGFs treated with P. gingivalis supernatant. Conclusions: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP‐1, and marginally increased TGF‐β1 from P. gingivalis–treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.     

Mechanism and implications of CXCR4‐mediated integrin activation by Porphyromonas gingivalis

In monocytes and macrophages, the interaction of Porphyromonas gingivalis with Toll‐like receptor 2 (TLR2) leads to the activation of a MyD88‐dependent antimicrobial pathway and a phosphatidylinositol‐3 kinase (PI3K) ‐dependent pro‐adhesive pathway, which activates the β₂‐integrin complement receptor 3 (CR3). By means of its fimbriae, P. gingivalis binds CXC‐chemokine receptor 4 (CXCR4) and induces crosstalk with TLR2 that inhibits the MyD88‐dependent antimicrobial pathway. In this paper, we investigated the impact of the P. gingivalis‐CXCR4 interaction on the pro‐adhesive pathway. Using human monocytes, mouse macrophages, or receptor‐transfected cell lines, we showed that the binding of P. gingivalis fimbriae to CXCR4 induces CR3 activation via PI3K, albeit in a TLR2‐independent manner. An isogenic strain of P. gingivalis expressing mutant fimbriae that do not interact with CXCR4 failed to efficiently activate CR3, leading to enhanced susceptibility to killing in vivo compared with the wild‐type organism. This in vivo observation is consistent with previous findings that activated CR3 mediates safe entry of P. gingivalis into macrophages. Taken together with our previous work, these results indicate that the interaction of P. gingivalis with CXCR4 leads to inhibition of antimicrobial responses and enhancement of pro‐adhesive responses, thereby maximizing its adaptive fitness in the mammalian host.     

Chemokine expression in Porphyromonas gingivalis–specific T‐cell lines

Autologous non‐T cells (monocytes and B cells) were added to Porphyromonas gingivalis–specific T cell lines established from 9 healthy adults together with P. gingivalis outer membrane antigens for 4–6, 16–18, 24 and 48 h. Flow cytometry was employed to analyze the CD4 and CD8 cells, monocytes and B cells for intracytoplasmic IP‐10 (interferon‐gamma inducible protein 10), MCP‐1 (monocyte chemoattractant protein 1), MIP‐1α (macrophage inflammatory protein 1α) and RANTES (regulated on activation normal T cell expressed and secreted) at the four time periods. All cell types were positive for each chemokine throughout the 48‐h time period. There were significantly fewer MCP‐1‐positive cells compared with the other 3 chemokines. However, the percentages of MCP‐1, MIP‐1α‐ and RANTES‐positive CD8 cells were significantly higher than the percentages of positive CD4 cells in all cultures. IP‐10‐positive CD4, CD14‐positive monocytes and CD19‐positive B cells were predominant compared with MIP‐1α‐ and RANTES‐positive cells at 24 h. In conclusion, the present study has shown that P. gingivalis–specific T cells, monocytes and B cells produce chemokines in response to P. gingivalis outer membrane antigens, IP‐10 being predominant, with MCP‐1 being significantly reduced in comparison with IP‐10, MIP‐1α and RANTES. Increased percentages of CD8 cells were induced to produce chemokines in comparison with CD4 cells, indicating a more preferential action on CD8 rather than CD4 cells.     

Establishment of Porphyromonas gingivalis–specific T‐cell lines from atherosclerosis patients

Porphyromonas gingivalis–specific T‐cell lines were established from atherosclerotic lesions from two patients. T‐cell lines were found to be a mixture of CD4⁺ and CD8⁺ T‐cells producing variable amount of interferon‐gamma, interleukin‐4 and interleukin‐10. The two patients have had a periodontal disease whose anti–P. gingivalis immunoglobulin G titers were significantly elevated, and P. gingivalis was the one of the predominantly cultivable microorganisms. This is the first report of the successful establishment of P. gingivalis–specific T‐cell lines from atherosclerotic patients.     

Prevalence of Porphyromonas gingivalis in relation to periodontal status assessed by real‐time PCR

Many studies have examined the presence of Porphyromonas gingivalis in periodontal pockets. However, monitoring the number of bacterial cells is difficult. In this study, we performed quantitative analyses of P. gingivalis to clarify the relationship between the numbers of this organism and periodontal status. Using the TaqMan real‐time PCR system, we found a significant positive correlation (P < 0.0001) between the number of P. gingivalis and pocket depth. The slope of the regression line indicated that for every 1‐mm increase in pocket depth, the number of P. gingivalis increased 10‐ fold. There was also a significant reduction (P < 0.01) in the numbers of P. gingivalis before and after treatment. These results suggest that the absolute and relative numbers of P. gingivalis are closely associated with periodontal status, and that quantitative analysis of this organism is important for the evaluation of periodontal therapy.