Autoimmunity in periodontal disease

Periodontal disease in characterized by the loss of the normal supporting tissues of the teeth and a humoral and cellular immune response to bacterial antigen of dental plaque which accumulates at the dento-gingival junction. This review considers the evidence for the existence of an autoimmune component of the host immune response, the possible origin of such a response and the way in which such a host response may contribute to the changes observed in the periodontium in the disease.     

Function of anti–Porphyromonas gingivalis immunoglobulin classes in immunized Macaca fascicularis

We previously reported that Macaca fascicularis immunized with formalin-killed Porphyromonas gingivalis were protected against the bone loss of periodontitis. To examine mechanisms of protection, we determined specific immunoglobulin G (IgG), IgM and IgA titers and opsonic capacities of sera from immunized and control animals. Serum IgG and IgA titers to P. gingivalis appeared early and persisted throughout the 36-week observation period. IgM titers were elevated until 6 to 12 weeks and then decreased through week 36. A significant association was observed between peak IgM titers prior to ligature placement and protection against bone loss (measured at week 30). In control monkeys, no significant IgG, IgA or IgM titers were seen. In sera from immunized animals, significant opsonic capacity was seen by 6–12 weeks and persisted throughout the study. In contrast, control sera showed only low opsonization capacity. Anti–P. gingivalis antibody titers in purified IgG, IgA and IgM fractions were determined by enzyme-linked immunosorbent assay, and opsonic activity was demonstrated only in the IgG fraction.     

Antigenic cross‐reactivity among Porphyromonas gingivalis serotypes

The goal of our research program is to develop a Porphyromonas gingivalis vaccine. Vaccine development requires identification of antigenic components shared by the many clonal types of P. gingivalis. The purpose of the present study was to evaluate the extent and nature of antigenic cross‐reactivity among serotypes of P. gingivalis and to identify shared antigenic components. Strains selected to represent serotypes A–D were 33277, A7A1‐28 W50 and 381, respectively. Using intact cells, antibodies were raised in rabbits. Titers were assessed by enzyme‐linked immunosorbent assay (ELISA) using intact cells as antigen, Western blots were prepared and biologic activity was measured as opsonization (chemiluminescence expressed as mV) and enhancement of phagocytosis and killing by polymorphonuclear leukocytes. Extensive cross‐reactivity that varied greatly among serotypes was observed by ELISA. The Western blots showed an even greater extent of cross‐reactivity, with shared protein components at approximately 140, 130, 37, 32 and 28 kDa and a shared variable molecular mass smear considered to be lipopolysaccharide and other carbohydrate. Additional protein components at 110, 85, 35 and 20 kDa appeared to be shared by some but not all serotypes. In the functional assays, strains 33277 and 381 were equally well opsonized by anti‐33277 and anti‐381 (500–650 mV) but opsonized to a much lesser extent by anti‐A7A1‐28 and anti‐W50 (roughly 125 mV and 350 mV respectively). A7A1‐28 and W50 were opsonized by all four immune sera almost equally but to a much lower extent (roughly 400 mV and 250 mV respectively). Enhancement of phagocytosis and killing in the presence of active complement mirrored opsonization with the exception that 381 was reasonably well opsonized by anti‐A7A1‐28 (400 mV) and anti‐W50 (350 mV), but poorly killed. The protein components at 140, 130, 37 and 28 kDa shared by all of the four serotypes appear to have potential as vaccine candidate antigens.     

Recognition and phagocytosis of multiple periodontopathogenic bacteria by anti‐Porphyromonas gingivalis heat‐shock protein 60 antisera

The present study has been performed to evaluate Porphyromonas gingivalis heat shock protein (HSP) 60 as a candidate vaccine to protect against multiple putative periodontopathic bacteria. Mouse anti‐P. gingivalis HSP antisera demonstrated the elevated IgG antibody titers against the multiple bacteria tested and cross‐reacted with heat‐induced bacterial proteins of the target bacteria. The antisera also demonstrated a significantly higher opsonophagocytosis function against all the target bacteria than the control sera (P < 0.01). We concluded that P. gingivalis HSP 60 could potentially be developed as a vaccine against multiple periodontopathic bacteria.     

Invasive differences among Porphyromonas gingivalis strains from healthy and diseased periodontal sites

Background and Objective:  The purpose of this study was to determine any difference between Porphyromonas gingivalis isolates from periodontally healthy sites as compared to those from diseased sites with respect to the ability to invade host cells.
Material and Methods:  Subgingival plaque samples were obtained from periodontally healthy and diseased sites using paper points. P .  gingivalis colonies were isolated and tested, using an antibiotic protection assay, for their ability to invade KB cells. P. gingivalis 381 and Escherichia coli MC1061 were used as controls.
Results:  Mean values of 16.79 ± 0.86 × 10³ colony-forming units/mL and 26.14 ± 2.11 × 10³ colony-forming units/mL were observed in invasion assays for isolates from periodontally healthy and diseased sites, respectively. P .  gingivalis present in diseased sites had significantly greater invasive abilities than strains isolated from healthy sites. No statistical difference was noted between male or female subjects concerning the degree of invasion; isolates from diseased sites from both genders had significantly greater invasion abilities than those from healthy sites. A significant correlation was found between the increased invasive capabilities of P .  gingivalis isolates vs. an increased probing depth.
Conclusion:  The increased invasion noted with P .  gingivalis isolates from diseased sites vs. healthy sites, and the increased invasive capabilities with increasing probing depth, indicate that P .  gingivalis isolates have a varying ability to invade host cells in the periodontal pocket.     

吸烟对牙周基础治疗效果影响的研究

目的评价吸烟与非吸烟慢性牙周炎患者牙周基础治疗1个月后的疗效差异。方法选择36例慢性牙周炎患者,吸烟组20例,非吸烟组16例,基线时两组牙周炎病情相似。从牙列的4个象限选取探诊深度在5～9mm范围的位点1～2个,吸烟组108个位点,非吸烟组88个位点,观察这些位点在牙周基础治疗前、治疗后1个月临床指标的变化,包括菌斑指数(PLI),牙龈出血指数(BI),牙周袋探诊深度(PPD)和附着丧失(AL);在作临床观察的同时,对治疗前后龈沟液白介素(IL)-1β进行检测。结果治疗前(基线时)两组PLI、BI、PPD、AL以及IL-1β差异不显著,牙周基础治疗1个月后,两组的各项指标均有明显的改善,但吸烟组改善程度明显低于非吸烟组(P<0.05)。结论慢性牙周炎患者,吸烟者牙周基础治疗的效果差于     

Effects of metronidazole on Porphyromonas gingivalis biofilms

Subgingival bacteria exist within a biofilm consisting of cells and extracellular matrix which may afford organisms protection from both antibiotics and components of the host immune system. MIC values for planktonic Porphyromonas gingivalis treated with metronidazole were compared with those obtained for the same strain in biofilms associated with hydroxyapatite (HA) surfaces. The treated biofilms were examined for growth and studied by scanning electron microscopy. A broth assay resulted in an MIC of 0.125 μg/ml for metronidazole against P. gingivalis . P. gingivalis biofilms exhibited growth after treatment with 20μg/ml metronidazole, which was 160 times the MIC for planktonic organisms. The results of this study indicate that biofilm-associated P. gingivalis may be resistant to metronidazole at concentrations which are usually attained by systemic administration.     

Prevalence of fimA genotypes of Porphyromonas gingivalis and periodontal health status in Chinese adults

Background and Objective:  Porphyromonas gingivalis fimbriae play a key role in colonization of the oral cavity. The fimA gene, which encodes fimbrillin ( FimA ), can be classified into six types (I–V and Ib) according to nucleotide sequence. In the present study, we investigated the relationship between the prevalence of P. gingivalis -specific fimA genotypes and periodontal health status in Chinese adults.
Material and Methods:  One-hundred and fifteen patients with chronic periodontitis and 136 periodontally healthy adults were selected. P. gingivalis detection, determination of fimA genotypes, and the co-existence of Actinobacillus actinomycetemcomitans and Tannerella forsythia with various fimA types, were assessed by the polymerase chain reaction. Odds ratios and 95% confidence intervals were calculated for associating the fimA -specific genes with periodontitis.
Results:  P. gingivalis was detected in 22.1% of healthy subjects and in 81.7% of the patients. A single fimA genotype was detected in most samples. In healthy adults, the most prevalent fimA genotype was type I (66.7%). However, type II was detected most frequently (43.6%) in the patient group, followed by type IV (30.9%). The frequency of co-existing A. actinomycetemcomitans and T. forsythia was highest in type II fimA -positive sites. Statistical analysis revealed that periodontitis was associated with occurrences of type I (odds ratio 0.97), Ib (odds ratio 13.26), II (odds ratio 36.62), III (odds ratio 4.57), IV (odds ratio 22.86) and V (odds ratio 1.19).
Conclusion:  P. gingivalis type II followed by type IV were considered as disease-associated strains that account for the pathogenesis of chronic periodontitis in Chinese adults.     

Serum IgG antibody response to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis: implications for periodontal diagnosis

Abstract. The relationship of the serum antibody titer and avidity to the putative periodontal pathogens Actinobacillus actinomycetemcomitans (Aa) strains Y4 and 29523 and Porphyromonas gingivalis (Pg) strain 381 were examined in relation to clinical parameters in 26 gingivitis and 28 periodontitis patients. The relationship of antibody titer and avidity to infection with the homologous organism was also examined in a subset of 30 patients. Antibody titer was determined by an enzyme-linked immunosorbent assay, and antibody avidity was assessed using a dissociation assay. Considering all patients, there was a significant negative correlation between mean probing depth and antibody titer (r=-0.28) and avidity (r=-0.28) to Aa Y4. There was a significant positive correlation of probing depth and antibody titer (r=0.46) and avidity (r=0.46) to Pg. The correlation of antibody titer and avidity to Aa and infection with Aa Y4 (r=-0.32, r=-0.21) and Aa 29523 (r=-0.35, r=-0.39) was negative, while the correlations of titer and avidity to Pg and presence of the organisms was strongly positive (r=0.40, r=0.35). These data indicate that the relationship of serum antibody titer and avidity to clinical parameters of periodontal disease severity and the level of infection with the homologous organism appears to be different for Aa and Pg. The development of an antibody response to Aa appears to protect the individual from infection with the organism. In contrast, the development of an antibody response to Pg was not able to eliminate the infection. These results should be considered when developing a diagnostic strategy for periodontal disease utilizing the humoral immune response.     

In vitro studies of lymphocyte apoptosis induced by the periodontal pathogen Porphyromonas gingivalis

Apoptosis has a physiological role in lymphocyte development and function serving to remove self-reactive T-cells in the thymus as well as activated peripheral T-cells when they are no longer required in the immune response. Evidence from the study of several pathogenic bacteria indicate that induction of premature cell death by apoptosis may be an important pathogenic mechanism promoting infection, inflammation and concomitant disease. In this paper we demonstrate that cultures of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis) promote lymphocyte apoptosis in peripheral blood mononuclear cells (PBMC). We have used assays designed to investigate the different molecular and cellular changes associated with apoptosis. Thus flow cytometry revealed that whole cultures of P. gingivalis promoted cell shrinkage in the lymphocyte fraction of PBMC and analysis of hypodiploidy confirmed that the cellular changes were associated with nuclear changes characteristic of apoptosis. We also found that apoptosis was promoted in PBMC exposed to both whole P. gingivalis cultures and culture supernatant but not washed bacterial cells; this indicates that molecule(s) secreted into the medium were responsible for this activity and not a factor intrinsic to the bacterial cell. Furthermore heat treatment has no effect on the ability of P. gingivalis cultures to induce lymphocyte apoptosis. In summary, a soluble heat stable component of the supernatant from P. gingivalis cultures promotes lymphocyte apoptosis. These data establish the principle that bacteria-induced apoptosis may be an important feature of the pathogenesis of periodontal disease.     

Activity of anti‐Porphyromonas gingivalis egg yolk antibody against gingipains in vitro

Introduction: We investigated the effect of anti‐Porphyromonas gingivalis egg yolk antibody against gingipains [immunoglobulin Y (IgY)‐GP] on gingipain activity in vitro. Methods: IgY‐GP was isolated from the yolks of White Leghorn hens immunized with purified gingipains. Control antibody (IgY) was isolated from the yolks of non‐immunized hens. Gingipain activity was assessed according to the rate of enzymatic substrate hydrolysis. Human epithelial cells were cultured with or without gingipains and with gingipains pretreated with either IgY‐GP or IgY. Results: Hydrolytic activity decreased in the presence of IgY‐GP. Cells incubated with gingipains showed a dose‐dependent loss of adhesion activity. Pretreatment of gingipains with IgY‐GP was associated with strong inhibition of cell detachment, whereas pretreatment with IgY was not. Conclusion: Our findings suggest that IgY‐GP may be an effective immunotherapeutic agent in the treatment of periodontitis.     

Enhanced monocyte migration and pro‐inflammatory cytokine production by Porphyromonas gingivalis infection

Pollreisz A, Huang Y, Roth GA, Cheng B, Kebschull M, Papapanou PN, Schmidt AM, Lalla E. Enhanced monocyte migration and pro‐inflammatory cytokine production by Porphyromonas gingivalis infection. J Periodont Res 2009; doi: 10.1111/j.1600‐0765.2009.01225.x. © 2009 The Authors. Journal compilation © 2009 Blackwell Munksgaard Background and Objective: Porphyromonas gingivalis, a major periodontal pathogen, has been reported to be involved in atherogenesis. In order to further understand this pathogen’s link with systemic inflammation and vascular disease, we investigated its influence on murine monocytes and macrophages from three different sources. Material and Methods: Concanavalin A‐elicited peritoneal macrophages, peripheral blood monocyte‐derived macrophages and WEHI 274.1 monocytes were infected with either P. gingivalis 381 or its non‐invasive fimbriae‐deficient mutant, DPG3. Results: Infection with P. gingivalis 381 markedly induced monocyte migration and significantly enhanced production of the pro‐inflammatory cytokines, tumor necrosis factor‐α and interleukin‐6. Consistent with a role for this pathogen’s major fimbriae and/or its invasive capacity, infection with DPG3 had a minimal effect on both monocyte attraction and pro‐inflammatory cytokine production. Conclusion: Since monocyte recruitment and activation are important steps in the development of vascular inflammation and atherosclerosis, these results suggest that P. gingivalis infection may be involved in these processes.     

Multiple restriction fragment length polymorphism genotypes of Porphyromonas gingivalis in single periodontal pockets

A total of 188 Porphyromonas gingivalis strains isolated from 13 periodontal pockets of 8 periodontitis patients were investigated by means of restriction fragment length polymorphism (RFLP) analysis using the fim A gene as a pobe. A total of 5 RFLP genotypes were identified, and over half of the isolates belonged to type I. Four of the 8 patients harbored only 1 RFLP genotype of P. gingivalis , and 1 patient harbored only 2 RFLP genotypes in different sites. On the other hand, in 3 other patients, multiple RFLP genotypes were found in single periodontal pockets. Further studies will be required to clarify whether multiple genotypes of P. gingivalis colonized a single periodontal pocket simultaneously or whether a mutation occurred in the fimbrilin gene locus of P. gingivalis .     

Identification of key determinants in Porphyromonas gingivalis host‐cell invasion assays

The periodontal pathogen Porphyromonas gingivalis can invade host cells, a virulence trait which may contribute to the persistence of infection at subgingival sites. Whilst the antibiotic protection assay has been commonly employed to investigate and quantify P. gingivalis invasion, data obtained have varied widely and a thorough investigation of the factors influencing this is lacking. We investigated the role of a number of bacterial and host‐cell factors and report that the growth phase of P. gingivalis, source (laboratory strain vs. clinical strain), host‐cell identity (cell line vs. primary), host‐cell lysis method, and host‐cell passage number had no significant effect on bacterial invasion. However, incubation time, host‐cell seeding density, method of quantification (viable count vs. DNA), and whether host cells were plated or in suspension, were shown to influence invasion. Also, cells isolated by rapid adhesion to fibronectin exhibited higher levels of P. gingivalis invasion, possibly as a result of increased levels of active α5β1 integrin. Interestingly, this may represent a population of cells with stem cell‐like properties. This study provides important new information by identifying the most important factors that influence P. gingivalis invasion assays and may help to explain variations in the levels previously reported.     

Interleukin‐1α stimulation in monocytes by periodontal bacteria: antagonistic effects of Porphyromonas gingivalis

Periodontal pathogenic bacteria are associated with elevated levels of interleukin‐1α (IL‐1α) but it is unclear if all species can induce cytokine production equally. Porphyromonas gingivalis may be able antagonize IL‐1α induced by other species through the activity of its proteases or lipopolysaccharide (LPS). Monomac‐6 cells and primary human monocytes were treated with culture supernatants from Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedius, Veillonella atypical and Prevotella nigrescens. IL‐1α protein levels were measured after 6 h of incubation. In addition, monocytes were co‐stimulated with supernatants from P. gingivalis and other bacteria. The role of P. gingivalis proteases was tested using Arg‐X and Lys‐X mutant strains. The role of LPS was investigated using purified P. gingivalis LPS and polymixin depletion. All species tested induced significant IL‐1α production, but P. gingivalis was the weakest. Co‐stimulation of monocytes with P. gingivalis antagonized the ability of other bacterial species to induce IL‐1α production. This effect was at its greatest with C. rectus (resulting in a 70% reduction). Gingipain mutant strains and chemical inhibition of protease activity did not reduce antagonistic activity. However, 100 ng/ml of P. gingivalis LPS can reproduce the antagonistic activity of P. gingivalis culture supernatants. Periodontitis‐associated bacterial species stimulate IL‐1α production by monocytes. P. gingivalis can antagonize this effect, and its LPS appears to be the crucial component. This study highlights the importance of mixed infections in the pathogenesis of periodontal disease because reduction of pro‐inflammatory cytokine levels may impair the ability of the host to tackle infection.