Cytomegalovirus periodontal presence is associated with subgingival Dialister pneumosintes and alveolar bone loss

Destructive periodontal disease is associated with human cytomegalovirus (HCMV), Epstein-Barr type 1 virus (EBV-1) and other members of the Herpesviridae family as well as with various gram-negative anaerobic bacteria, including the Dialister pneumosintes species. This study aimed to determine possible interrelationships between periodontal HCMV, EBV-1, herpes simplex virus and D. pneumosintes, and relate the microbiological findings to periodontitis clinical status. Sixteen subjects each contributed paper point samples from two progressing and two stable periodontitis lesions, as determined by ongoing loss of probing attachment. Polymerase chain reaction methodology was used to identify the study herpesviruses and D. pneumosintes. Chi-squared tests, Fisher exact tests and multivariate logistic regression were employed to identify statistical associations among herpesviruses, bacteria and clinical variables. HCMV, and no other virus or combination of viruses, was positively associated with the presence of D. pneumosintes, and the relationship was specific for individual periodontitis sites with no detectable subject effect. D. pneumosintes was in turn positively associated with periodontal pocket depth and disease-active periodontitis. When the average percentage of alveolar bone loss in all teeth was treated as a response, HCMV remained significant even after D. pneumosintes was included in the model, suggesting that both HCMV and D. pneumosintes affected bone loss or, alternatively, HCMV affected factors not studied that themselves can induce bone loss. We hypothesize that periodontal HCMV sets the stage for subgingival proliferation of D. pneumosintes and subsequent periodontal disease progression. Studies on herpesviral-bacterial interactions may hold great promise for delineating important etio-pathogenic aspects of destructive periodontal disease.     

Periodontopathic bacteria and herpesviruses in chronic periodontitis

Periodontal disease involves complex interactions of microorganisms and host defenses. This work investigated the associations between putative bacterial pathogens, herpesviruses and chronic periodontitis. Subgingival samples were collected from 40 periodontally healthy individuals and from 40 patients with chronic periodontitis with probing depths of ≤3 mm or ≥6 mm. Multiplex and nested polymerase chain reactions were used to identify bacterial pathogens and herpesviruses. Porphyromonas gingivalis, Tannerella forsythia, Epstein–Barr virus (EBV) type 1, cytomegalovirus (CMV), Aggregatibacter actinomycetemcomitans and EBV type 2 were detected in, respectively, 95, 75, 72.5, 50, 12.5 and 10% of sites with probing depths ≥6 mm. P. gingivalis, T. forsythia, EBV‐1 and CMV were statistically associated with probing depths ≥6 mm. A. actinomycetemcomitans and EBV‐2 showed no association with periodontitis sites, and no significant associations were found for any of the test infectious agents and probing depths ≤3 mm. Our results confirm an association between P. gingivalis, T. forsythia, EBV‐1 and CMV, and chronic periodontitis. These infectious agents may play an important synergistic role in the pathogenesis of chronic periodontitis.     

Impact of Porphyromonas gingivalis inoculation on ligature-induced alveolar bone loss. A pilot study in rats

Meulman T, Peruzzo DC, Stipp RN, Gonçalves PF, Sallum EA, Casati MZ, Goncalves RB, Nociti FH Jr. Impact of Porphyromonas gingivalis inoculation on ligature‐induced alveolar bone loss. A pilot study in rats. J Periodont Res 2011; 46: 629–636.©2011 John Wiley & Sons A/S Background and Objective: Periodontitis is a polymicrobial infection characterized by the loss of connective tissue attachment, periodontal ligament and alveolar bone. The aim of this study was to evaluate the impact of Porphyromonas gingivalis inoculation on the ligature‐induced alveolar bone loss (ABL) model in rats. Material and Methods: Forty male Wistar rats were randomly assigned to the following groups: G1, control (n = 10); G2, ligature‐induced ABL (n = 15); and G3, ligature‐induced ABL + P. gingivalis inoculation (n = 15). Rats in G2 and G3 were killed 15, 21 and 30 d after ligature placement, and the following parameters were assessed: microbiological load; ABL; and interleukin (IL)‐1β (Il1beta)/Il1ra, Il6/Il10 and Rankl/osteoprotegerin (Opg) mRNA ratios in the gingival tissues, as determined by quantitative PCR. Results: Microbiological analyses demonstrated that rats in G1, G2 and G3 were positive for the presence of bacteria (determined using PCR amplification of the 16S gene), but that only the treatment sites of rats in G3 were positive for P. gingivalis at all time‐points investigated. Histometrically, significant bone loss (p < 0.001) was observed for both ligated groups (G2 and G3) compared with the nonligated group (G1), with higher ABL observed for G2 at all the experimental time‐points. Furthermore, gene‐expression analysis demonstrated that the presence of P. gingivalis in the dentogingival area significantly decreased the Il1β/Il1ra, Il6/Il10 and Rankl/Opg mRNA ratios compared with ligature alone. Conclusion: Within the limits of this pilot study, it was concluded that inoculation of P. gingivalis affected the ligature‐induced ABL model by the induction of an anti‐inflammatory and antiresorptive host response.     

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.     

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.     

Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway,but not through the p38 MAPK pathway

Zhang D, Zheng H, Zhao J, Lin L, Li C, Liu J, Pan Y. Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway, but not through the p38 MAPK pathway. J Periodont Res 2011; 46: 31–38. © 2010 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule‐1 (ICAM‐1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. Material and Methods: We examined the expression of ICAM‐1, IκBα, phospho‐p38 MAPK and nuclear factor‐kappaB (NF‐κB) p65 in an umbilical vein endothelial cell line (ECV‐304) treated with ATCC 33277 and W83, with or without the NF‐κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real‐time PCR, western blotting and immunofluorescence. Results: Both strains could induce ICAM‐1 expression; additionally W83 was able to increase ICAM‐1 expression more significantly than ATCC 33277. In P. gingivalis‐infected endothelial cells, both p38 MAPK and NF‐κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF‐κB. It was found that ICAM‐1 production in endothelial cells was abrogated by inhibition of the NF‐κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. Conclusion: The induction of ICAM‐1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF‐κB pathway, but not by the p38 MAPK pathway.     

牙龈卟啉单胞菌诱导的耐受对小鼠牙槽骨吸收的影响

目的:研究牙龈卟啉单胞菌(P.gingivalis)诱导的耐受对小鼠骨吸收相关因子白细胞介素-1β(IL-1β)、骨保护素(OPG)/NF-κB受体激活蛋白配体(RANKL)表达水平,以及牙槽骨吸收的影响。方法:采用1×10~9CFU P.gingivalis口内涂布给菌5 d,磷酸盐缓冲液(PBS)洗脱后,再次口内涂布P.gingivalis给菌2 d,构建Balb/c小鼠牙周组织耐受模型。10 d后收集新鲜牙龈组织,采用western bolt检测牙龈组织中IL-1β、OPG和RANKL表达水平的变化。6周后,收集上颌骨,采用亚甲基蓝染色法检测牙槽骨吸收水平的改变。结果:耐受组小鼠牙龈组织中IL-1β表达水平较非耐受组降低,OPG/RANKL比值较非耐受组升高。亚甲基蓝染色结果显示,耐受组小鼠釉牙骨质界至牙槽嵴顶(ABC-CEJ)的距离小于非耐受组(P<0.01),说明耐受组小鼠牙槽骨吸收较非耐受组降低。结论:P.gingivalis诱导小鼠牙周组织耐受后,小鼠牙龈组织中骨吸收相关因子IL-1β分泌降低,OPG/RANKL比值升高,抑制了牙槽骨吸收。     

The immunogenicity of outer membrane proteins of haemin-depleted Porphyromonas (Bacteriodes) Gingvilis W50 in periodontal desease

The antigens from outer membrane protein extracts of Porphyromonas gingivalis (W50), grown under different haemin concentrations, were examined for binding with serum antibodies from patients with severe progressive periodontitis or from periodontally healthy control subjects. P. gingivalis was grown under haemin limitation (0.33 micrograms/ml) and haemin excess (2.5 micrograms/ml) conditions in a chemostat at a mean generation time of 6.9 h, at pH 7.5. Sarkosyl-insoluble fractions of outer membrane proteins from P. gingivalis were prepared, and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot techniques. The SDS-PAGE analysis of the outer membrane of haemin-limited P. gingivalis identified several new protein components, or changed expression of bands compared with cells grown under haemin excess. Immunoblot analysis showed IgG antibodies to 2 haemin deprivation-induced proteins in patients with severe progressive periodontitis, but not in the control sera. These results confirm the immunogenicity of some of the haemin-regulated outer membrane proteins of P. gingivalis in severe progressive periodontitis.     

Viruses in periodontal disease - a review

The purpose of this review was to evaluate the evidence supporting the hypothesis that viral infection plays a role in the development of periodontitis. An involvement in periodontal diseases has been suspected specifically for human immunodeficiency virus (HIV) and herpes viruses. An association has been demonstrated between HIV infection and some distinct forms of periodontal infection, i.e. necrotizing lesions. Furthermore, reports of increased prevalence and severity of chronic periodontitis in HIV-positive subjects suggests that HIV infection predispose to chronic periodontitis. Several studies, most of them from the same research group, have demonstrated an association of herpesviruses with periodontal disease. Viral DNA have been detected in gingival tissue, gingival cervicular fluid (GCF) and subgingival plaque from periodontaly diseased sites. In addition markers of herpesviral activation have been demonstrated in the GCF from periodontal lesions. Active human cytomegalovirus (HCMV) replication in periodontal sites may suggest that HCMV re-activation triggers periodontal disease activity. Concerns regarding sampling, methods and interpretation cast doubts on the role of viruses as causes of periodontal disease.     

Herpesviruses: a unifying causative factor in periodontitis?

Human cytomegalovirus and Epstein-Barr virus type 1 are discussed in this review as they relate to destructive periodontal disease in humans. Genomes of the two herpesviruses occur frequently in severe adult periodontitis, localized and generalized juvenile periodontitis, Papillon-Lefèvre syndrome periodontitis, Down's syndrome periodontitis, HIV-associated periodontitis and acute necrotizing ulcerative gingivitis. Herpesvirus infections generally involve a mild or asymptomatic primary phase followed by an asymptomatic latent phase interrupted sporadically by periods of activation, where viral replication and possibly clinical disease become manifest. Herpesvirus reactivation is triggered by a number of immunosuppressing factors, some of which have also been shown to be risk indicators of periodontal disease. Available evidence argues for the involvement of active cytomegalovirus infection in the initiation and progression of localized juvenile periodontitis and possibly other types of periodontal disease. In periodontal disease, herpesviruses may cause release of tissue-destructive cytokines, overgrowth of pathogenic periodontal bacteria, and initiation of cytotoxic or immunopathogenic events. Understanding the significance of herpesviruses in the causation and pathogenesis of destructive periodontal diseases may have important implications in future prevention and treatment of the diseases.     

The proportion of interleukin-4, interferon-gamma and interleukin-10-positive cells in Porphyromonas gingivalis–specific T-cell lines established from P. gingivalis–positive subjects

T-cell cytokine profiles in ten adult periodontitis and seven age-matched healthy or gingivitis subjects were determined. Porphyromonas gingivalis-specific T-cell lines were established from the peripheral blood of these individuals all of whom had past or present evidence of P. gingivalis infection. FACS analysis was used to determine the percentage of CD4- and CD8-positive cells in each line staining for cytoplasmic interleukin (IL)-4, interferon-gamma and IL-10. There were no differences in the mean percentage of IL-4-, interferon-gamma- or IL-10-positive T cells between the two groups. However, the individual profiles showed that the CD4 cells in five of the seven healthy or gingivitis lines had a higher proportion of interferon-gamma-positive cells, with two lines demonstrating higher percentages of IL-10- and/or IL-4-positive CD4 cells. Five of the ten adult periodontitis lines demonstrated either equal or higher percentages of IL-4-positive and/or IL-10-positive CD4 cells. With respect to the CD8 cells, two of the seven lines established from the healthy or gingivitis subjects and six of the ten adult periodontitis lines showed profiles with a higher percentage IL-4- and/or IL-10-positive cells. When the total T-cell contribution (CD4 plus CD8) for each T-cell line was determined from the individual CD4:CD8 ratios, only one of the healthy or gingivitis lines showed a profile with a higher proportion of IL-10-positive cells, while the results for the adult periodontitis lines were the same as indicated for the CD4 cell profiles, with five lines showing a higher percentage of IL-4- and/or IL-10-positive cells. In conclusion, this study has shown that in P. gingivalis-responsive T-cell lines established from adult periodontitis and healthy or gingivitis subjects, there was a predominant trend towards a higher percentage of interferon-gamma positive cells than either IL-4- or IL-10-positive cells. However, there were variations from this trend, although whether these variations indicate true susceptibility to progressive disease has yet to be determined.     

Mammalian viruses in human periodontitis

A prior investigation has demonstrated a higher prevalence of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in subgingival specimens from periodontitis patients than from gingivitis patients. This study aimed to determine the frequency of HCMV, EBV-1, EBV-2, herpes simplex virus (HSV) and human immunodeficiency virus (HIV) in subgingival samples from 27 adults who each contributed both a periodontitis and a gingivitis site. Viral detection was performed using a nested-polymerase chain reaction method. Twenty-four subjects (89%) yielded at least one of the five test viruses from deep periodontal pockets, wheras only 15 (56%) showed viruses from shallow periodontal sites ( P =0.015; chi-square test). Viral co-infection occurred more frequently in deep than in shallow periodontal sites ( P =0.015). HCMV was detected with higher frequency in deep than in shallow periodontal sites ( P =0.023). The possible periodontopathogenic mechanisms of mammalian viruses in human periodontitis are discussed. The role and importance of HCMV and other mammalian viruses in the initiation and progression of destructive periodontal disease merits further investigation.     

The proportion of interleukin-4, interferon-gamma and interleukin-10-positive cells in Porphyromonas gingivalis–specific T-cell lines established from P. gingivalis–positive subjects

T-cell cytokine profiles in ten adult periodontitis and seven age-matched healthy or gingivitis subjects were determined. Porphyromonas gingivalis–specific T-cell lines were established from the peripheral blood of these individuals all of whom had past or present evidence of P. gingivalis infection. FACS analysis was used to determine the percentage of CD4- and CD8-positive cells in each line staining for cytoplasmic interleukin (IL)-4, interferon-gamma and IL-10. There were no differences in the mean percentage of IL-4-, interferon-gamma- or IL-10-positive T cells between the two groups. However, the individual profiles showed that the CD4 cells in five of the seven healthy or gingivitis lines had a higher proportion of interferon-gamma-positive cells, with two lines demonstrating higher percentages of IL-10- and/or IL-4-positive CD4 cells. Five of the ten adult periodontitis lines demonstrated either equal or higher percentages of IL-4-positive and/or IL-10-positive CD4 cells. With respect to the CD8 cells, two of the seven lines established from the healthy or gingivitis subjects and six of the ten adult periodontitis lines showed profiles with a higher percentage IL-4- and/or IL-10-positive cells. When the total T-cell contribution (CD4 plus CD8) for each T-cell line was determined from the individual CD4:CD8 ratios, only one of the healthy or gingivitis lines showed a profile with a higher proportion of IL-10-positive cells, while the results for the adult periodontitis lines were the same as indicated for the CD4 cell profiles, with five lines showing a higher percentage of IL-4- and/or IL-10-positive cells. In conclusion, this study has shown that in P. gingivalis–responsive T-cell lines established from adult periodontitis and healthy or gingivitis subjects, there was a predominant trend towards a higher percentage of interferon-gamma positive cells than either IL-4- or IL-10-positive cells. However, there were variations from this trend, although whether these variations indicate true susceptibility to progressive disease has yet to be determined.     

Effect of chlorhexidine on the adherence properties of Porphyromonas gingivalis

Abstract Chlorhexidine is a bisbiguanide compound that possesses substantive and antibacterial properties. The aim of the present investigation was to evaluate the effect of chlorhexidine on the adherence of Porphyromonas gingivalis to epithelial cells and erythrocytes. Both pretreatment of bacterial cells with chlorhexidine (>20 μg/ml) and incorporation of chlorhexidine in an adherence assay markedly affected the ability of P. gingivalis to adhere to epithelial cells. Chlorhexidine also strongly inhibited hemagglotination by P. gingivalis. It is suggested that chlorhexidine binds to cells, altering the structural conformation of the outer membrane and reducing adherence. This ability of chlorhexidine to prevent bacterial adherence may represent an additional mechanism by which this antimicrobial agent exerts its beneficial clinical effect when used as an adjunct to periodontal therapy.     

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.     

The effect of incubation temperature on the specificity of the BANA (N-benzoyl-DL-arginine-naphthylamide) test

The hydrolysis of BANA by subgingival plaque samples is associated with the presence of either Treponema denticola, Porphyromonas gingivalis , and/or Bacteroides farsythus. A protocol in which pure cultures were incubated for 15 min at 55°C detected about 5 × 10⁵ CPU of P. gingivalis and 1 × 10⁶ CPU of T. denticola. Clinical studies indicated that the BANA test in this configuration will detect about 10⁴ organisms in vivo as compared with the 10⁵ to 10⁶ organisms found with in vitro grown cells. The BANA test can be made less sensitive by decreasing the time and/or temperature of incubation, which could improve the specificity of the test. In the present study we determined the incubation parameters that would give optimal specificity when the plaque samples were removed from sites of gingival health. Twenty-six approximal plaque samples were taken from each of 90 clinically healthy subjects and incubated with the BANA substrate on PerioScan cards (Oral-B Laboratories) for 5 and 15 min at 35°, 45°, and 55°C. Subjects were randomly assigned to the various temperatures. Wooden toothpicks were inserted interproximally in all sites anterior to distal of the first molars and then each side of the toothpick was wiped onto the PerioScan card. The specificity of the BANA test relative to clinical health was 96% when the cards were incubated for 5 min at 35°C, but decreased to 50–70% when the cards were incubated for 15 min at 35°C or for 5 and 15 min at 45°C and 55°C. These findings indicate that the specificity of the BANA test can be improved by shortening the incubation period to 5 min and by reducing the incubation temperature to 35°C.