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1.  Antigenically Diverse Reference Strains and Autologous Strains of Actinobacillus actinomycetemcomitans Are Equally Efficient Antigens in Enzyme-Linked Immunosorbent Assay Analysis 
Journal of Clinical Microbiology  2002;40(12):4640-4645.
Actinobacillus actinomycetemcomitans is a major pathogen in periodontitis. Data on the clinical relevance of serum immunoglobulin G (IgG) antibody levels against this species are controversial. The aim of the present study was to elucidate how different strains used as antigens in enzyme-linked immunosorbent assay (ELISA) influence the detection of individuals with elevated serum IgG levels against A. actinomycetemcomitans. We hypothesized that the highest antibody levels are targeted to the autologous strains. A total of 19 strains—six antigenically diverse reference strains (serotypes a through e and a nonserotypeable strain) and 13 serotyped autologous strains—were used as whole-cell antigens in ELISA. Serum samples were from 26 untreated adult patients with periodontitis, whose subgingival bacterial samples were either culture positive (n = 13) or culture negative (n = 13) for A. actinomycetemcomitans, and from 10 culture-negative nonperiodontitis subjects. The highest individual (P < 0.05) IgG levels against the reference strains were most commonly against serotypes a and b in patients and against serotype c in nonperiodontitis subjects. The culture-positive patients had the highest (P < 0.05) IgG antibody levels against their autologous strains and against the reference strains of the same serotype. On the contrary, for these patients the levels of antibody against the reference strains of other serotypes were comparable to those of the nonperiodontitis subjects. The results indicated that the serum IgG antibody levels against A. actinomycetemcomitans strongly depend on the strains used as antigens in the ELISA. Elevated serum IgG levels against A. actinomycetemcomitans can be detected equally well using either the autologous strains or a variety of antigenically diverse reference strains as antigens.
doi:10.1128/JCM.40.12.4640-4645.2002
PMCID: PMC154631  PMID: 12454165
2.  Improved Multiplex PCR Using Conserved and Species-Specific 16S rRNA Gene Primers for Simultaneous Detection of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis 
Journal of Clinical Microbiology  1999;37(11):3504-3508.
Among putative periodontal pathogens, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, and Porphyromonas gingivalis are most convincingly implicated as etiological agents in periodontitis. Therefore, techniques for detection of those three species would be of value. We previously published a description of a multiplex PCR that detects A. actinomycetemcomitans and P. gingivalis. The present paper presents an improvement on that technique, which now allows more sensitive detection of all three periodontal pathogens. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans, B. forsythus, and P. gingivalis cells. Primer specificity was tested against (i) all gene sequences from the GenBank-EMBL database, (ii) six A. actinomycetemcomitans, one B. forsythus, and four P. gingivalis strains, (iii) eight different species of oral bacteria, and (iv) supra- and subgingival plaque samples from 20 healthy subjects and subgingival plaque samples from 10 patients with periodontitis. The multiplex PCR had a detection limit of 10 A. actinomycetemcomitans, 10 P. gingivalis, and 100 B. forsythus cells. Specificity was confirmed by the fact that (i) none of our forward primers were homologous to the 16S rRNA genes of other oral species, (ii) amplicons of predicted size were detected for all A. actinomycetemcomitans, B. forsythus, and P. gingivalis strains tested, and (iii) no amplicons were detected for the eight other bacterial species. A. actinomycetemcomitans, B. forsythus, and P. gingivalis were detected in 6 of 20, 1 of 20, and 11 of 20 of supragingival plaque samples, respectively, and 4 of 20, 7 of 20, and 13 of 20 of subgingival plaque samples, respectively, from periodontally healthy subjects. Among patients with periodontitis, the organisms were detected in 7 of 10, 10 of 10, and 7 of 10 samples, respectively. The simultaneous detection of three periodontal pathogens is an advantage of this technique over conventional PCR assays.
PMCID: PMC85679  PMID: 10523542
3.  Multiplex PCR using conserved and species-specific 16S rRNA gene primers for simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. 
Journal of Clinical Microbiology  1996;34(11):2674-2678.
Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis are strongly associated with periodontitis. However, little is known about their distribution in periodontally healthy individuals, because culturing techniques are not sufficiently sensitive. A modified multiplex PCR was developed to address that question. Our method uses two species-specific forward primers in combination with a single reverse primer. These primers target variable and conserved regions of the 16S rRNA gene. Sensitivity was determined by testing serial dilutions of A. actinomycetemcomitans and P. gingivalis cells. Primer specificity was tested against (i) six A. actinomycetemcomitans strains and four P. gingivalis strains, (ii) seven different species of oral bacteria, and (iii) supra- and subgingival plaque from 20 subjects. The multiplex PCR had a lower limit of detection of 2 A. actinomycetemcomitans and 30 P. gingivalis cells. Species-specific amplicons were obtained for all A. actinomycetemcomitans and P. gingivalis strains tested and did not occur with seven other bacterial species unless A. actinomycetemcomitans and P. gingivalis were added. Neither target species was detected in supragingival plaque; A. actinomycetemcomitans was detected in one subgingival specimen, and P. gingivalis was detected in another. When plaque samples were spiked with 10 A. actinomycetemcomitans cells and 100 P. gingivalis cells, species-specific amplicons were detected. These findings show our multiplex PCR to be highly sensitive and specific while allowing simultaneous detection of A. actinomycetemcomitans and P. gingivalis. This assay has potential applications in epidemiological studies, diagnosis, treatment planning, and monitoring of periodontal pathogens.
PMCID: PMC229384  PMID: 8897163
4.  Tracking of plasma antibodies against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis during 15 years 
Journal of Oral Microbiology  2009;1:10.3402/jom.v1i0.1979.
Background
Plasma antibody measurements of antibody levels to periodontal pathogens may be used to support diagnosis, disease activity, classification, and prognosis of periodontitis.
Objective
The aim of this study was to investigate the long-term stability of plasma antibody levels against Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis.
Design
Plasma immunoglobulin G (IgG) antibody levels against the pathogens were analyzed annually during 15 years from 21 voluntary subjects, whose periodontal status was not known at the point of selection. The total number of plasma samples was 315. In connection of the last sampling, the clinical and radiographic periodontal status was examined. Pooled bacterial samples from periodontal pockets, as well as salivary samples were collected for A. actinomycetemcomitans and P. gingivalis detection, and antibody determinations, respectively. According to the clinical status, six subjects had periodontitis, whereas 15 did not.
Results
Plasma IgG-class antibody levels to periodontal pathogens remained extremely stable during the 15-year period and no significant (p>0.05) intra-individual variations were observed. Retrospectively, the average plasma IgG antibody levels against A. actinomycetemcomitans and P. gingivalis were 1.6–2.3 (p<0.05) and 1.4–1.7 (p<0.05) fold higher in the subjects with periodontitis than those without, respectively, during the whole 15-year tracking. As expected, at the time of the periodontal examination the plasma and salivary IgG antibody levels were associated both with periodontitis and bacterium-positivity.
Conclusions
Plasma IgG levels against A. actinomycetemcomitans and P. gingivalis are extremely stable during 15 years both in subjects with and without periodontitis.
doi:10.3402/jom.v1i0.1979
PMCID: PMC3077000  PMID: 21523211
Aggregatibacter actinomycetemcomitans; longitudinal studies; periodontitis; plasma; oral infections; Porphyromonas gingivalis; saliva
5.  Periodontitis Is Associated with a Low Concentration of Vitamin C in Plasma 
This study aimed to clarify how concentrations of vitamin C in plasma relate to the serology of periodontitis. The random sample used comprised 431 men, 194 from Finland and 237 from Russia. The plasma vitamin C concentration was determined by o-phtaldialdehyde-fluorometry, and serum immunoglobulin G antibodies to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis were determined by a multiserotype enzyme-linked immunosorbent assay (ELISA). The mean plasma vitamin C concentration was higher (P < 0.001) in Finnish subjects (mean ± standard deviation, 4.5 ± 2.8 mg/liter) than in Russian subjects (1.4 ± 1.8 mg/liter). Mean antibody levels to both A. actinomycetemcomitans (4.7 ± 3.6 versus 5.2 ± 3.1 ELISA units [P = 0.05]) and P. gingivalis (5.7 ± 2.5 versus 7.6 ± 2.9 ELISA units [P < 0.001]) were lower in Finnish men than in Russian men. In the combined Finnish and Russian population, the antibody levels to P. gingivalis were negatively correlated with vitamin C concentrations (r = −0.22; P < 0.001); this association remained statistically significant (P = 0.010) in a linear regression model after adjustment for confounding factors. The proportion of P. gingivalis-seropositive subjects decreased with increasing vitamin C concentrations (P for trend, <0.01), but no trend was seen among A. actinomycetemcomitans-seropositive subjects. In conclusion, P. gingivalis infection is associated with low concentrations of vitamin C in plasma, which may increase colonization of P. gingivalis or disturb the healing of the infected periodontium.
doi:10.1128/CDLI.10.5.897-902.2003
PMCID: PMC193894  PMID: 12965924
6.  Periodontitis in established rheumatoid arthritis patients: a cross-sectional clinical, microbiological and serological study 
Arthritis Research & Therapy  2012;14(5):R222.
Introduction
The association between rheumatoid arthritis (RA) and periodontitis is suggested to be linked to the periodontal pathogen Porphyromonas gingivalis. Colonization of P. gingivalis in the oral cavity of RA patients has been scarcely considered. To further explore whether the association between periodontitis and RA is dependent on P. gingivalis, we compared host immune responses in RA patients with and without periodontitis in relation to presence of cultivable P. gingivalis in subgingival plaque.
Methods
In 95 RA patients, the periodontal condition was examined using the Dutch Periodontal Screening Index for treatment needs. Subgingival plaque samples were tested for presence of P. gingivalis by anaerobic culture technique. IgA, IgG and IgM antibody titers to P. gingivalis were measured by ELISA. Serum and subgingival plaque measures were compared to a matched control group of non-RA subjects.
Results
A higher prevalence of severe periodontitis was observed in RA patients in comparison to matched non-RA controls (27% versus 12%, p < 0.001). RA patients with severe periodontitis had higher DAS28 scores than RA patients with no or moderate periodontitis (p < 0.001), while no differences were seen in IgM-RF or ACPA reactivity. Furthermore, RA patients with severe periodontitis had higher IgG- and IgM-anti P. gingivalis titers than non-RA controls with severe periodontitis (p < 0.01 resp. p < 0.05), although subgingival occurrence of P. gingivalis was not different.
Conclusions
Severity of periodontitis is related to severity of RA. RA patients with severe periodontitis have a more robust antibody response against P. gingivalis than non-RA controls, but not all RA patients have cultivable P. gingivalis.
doi:10.1186/ar4061
PMCID: PMC3580533  PMID: 23075462
7.  Immune response to cytolethal distending toxin of Aggregatibacter actinomycetemcomitans in periodontitis patients 
Journal of periodontal research  2010;45(4):471-480.
Background and Objective
Cytolethal distending toxin (CDT) is a genotoxin produced by Aggregatibacter actinomycetemcomitans. In spite of its association with pathogenesis, little is known about the humoral immune response against the CDT. This study aimed to test whether subgingival colonization and humoral response to A. actinomycetemcomitans would lead to a response against CDT.
Material and Methods
Sera from periodontally healthy, localized and generalized aggressive periodontitis and chronic periodontitis subjects (n = 80) were assessed for immunoglobulin G titers to A. actinomycetemcomitans serotypes a/b/c and to each CDT subunit (CdtA, CdtB and CdtC) by ELISA. A. actinomycetemcomitans subgingival levels and neutralization of CDT activity were also analyzed.
Results
Sera from 75.0% localized and 81.8% generalized aggressive periodontitis patients reacted to A. actinomycetemcomitans. A response to serotype b was detected in localized (66.7%) and generalized aggressive periodontitis (54.5%). Reactivity to A. actinomycetemcomitans correlated with subgingival colonization (R = 0.75, p < 0.05). There was no correlation between A. actinomycetemcomitans colonization or response to serotypes and the immunoglobulin G response to CDT subunits. Titers of immunoglobulin G to CdtA and CdtB did not differ among groups; however, sera of all generalized aggressive periodontitis patients reacted to CdtC. Neutralization of CDT was not correlated with levels of antibodies to CDT subunits.
Conclusion
Response to CdtA and CdtB did not correlate with the periodontal status of the subject in the context of an A. actinomycetemcomitans infection. However, a response to CdtC was found in sera of generalized but not of localized aggressive periodontitis subjects. Differences in response to CdtC between generalized and localized aggressive periodontitis subjects indicate that CDT could be expressed differently by the infecting strains. Alternatively, the antibody response to CdtC could require the colonization of multiple sites.
doi:10.1111/j.1600-0765.2009.01260.x
PMCID: PMC3534802  PMID: 20337882
Aggregatibacter actinomycetemcomitans; cytolethal distending toxin; enzyme-linked immunosorbent assay (ELISA); immunoglobulin G; serotype
8.  Immunoglobulin G response to subgingival gram-negative bacteria in human subjects. 
Infection and Immunity  1984;45(1):47-51.
Serum and gingival crevicular fluid from normal healthy adults and patients with periodontitis were screened for immunoglobulin G antibodies to antigens from Bacteroides gingivalis 381, Bacteroides intermedius 24, Bacteroides loescheii ATCC 15930, Fusobacterium nucleatum ATCC 25586, Eikenella corrodens 1073, Actinobacillus actinomycetemcomitans ATCC 29522, and Capnocytophaga sp. strain M-12. Immunoglobulin G antibody titers to the antigens were measured by an enzyme-linked immunosorbent assay. The antibody levels to B. gingivalis in serum and gingival crevicular fluid were significantly higher in the samples from patients with periodontitis than in samples from healthy individuals. Although there were individual differences within patient groups, a positive correlation (P less than 0.01) was found between the serum immunoglobulin G levels to B. gingivalis and the development of periodontitis. The antibodies to F. nucleatum (P less than 0.05), E. corrodens (P less than 0.05), and A. actinomycetemcomitans were slightly higher in patients with periodontitis than in normal subjects. There were no remarkable differences between the two groups in titers to B. intermedius, B. loescheii, and Capnocytophaga sp.
PMCID: PMC263260  PMID: 6735473
9.  Serology of oral Actinobacillus actinomycetemcomitans and serotype distribution in human periodontal disease. 
Infection and Immunity  1983;41(1):19-27.
Actinobacillus actinomycetemcomitans from the human oral cavity was serologically characterized with rabbit antisera to the type strain NCTC 9710; a number of reference strains, including Y4, ATCC 29522, ATCC 29523, ATCC 29524, NCTC 9709; and our own isolates representative of each of 10 biotypes. Using immunoabsorbed antisera, we identified three distinct serotypes by immunodiffusion and indirect immunofluorescence. Serotype a was represented by ATCC 29523 and SUNYaB 75; serotype b was represented by ATCC 29522 and Y4; and serotype c was represented by NCTC 9710 and SUNYaB 67. Indirect immunofluorescence revealed no reaction between the three A. actinomycetemcomitans serotype-specific antisera and 62 strains representing 23 major oral bacterial species. Distinct from the serotype antigens were at least one A. actinomycetemcomitans species common antigen and an antigen shared with other Actinobacillus species, Haemophilus aphrophilus, and Haemophilus paraphrophilus. All serotype a A. actinomycetemcomitans strains failed to ferment xylose, whereas all serotype b organisms fermented xylose. Serotype c included xylose-positive as well as xylose-negative strains. A total of 301 isolates of A. actinomycetemcomitans from the oral cavity of 74 subjects were serologically categorized by indirect immunofluorescence with serotype-specific rabbit antisera. Each patient harbored only one serotype of A. actinomycetemcomitans. Fourteen healthy subjects, five diabetics, and seventeen adult periodontitis patients exhibited serotypes a and b in approximately equal frequency, whereas serotype c was found less frequently. In contrast, in 29 localized juvenile periodontitis patients, the incidence of serotype b was approximately two times higher than that of serotypes a or c, suggesting a particularly high periodontopathic potential of A. actinomycetemcomitans serotype b strains. In subjects infected with A. actinomycetemcomitans, serum antibodies were detected to the serotype antigens, indicating that these antigens may play a role in the pathogenesis of periodontal disease.
Images
PMCID: PMC264736  PMID: 6407997
10.  Assessment of serum antibody patterns and analysis of subgingival microflora of members of a family with a high prevalence of early-onset periodontitis. 
Infection and Immunity  1985;49(3):742-750.
In a study of members of a large family with a high prevalence of early-onset periodontitis, we sampled the subgingival microflora and characterized 40 isolates from each sample. We surveyed serum samples by enzyme-linked immunosorbent assay for antibodies reacting with any of a panel of 21 periodontal bacteria. The mother and 7 of her 13 children had early-onset periodontitis. Bacteroides gingivalis was not detected in the subgingival flora of any affected or unaffected family member, and Actinobacillus actinomycetemcomitans was isolated from only one affected child. Capnocytophaga ochracea was isolated from five of seven affected children and from none of their normal siblings. We found no significant differences among the floras from family members who had rapidly progressive, juvenile, and prepubertal forms of periodontitis. Elevated levels of serum antibody reacting with one or more of the bacteria tested were found in all family members with disease, but in only one periodontally normal family member. Both children with prepubertal periodontitis had antibodies reacting with C. sputigena, a species not found in their subgingival floras, but with none of the other bacteria tested. All remaining affected family members had antibodies to one or more serotypes of A. actinomycetemcomitans, and four had antibodies reacting with additional bacteria, including C. sputigena, Eikenella corrodens, Fusobacterium nucleatum, and Haemophilus aphrophilus. Sera from patients contained antibodies specific for putative periodontal pathogens not found in their pocket flora, and conversely, putative periodontal pathogens for which no serum antibodies were found frequently comprised a large proportion (10% or more) of the pocket flora. In no case were both the bacterium and its antibody found. These observations are suggestive of sequential infection in the early-onset forms of periodontitis and of induction of protective immunity against reinfection by the same microorganism.
PMCID: PMC261261  PMID: 4030102
11.  Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis. 
Molecular Pathology  1997;50(6):291-297.
AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in periodontitis, owing to the release of proteinases from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence.
Images
PMCID: PMC379662  PMID: 9536278
12.  Association between Polycystic Ovary Syndrome, Oral Microbiota and Systemic Antibody Responses 
PLoS ONE  2014;9(9):e108074.
Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.
doi:10.1371/journal.pone.0108074
PMCID: PMC4169459  PMID: 25232962
13.  Prevalence and distribution of Aggregatibacter actinomycetemcomitans and its cdtB gene in subgingival plaque of Chinese periodontitis patients 
BMC Oral Health  2014;14:37.
Background
Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans) is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. This study was to elucidate the distribution of A.actinomycetemcomitans and the prevalence of its cdtB gene in Chinese subjects.
Methods
A total of 255 subgingival samples were obtained from 30 subjects. Samples were collected from periodontal healthy sites as well as shallow, moderate and deep pockets. The absolute quantity of A.actinomycetemcomitans and cdtB gene were determined by real-time polymerase chain reaction.
Results
A.actinomycetemcomitans was detected in 92 of 105 (87.6%) samples of aggressive periodontitis (AgP) patients, in 73 of 79 (92.4%) samples of chronic periodontitis ( CP) patients and in 5 of 71 (7.0%) samples of periodontal healthy subjects. The cdtB gene was detected in 72 sites (78.3%) with AgP infected with A.actinomycetemcomitans, 54 sites (74.0%) with CP infected with A.actinomycetemcomitans and none in healthy sites infected with A.actinomycetemcomitans. In addition, quantity of A.actinomycetemcomitans and cdt gene in samples from deep pockets were significant larger than moderate, shallow and healthy sites (P < 0.05). In comparison to CP, AgP patients were infected with increased numbers of cdt genotype in deep pockets (P < 0.05).
Conclusion
This study suggests that the cdtB gene are prevalent in A.actinomycetemcomitans, and the distribution of cdt genotype strain may be correlated with AgP and serious periodontal inflammation.
doi:10.1186/1472-6831-14-37
PMCID: PMC4002197  PMID: 24725913
Aggregatibacter actinomycetemcomitans; Cytolethal distending toxin; Subgingival plaque; Real-time PCR
14.  Serum Antibodies to Porphyromonas gingivalis Chaperone HtpG Predict Health in Periodontitis Susceptible Patients 
PLoS ONE  2008;3(4):e1984.
Background
Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis.
Methodology/Principal Findings
ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043) but not anti-Fusobacterium nucleatum (an oral opportunistic commensal) HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018). There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG.
Conclusions/Significance
Our results suggest: 1) anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2) may augment the host defence to periodontitis and 3) a unique peptide of P. gingivalis HtpG offers significant potential as an effective diagnostic target and vaccine candidate. These results are compatible with a novel immune control mechanism unrelated to direct binding of bacteria.
doi:10.1371/journal.pone.0001984
PMCID: PMC2291562  PMID: 18431474
15.  Association between infection of different strains of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in subgingival plaque and clinical parameters in chronic periodontitis*  
Objective: The aim of this study was to investigate subgingival infection frequencies of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans strains with genetic variation in Chinese chronic periodontitis (CP) patients and to evaluate its correlation with clinical parameters. Methods: Two multiplex polymerase chain reaction (PCR) assays were developed to detect the 16SrDNA, collagenase (prtC) and fimbria (fimA) genes of P. gingivalis and the 16SrDNA, leukotoxin (lktA) and fimbria-associated protein (fap) genes of A. actinomycetemcomitans in 60 sulcus samples from 30 periodontal healthy subjects and in 122 subgingival plaque samples from 61 patients with CP. The PCR products were further T-A cloned and sent for nucleotide sequence analysis. Results: The 16SrDNA, prtC and fimA genes of P. gingivalis were detected in 92.6%, 85.2% and 80.3% of the subgingival plaque samples respectively, while the 16SrDNA, lktA and fap genes of A. actinomycetemcomitans were in 84.4%, 75.4% and 50.0% respectively. Nucleotide sequence analysis showed 98.62%~100% homology of the PCR products in these genes with the reported sequences. P. gingivalis strains with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ were predominant in deep pockets (>6 mm) or in sites with attachment loss ≥5 mm than in shallow pockets (3~4 mm) or in sites with attachment loss ≤2 mm (P<0.05). P. gingivalis strains with prtC+/fimA+ also showed higher frequency in gingival index (GI)=3 than in GI=1 group (P<0.05). Conclusion: Infection of P. gingivalis with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ correlates with periodontal destruction of CP in Chinese. Nonetheless P. gingivalis fimA, prtC genes and A. actinomycetemcomitans lktA gene are closely associated with periodontal destruction, while A. actinomycetemcomitans fap gene is not.
doi:10.1631/jzus.2007.B0121
PMCID: PMC1791058  PMID: 17266188
Porphyromonas gingivalis; Actinobacillus actinomycetemcomitans; Strain; Periodontitis; PCR
16.  Humoral immunity of older adults with periodontal disease to Porphyromonas gingivalis. 
Infection and Immunity  1991;59(12):4363-4370.
The effect of age on the humoral response to Porphyromonas gingivalis was assessed in groups of adults (25 to 54 years and 55 to 74 years) with periodontal disease and compared with that in age-matched healthy controls. To determine whether there was an antibody response against P. gingivalis, we measured serum antibodies against whole cells of P. gingivalis 381, A7A1-28, and W50. In addition, antibody levels against purified P. gingivalis outer membrane proteins (i.e., the 43-kDa fimbrial protein and a 75-kDa protein) were also evaluated. Elderly subjects showed the same response to P. gingivalis as younger subjects. Immunoglobulin G (IgG) antibodies to both purified proteins were also elevated in both diseased groups as compared with the normal groups. Total serum IgG, IgA, and IgM levels were also determined by an enzyme-linked immunosorbent assay for all four groups. Total serum IgG levels were elevated in older adults with periodontitis and total IgA levels were elevated in both groups of older adults compared with the younger groups of similar disease status. Total serum IgM levels were comparable for the four groups. Antinuclear antibody titers were assessed in the two groups of older adults and were also found to be higher for the group with periodontitis. These studies show that older adults as well as younger adults have markedly elevated specific antibodies of the IgG and IgA classes to antigens of P. gingivalis, a putative pathogen in both groups. Furthermore, older adults with periodontitis have significantly elevated levels of total serum IgG which may possibly be related to higher levels of autoantibodies.
PMCID: PMC259050  PMID: 1682261
17.  Cleavage of IgG1 in GCF is associated with presence of Porphyromonas gingivalis 
Journal of periodontal research  2012;48(4):458-465.
Background and Objectives
Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of P. gingivalis and other periodontopathogens.
Material and methods
GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis, and five periodontally-healthy individuals. The bacterial loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia, and Tannerella forsythia were analysed by real-time PCR, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA.
Results
Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in the healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. P. gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (p < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of T. forsythia and P. intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of P. gingivalis (r = 0.425, p < 0.01). The presence of Kgp (range 0.07–10.98 ng/ml) was associated with proteolytic fragments of IgG1 (p < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp.
Conclusion
In patients with periodontitis cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by P. gingivalis.
doi:10.1111/jre.12027
PMCID: PMC3566341  PMID: 23116446
IgG; GCF; periodontitis Porphyromonas gingivalis; gingipains
18.  Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis. 
Infection and Immunity  1996;64(10):4226-4230.
The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP).
PMCID: PMC174360  PMID: 8926092
19.  High incidence of Aggregatibacter actinomycetemcomitans infection in patients with cerebral infarction and diabetic renal failure: a cross-sectional study 
BMC Infectious Diseases  2013;13:557.
Background
Recent epidemiological studies suggest that periodontitis is a major risk factor for renal failure and cerebral infarction. The aim of this study was to evaluate the association among periodontitis, renal failure, and cerebral infarction, focusing on microbiological and immunological features.
Methods
Twenty-one patients treated with hemodialysis (HD) were enrolled in this study. They were 8 with diabetic nephropathy and 13 with non-diabetic nephropathy. Blood examination, periodontal examination, brain magnetic resonance image (MRI), and dental radiography were performed on all patients. Subgingival plaque, saliva, and blood samples were analyzed for the periodontal pathogens, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), Porphyromonas gingivalis (P. gingivalis), and Prevotella intermedia (P. intermedia) using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA).
Results
We found that the patients with diabetic nephropathy had more A. actinomycetemcomitans compared with non-diabetic nephropathy (P = 0.038) in dental plaque. Furthermore, the patients with diabetic nephropathy showed a significantly higher incidence of cerebral infarction compared with those with non-diabetic nephropathy (P = 0.029). Clinical oral and radiographic scores tended to be higher among patients in the diabetic nephropathy group than in the non-diabetic nephropathy group.
Conclusions
Periodontal pathogens, particularly A. actinomycetemcomitans, may play a role, at least a part, in the development of cerebral infarction in Japanese HD patients with diabetic nephropathy.
doi:10.1186/1471-2334-13-557
PMCID: PMC4222637  PMID: 24267704
Periodontitis; Aggregatibacter actinomycetemcomitans; Diabetic nephropathy; Cerebral infarction
20.  Double-blind analysis of the relation between adult periodontitis and systemic host response to suspected periodontal pathogens. 
Infection and Immunity  1986;52(3):768-776.
By using a sensitive enzyme-linked immunosorbent assay, 200 randomly selected sera from Red Cross blood donors were screened for immunoglobulin G (IgG), IgA, and IgM levels against Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, and Bacteroides intermedius. A subgroup of 79 blood donors was clinically examined for type and extent of periodontal destruction, and serological and clinical data were subjected in all possible dual combinations to correlation analyses. The results revealed that the majority of the blood donors suffered from moderate to severe adult periodontitis, often coupled with severe gingival inflammation. No cases of localized juvenile periodontitis or rapidly progressive periodontitis were observed. The extent of periodontal destruction proved to be significantly correlated only to the IgG response levels against B. gingivalis. Corresponding correlation tests assessing the relationships of loss of attachment, bone loss, pocket depth, and papillary bleeding index with the IgG responses to A. actinomycetemcomitans were of marginal significance, while the IgG responses to B. intermedius revealed no relationship to the periodontal health status. The specific IgM responses proved to be unrelated to the clinical parameters, but interestingly, they were found to be highly correlated with each other. Specific IgA levels were frequently too low for enzyme-linked immunosorbent assay testing and, therefore, had to be exempted from statistical analyses. Assessments of the serotype specificity of strongly elevated IgG responses to A. actinomycetemcomitans disclosed no evidence for an association of a particular serotype-specific IgG response with the occurrence of adult periodontal destruction. In contrast to results of earlier studies, a number of sera were found to contain strongly elevated IgG levels against two or even all three serotypes. Although derived by an alternative approach, the reported results largely corroborate earlier observations linking only the occurrence of elevated anti-B. gingivalis IgG responses to the presence of marked periodontal lesions in adults.
PMCID: PMC260925  PMID: 3710586
21.  Actinobacillus actinomycetemcomitans Serotype d-Specific Antigen Contains the O Antigen of Lipopolysaccharide  
Infection and Immunity  2003;71(9):5005-5011.
Actinobacillus actinomycetemcomitans is a gram-negative, facultatively anaerobic bacterium which is associated especially with aggressive forms of periodontitis. Contradictory results on the localization of the A. actinomycetemcomitans serotype-specific antigen have been reported. The aim of the present study was to characterize the A. actinomycetemcomitans serotype d-specific antigen. The antigen was isolated by affinity chromatography. The affinity column was prepared from immunoglobulin G isolated from rabbit antiserum raised against A. actinomycetemcomitans serotype d. The isolated antigen was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and silver staining, all of which revealed a ladder-like structure typical for the O antigen of lipopolysaccharide (LPS). In a displacement enzyme-linked immunosorbent assay (ELISA), the isolated antigen displaced in a concentration-dependent manner the binding of the polyclonal rabbit antiserum raised against A. actinomycetemcomitans serotype d to the competing whole-cell serotype d antigen. The isolated antigen contained LPS, and an equal concentration of LPS isolated from A. actinomycetemcomitans serotype d gave a similar displacement curve in the ELISA. In order to test the immunogenic properties of the isolated antigen, it was used to immunize a rabbit. The antiserum raised against the isolated antigen displayed specificity in Western blotting and ELISA similar to that of antibody raised against LPS isolated from A. actinomycetemcomitans serotype d. In conclusion, our results show that the A. actinomycetemcomitans serotype d-specific antigen contains the O-antigenic structure of LPS.
doi:10.1128/IAI.71.9.5005-5011.2003
PMCID: PMC187330  PMID: 12933843
22.  Serum antibodies to oral Bacteroides asaccharolyticus (Bacteroides gingivalis): relationship to age and periondontal disease. 
Infection and Immunity  1981;31(1):182-192.
An enzyme-linked immunosorbent assay microplate method was used for measuring levels of antibody specific for the oral serotype of Bacteroides asaccharolyticus (Bacteroides gingivalis) in serum samples obtained from umbilical cords, infants, children, periodontally normal adults, and edentulous adults. Serum from patients with various periodontal diseases, including adult periodontitis, localized juvenile periodontitis, generalized juvenile periodontitis, post-localized juvenile periodontitis, and acute necrotizing ulcerative gingivitis, were also studied. A positive correlation between increase in age and increase in both prevalence and level of specific antibody in the G, A, and M classes of immunoglobulins was observed. This indicates that antibodies reactive with oral B. asaccharolyticus found in up to 84% of normal adults are natural antibodies, presumably with a protective role. Among the patient groups, those with adult periodontitis were found to have levels of immunoglobulin G antibodies to oral B. asaccharolyticus that were five times higher than the antibody levels found in control subjects. The levels of IgG antibodies to this organism in the other patient groups were comparable to the levels found in the control group. However, 50% of the individuals in the generalized juvenile periodontitis group had high levels of immunoglobulin G antibodies to B. asaccharolyticus, suggesting heterogeneity with respect to immune response in these patients. These results indicate that antibodies to oral B. asaccharolyticus (B. gingivalis) occur at low levels in most normal children and adults and that the rise in titer of the specific antibodies of each major class of immunoglobulins parallels the ontogenic change in serum levels of that isotype. In contrast, there is a marked increase in titer of immunoglobulin G antibodies to oral B. asaccharolyticus in the group of patients with adult periodontitis and in patients with the generalized form of juvenile periodontitis.
Images
PMCID: PMC351768  PMID: 7216444
23.  Isolation of Porphyromonas gingivalis and Detection of Immunoglobulin A Specific to Fimbrial Antigen in Gingival Crevicular Fluid 
Journal of Clinical Microbiology  1998;36(8):2322-2325.
The present study evaluated the prevalence of Porphyromonas gingivalis and the correlation between the bacterial culture method and the detection of immunoglobulin A (IgA) specific to the P. gingivalis fimbrial antigen in gingival crevicular fluid (GCF). P. gingivalis was isolated from 78.3% of subgingival plaque samples obtained from active sites and 34.7% of those from inactive sites of periodontal patients. P. gingivalis was isolated from only 4.7% of healthy subjects (control group). Immunoglobulins specific to the P. gingivalis fimbrial antigen were detected by enzyme-linked immunosorbent assay (ELISA). The overall agreement between the results of the P. gingivalis culture method and the results of specific IgA detection in periodontal patients was 71.7% for active sites and 58.7% for inactive sites. IgA specific to P. gingivalis was absent in GCF from all of the sites of healthy subjects. The results suggest that P. gingivalis is associated with the local production of specific IgA. The detection of IgA antibodies specific to P. gingivalis in GCF by ELISA may be used as a predictive parameter to reveal the early phase of the activation of recurrent periodontal infections.
PMCID: PMC105039  PMID: 9666013
24.  Immunoglobulin G (IgG) Class, but Not IgA or IgM, Antibodies to Peptides of the Porphyromonas gingivalis Chaperone HtpG Predict Health in Subjects with Periodontitis by a Fluorescence Enzyme-Linked Immunosorbent Assay▿  
Clinical and Vaccine Immunology : CVI  2009;16(12):1766-1773.
Chaperones are molecules found in all cells and are critical in stabilization of synthesized proteins, in repair/removal of defective proteins, and as immunodominant antigens in innate and adaptive immunity. Subjects with gingivitis colonized by the oral pathogen Porphyromonas gingivalis previously demonstrated levels of anti-human chaperone Hsp90 that were highest in individuals with the best oral health. We hypothesized that similar antibodies to pathogen chaperones might be protective in periodontitis. This study examined the relationship between antibodies to P. gingivalis HtpG and clinical statuses of healthy and periodontitis-susceptible subjects. We measured the humoral responses (immunoglobulin G [IgG], IgA, and IgM) to peptides of a unique insert (P18) found in Bacteroidaceae HtpG by using a high-throughput, quantitative fluorescence enzyme-linked immunosorbent assay. Indeed, higher levels of IgG class anti-P. gingivalis HtpG P18 peptide (P < 0.05) and P18α, consisting of the N-terminal 16 amino acids of P18 (P < 0.05), were associated with better oral health; these results were opposite of those found with anti-P. gingivalis whole-cell antibodies and levels of the bacterium in the subgingival biofilm. When we examined the same sera for IgA and IgM class antibodies, we found no significant relationship to subject clinical status. The relationship between anti-P18 levels and clinical populations and individual subjects was found to be improved when we normalized the anti-P18α values to those for anti-P18γ (the central 16 amino acids of P18). That same ratio correlated with the improvement in tissue attachment gain after treatment (P < 0.05). We suggest that anti-P. gingivalis HtpG P18α antibodies are protective in periodontal disease and may have prognostic value for guidance of individual patient treatment.
doi:10.1128/CVI.00272-09
PMCID: PMC2786377  PMID: 19793900
25.  Association of Three Bacterial Species and Periodontal Status in Chinese Adults: an Epidemiological Approach ▿  
Journal of Clinical Microbiology  2010;49(1):184-188.
Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia are oral pathogens associated with periodontitis. The association between these three bacteria and periodontal disease has been reported in populations of many countries. However, corresponding data in Chinese populations are still lacking. The aim of this study was to detect these pathogens in subgingival plaque collected from 468 subjects with chronic periodontitis in a group of Chinese adults by using a PCR method and to determine the degree of association between the target bacteria and periodontal status based on logistic regression analysis. A. actinomycetemcomitans, P. gingivalis, and T. forsythia were found in 20.5%, 70.7%, and 77.1% of the subjects, respectively. About one-third (36.1%) of subjects had chronic periodontitis. Upon univariate analysis, age, male gender, current smoking status, diabetes, and the presence of A. actinomycetemcomitans or P. gingivalis were positively associated with chronic periodontitis, whereas education and income exhibited inverse associations with chronic periodontitis. Upon multivariate analysis, education, current smoking status, diabetes, and the presence of A. actinomycetemcomitans and P. gingivalis remained significant. The adjusted odds ratios for having chronic periodontitis were 2.5 and 3.4 in subjects positive for A. actinomycetemcomitans and P. gingivalis, respectively. However, no significant association was observed between the presence of T. forsythia and periodontal status. This study assesses the prevalence of periodontal pathogens and chronic periodontitis and the associations with sociodemographic characteristics among this group of Chinese adults. These findings also suggest that PCR should be considered for field oral epidemiologic studies and may be necessary in investigations presenting major logistic challenges.
doi:10.1128/JCM.01819-10
PMCID: PMC3020465  PMID: 21106792

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