Single nucleotide polymorphisms of pattern recognition receptors and chronic periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is a multifactorial disease influenced partly by genetics. Activation of pattern recognition receptors (PRRs) can lead to the up-regulation of inflammatory pathways, resulting in periodontal tissue destruction. Hence, functional polymorphisms located in PRRs can explain differences in host susceptibility to periodontitis. This study investigated single nucleotide polymorphisms of PRRs including toll-like receptor (TLR)2 (G2408A), TLR4 (A896G), TLR9 (T1486C), TLR9 (T1237C) and CD14 (C260T) in patients with chronic periodontitis and in periodontally healthy subjects. METHODS: One-hundred and fourteen patients with chronic periodontitis and 77 periodontally healthy subjects were genotyped using TaqMan® allelic discrimination assays. Fisher's exact test and chi-square analyses were performed to compare genotype and allele frequencies. RESULTS: The frequency of subjects with the CC genotype of CD14 (C260T) (24.6% in the chronic periodontitis group vs. 13% in the periodontally healthy group) and those expressing the T allele of CD14 (C260T) (CT and TT) (75.4% in the chronic periodontitis group vs. 87% in the periodontally healthy group) was statistically different among groups (p = 0.04). Homozygocity for the C allele of the CD14 (C260T) polymorphism (CC) was associated with a two--fold increased susceptibility to periodontitis (p = 0.04; odds ratio, 2.49; 95% confidence interval, 1.06-6.26). Individuals with the CC genotype of TLR9 (T1486C) (14.9% in the chronic periodontitis group vs. 28.6% in the periodontally healthy group) and those expressing the T allele of TLR9 (T1486C) (CT and TT) (85.1% in the chronic periodontitis group vs. 71.4% in the periodontally healthy group) were also significantly differently distributed between groups without adjustment (p = 0.03). Further analysis of nonsmokers revealed a significant difference in the distribution of genotypes between groups for TLR9 (T1486C; p = 0.017) and CD14 (C260T; p = 0.03), polymorphisms again without adjustment. CONCLUSION: The CC genotype of CD14 (C260T) is related to susceptibility to chronic periodontitis in Caucasians. In addition, differences observed in the distribution of TLR9 (T1486C) genotypes between groups warrant further investigation.

The Subgingival Microbiome Relationship to Periodontal Disease in Older Women.

Understanding of the oral microbiome in relation to periodontal disease in older adults is limited. The composition and diversity of the subgingival microflora and their oligotypes in health and levels of periodontal disease were investigated in this study on older postmenopausal women. The 16S rRNA gene was sequenced using the Illumina MiSeq platform in 1,206 women aged 53 to 81 y. Presence and severity of periodontal disease were defined by Centers for Disease Control and Prevention/American Academy of Periodontology criteria. Composition of the microbiome was determined by 16S rRNA amplicon sequencing and the abundance of taxa described by the centered log2-ratio (CLR) transformed operational taxonomic unit (OTU) values. Differences according to periodontal disease status were determined by analysis of variance with Bonferroni correction. Bacteria oligotypes associated with periodontal disease and health were determined by minimum entropy decomposition and their functions estimated in silico using PICRUSt. Prevalence of none/mild, moderate, and severe periodontal disease was 25.1%, 58.3%, and 16.6%, respectively. Alpha diversity of the microbiome differed significantly across the 3 periodontal disease categories. ß-Diversity differed between no/mild and severe periodontal disease, although considerable overlap was noted. Of the 267 bacterial species identified at ≥0.02% abundance, 56 (20.9%) differed significantly in abundance according to periodontal disease status. Significant linear correlations for pocket depth and clinical attachment level with bacterial amounts were observed for several taxa. Of the taxa differing in abundance according to periodontal disease status, 53% had multiple oligotypes appearing to differ between none/mild and severe periodontal disease. Among older women, taxonomic differences in subgingival microbiome composition and diversity were observed in relation to clinical periodontal disease measures. Potential differences in bacterial subspecies (oligotypes) and their function were also identified in periodontal disease compared with health.

Osteoporosis and oral infection: independent risk factors for oral bone loss.

Studies have suggested that oral bone loss is independently influenced by local and systemic factors, including osteoporosis. This cross-sectional study of 1256 post-menopausal women, recruited from the Buffalo center of the Women's Health Initiative Observational Study, evaluated the influence of oral infection and age on the associations between osteoporosis and oral bone loss. Systemic bone density was measured by dual-energy x-ray absorptiometry. Alveolar crestal height was measured from standardized dental radiographs. Oral infection was assessed from subgingival plaque samples. Total forearm density [beta (SE)= -0.931 (0.447), p=0.038] and presence of Tannerella forsythensis [beta (SE)=0.125 (0.051), p=0.015] were independently associated with mean alveolar height among women aged <70 years after confounder adjustment. Women aged 70+ years had worse oral bone loss, in general, but neither bone density nor oral infection was significantly associated with mean alveolar height in this age group. Systemic bone density and oral infection independently influenced oral bone loss in post-menopausal women aged <70 years.

Tannerella forsythia-produced methylglyoxal causes accumulation of advanced glycation endproducts to trigger cytokine secretion in human monocytes.

The periodontal pathogen Tannerella forsythia has the unique ability to produce methylglyoxal (MGO), an electrophilic compound which can covalently modify amino acid side chains and generate inflammatory adducts known as advanced glycation endproducts (AGEs). In periodontitis, concentrations of MGO in gingival-crevicular fluid are increased and are correlated with the T. forsythia load. However, the source of MGO and the extent to which MGO may contribute to periodontal inflammation has not been fully explored. In this study we identified a functional homolog of the enzyme methylglyoxal synthase (MgsA) involved in the production of MGO in T. forsythia. While wild-type T.forsythia produced a significant amount of MGO in the medium, a mutant lacking this homolog produced little to no MGO. Furthermore, compared with the spent medium of the T. forsythia parental strain, the spent medium of the T. forsythia mgsA-deletion strain induced significantly lower nuclear factor-kappa B activity as well as proinflammogenic and pro-osteoclastogenic cytokines from THP-1 monocytes. The ability of T. forsythia to induce protein glycation endproducts via MGO was confirmed by an electrophoresis-based collagen chain mobility shift assay. Together these data demonstrated that T. forsythia produces MGO, which may contribute to inflammation via the generation of AGEs and thus act as a potential virulence factor of the bacterium.

A Randomized Clinical Trial Evaluating rh-FGF-2/ß-TCP in Periodontal Defects.

Biological mediators have been used to enhance periodontal regeneration. The aim of this prospective randomized controlled study was to evaluate the safety and effectiveness of 3 doses of fibroblast growth factor 2 (FGF-2) when combined with a ß-tricalcium phosphate (ß-TCP) scaffold carrier placed in vertical infrabony periodontal defects in adult patients. In this double-blinded, dose-verification, externally monitored clinical study, 88 patients who required surgical intervention to treat a qualifying infrabony periodontal defect were randomized to 1 of 4 treatment groups-ß-TCP alone (control) and 0.1% recombinant human FGF-2 (rh-FGF-2), 0.3% rh-FGF-2, and 0.4% rh-FGF-2 with ß-TCP-following scaling and root planing of the tooth prior to a surgical appointment. Flap surgery was performed with EDTA conditioning of the root prior to device implantation. There were no statistically significant differences in patient demographics and baseline characteristics among the 4 treatment groups. When a composite outcome of gain in clinical attachment of 1.5 mm was used with a linear bone growth of 2.5 mm, a dose response pattern detected a plateau in the 0.3% and 0.4% rh-FGF-2/ß-TCP groups with significant improvements over control and 0.1% rh-FGF-2/ß-TCP groups. The success rate at 6 mo was 71% in the 2 higher-concentration groups, as compared with 45% in the control and lowest treatment groups. Percentage bone fill in the 2 higher-concentration groups was 75% and 71%, compared with 63% and 61% in the control and lowest treatment group. No increases in specific antibody to rh-FGF-2 were detected, and no serious adverse events related to the products were reported. The results from this multicenter trial demonstrated that the treatment of infrabony vertical periodontal defects can be enhanced with the addition of rh-FGF-2/ß-TCP (ClinicalTrials.gov NCT01728844).

Recombinant expression and partial characterization of the human formyl peptide receptor.

FMLP-receptor DNA was expressed in Escherichia coli. The expressed product could specifically bind FMLP. This is the first-reported expression of a functional FMLP receptor in Escherichia coli. We confirm that receptor glycosylation is not essential for ligand binding. A deletion mutant did not bind FMLP, suggesting that the deleted portion plays a role in ligand binding.

Periodontal disease and risk of cerebrovascular disease: the first national health and nutrition examination survey and its follow-up study.

BACKGROUND: Periodontal disease has been found to be a potential risk factor for coronary heart disease. However, its association with cerebrovascular accidents (CVAs) is much less studied. METHODS: This study examines the association between periodontal disease and CVA. The study cohort comprises 9962 adults aged 25 to 74 years who participated in the First National Health and Nutrition Examination Survey and its follow-up study. Baseline periodontal status was categorized into (1) no periodontal disease, (2) gingivitis, (3) periodontitis, and (4) edentulousness. All CVAs (International Classification of Diseases, Ninth Revision [ICD-9], codes 430-438) were ascertained by hospital records for nonfatal events and death certificates for fatal events. The first CVA, nonfatal or fatal, was used to define incidence. Relative risks were estimated by hazard ratios from the Cox proportional hazard model with adjustment for several demographic variables and well-established cardiovascular risk factors. Weights were used to generate risk estimates. RESULTS: Periodontitis is a significant risk factor for total CVA and, in particular, nonhemorrhagic stroke (ICD-9, 433-434 and 436-438). Compared with no periodontal disease, the relative risks (95% confidence intervals) for incident nonhemorrhagic stroke were 1.24 (0.74-2.08) for gingivitis, 2.11 (1.30-3.42) for periodontitis, and 1.41 (0.96-2.06) for edentulousness. For total CVA, the results were 1.02 (0.70-1.48) for gingivitis, 1.66 (1.15-2.39) for periodontitis, and 1.23 (0.91-1.66) for edentulousness. Increased relative risks for total CVA and nonhemorrhagic stroke associated with periodontitis were also seen in white men, white women, and African Americans. Similar results were found for fatal CVA. CONCLUSION: Periodontal disease is an important risk factor for total CVA and, in particular, nonhemorrhagic stroke.

Periodontal disease and NIDDM in Pima Indians.

The goal of this study was to determine the prevalence and incidence of periodontal disease and its relationship with non-insulin-dependent diabetes mellitus (NIDDM). Two thousand two hundred seventy-three Pima Indians (949 men, 1324 women) aged greater than or equal to 15 yr from the Gila River Indian Community in Arizona were examined between 1983 and 1989. Periodontal disease was diagnosed by tooth loss and by percentage of interproximal crestal alveolar bone loss ascertained from panoramic radiography. Subjects with little or no evidence of periodontal disease were classified as nondiseased. Thus, the incidence of advanced periodontal disease was determined. The age- and sex-adjusted prevalence of periodontal disease at first dental examination was 60% in subjects with NIDDM and 36% in those without. Twenty-two new cases developed in a subset of 701 subjects (272 men, 429 women) aged 15-54 yr who initially had little or no evidence of periodontal disease and had at least one additional dental examination. The incidence of periodontal disease in this group was similar in men and women (incidence-rate ratio 1.0, 95% confidence interval [Cl] 0.5-1.9, controlled for age and diabetes). Higher age predicted a greater incidence of periodontal disease (chi 2 = 30.6, df = 3, P less than 0.001, controlled for sex and diabetes). The rate of periodontal disease in subjects with diabetes was 2.6 times (95% Cl 1.0-6.6, controlled for age and sex) that observed in those without. Although periodontal disease was common in nondiabetic Pima Indians, in whom most of the incident cases occurred, diabetes clearly conferred a substantially increased risk. Thus, periodontal disease should be considered a nonspecific complication of NIDDM.

Structures involved in the interaction of Porphyromonas gingivalis fimbriae and human lactoferrin.

The ability of laboratory and clinical strains of Porphyromonas gingivalis to bind lactoferrin has been assessed (FEMS Immunology and Medical Microbiology, 1996, 14, 135-143). Relative binding for P. gingivalis to lactoferrin varies among strains from 3.78 to 26.62%. We also observed that fimbriated strains of P. gingivalis bind more strongly to lactoferrin as compared to nonfimbriated strains of P. gingivalis. This observation led us to study fimbrial interaction with human lactoferrin and the fine structure of these interactions. Binding of iodinated purified fimbriae was studied using an overlay assay. Iodinated fimbriae bind specifically and strongly to human lactoferrin. When various sugars were used to inhibit binding, only N-acetylgalactosamine and fucose were inhibitory. To confirm further that oligosaccharide of lactoferrin is involved in the interaction, lactoferrin was chemically deglycosylated, and fimbriae failed to bind deglycosylated lactoferrin. Antifimbriae, as well as four antipeptide antibodies against different regions of the P. gingivalis fimbrillin, were used to inhibit the interaction. Antipeptide E, directed against amino acids 81-98 (AAGLIMTAEPKTIVLKAG-C), was found to be the most effective inhibitor for the lactoferrin-fimbriae interaction. These results suggest that the binding of P. gingivalis cells to lactoferrin is lectin like, directed to a oligosaccharide of lactoferrin. Furthermore, these studies suggest that the region of fimbriae that binds to lactoferrin is the N-terminus of the molecule. It is likely that binding of lactoferrin to P. gingivalis cells results in antimicrobial activity directed against these cells by virtue of its ability to deprive the bacterial cell of needed iron.

A rapid, semi-automated procedure for the evaluation of leukocyte locomotion in the micropore filter assay.

A commerically available bacterial colony counter has been modified to allow rapid, accurate, semi-automated evaluation of cell numbers in the micropore filter assay for chemotaxis. The method is valuable for objective, rapid evaluation of cell counts at various levels through the filter, as well as counts on the distal surface of the filter. Coupled with a programmable calculator, this instrument had made feasible a new method of assessing random migration by the regression line analysis, which discriminates between migration rate and mass migration of cells. This combination of equipment may thus serve as a considerable time saving accessory to laboratories engaged in cell locomotion research, but also will allow more rigorous assessment of differences among specific populations of cells.

In vitro antiplaque properties of a series of alkyl bis(biguanides).

A series of eight alkyl bis(biguanide) analogues of alexidine, N,N''''-1,6-hexanediyl bis[N'-(2-ethylhexyl)imidodicarbonimidic diamide] (1), was prepared. Five of these analogues constituted a series isolipophilic with 1 but with varying bridge length between biguanide moieties. The compounds were evaluated in vitro for antibacterial and antiplaque properties against Streptococcus mutans, Actinomyces viscosus, and Actinomyces naesludii. One analogue, N N,''''-1,6-hexanediyl bis[N'(n-octyl)imidodicarbonimidic diamide], appeared to be more effective than either 1 or chlorhexidine against this spectrum of dental plaque forming microorganisms.

Potential salicylamide antiplaque agents: in vitro antibacterial activity against Actinomyces viscosus.

A series of 55 salicylamides, including 3,5-dibromo-, 5-n-alkyl-, and 5-n-acylsalicyloyl derivatives of various anilines, heterocyclic amines, benzylamines, and alkylamines, was synthesized and evaluated for in vitro antibacterial activity against Actinomyces viscosus, an adherent oral microorganism implicated in periodontal disease. The in vitro minimum inhibitory concentrations of 15 4'-bromosalicylanilides were found to correlate (r = 0.92) with estimated log D values. Several nonhalogenated salicylanilides, such as 5-n-hexyl- (40) and 5-n-decanoyl-4'-nitrosalicylanilide (47), were found to exhibit higher levels of in vitro antibacterial activity against a number of Actinomycetes than did tribromsalan (1) or fluorophene (2).

5-(Alkylsulfonyl)salicylanilides as potential dental antiplaque agents.

A series of 22 5-(alkylsulfonyl)salicylanilides was synthesized and evaluated for in vitro antibacterial and antiplaque activity against Actinomyces viscosus and Streptococcus mutans, adherent microorganisms implicated in periodontal disease and dental caries. The minimum inhibitory concentrations of 25 salicylanilides (including 5-acyl-, 5-alkyl-, and 5-(alkylsulfonyl)-4'-bromo- and -4'-(trifluoromethyl)salicylanilides) were found to correlate (r = 0.94) with estimated log D values. Several salicylanilides, such as 5-(decylsulfonyl)- and 5-(dodecylsulfonyl)-4'-(trifluoromethyl)salicylanilides (15 and 19) were found to exhibit high levels of in vitro antibacterial and antiplaque activity against A. viscosus and S. mutans.

Substituted 2-(2-hydroxyphenyl)benzimidazoles as potential agents for the control of periodontal diseases.

A series of 16 substituted 2-(2-hydroxphenyl)benzimidazoles was synthesized and evaluated in vitro for antibacterial activity against bacteria associated with periodontal diseases. Several compounds demonstrated a high level of activity, in tube dilution assay, against Actinomycetes viscosus and Bacteriodes gingivalis. These results indicate that several of these compounds may serve as topical antibacterial agents for the control of acute marginal inflammatory gingivitis and periodontitis.

Porphyromonas gingivalis surface components induce interleukin-1 release and tyrosine phosphorylation in macrophages.

The aim of the present study was to characterize the responses of macrophages to surface antigens of Porphyromonas gingivalis. Native fimbriae, full-length recombinant fimbrillin, and a lectin-like 12-kDa antigen all stimulated BALB/c peritoneal macrophages to secrete interleukin (IL)-1 beta. The antigens induced similar patterns of tyrosine phosphorylation; proteins in approximately 35-46 kDa range of undetermined identities were phosphorylated in the macrophages. The abilities of the surface antigens to induce IL-1 beta were markedly attenuated by tyrosine kinase inhibitors. This inhibition correlated with inhibition of the induced phosphorylation of specific macrophage proteins at tyrosine. The data suggest that tyrosine kinase(s) plays an important role in the regulatory intracellular signaling mechanisms by which P. gingivalis surface antigens can mediate certain responses in macrophages.

Host responses in periodontal diseases.

Great progress has been made in our understanding of the pathogenesis of periodontal disease, the primary role of bacteria as etiologic agents, and the critical modifying role of host responses. It is useful to consider several stages in the pathogenesis of periodontal disease - (a) colonization, (b) invasion, (c) destruction, and (d) healing - and to place into perspective the various host responses as they may affect each of these four stages (Table 5). With respect to colonization, although very little direct evidence is available, it is reasonable to suggest that antibodies, either secretory or serum-derived, acting by virtue of their ability to block attachment, could inhibit colonization by immune reduction of adherence mechanisms. With respect to invasion of the tissue, it appears that phagocytes, particularly the neutrophils, are important, acting in concert with opsonic antibody and complement in ingesting and killing the periodontal microflora before or during the early invasive process. A major advance in our understanding of the pathogenesis of periodontal diseases is the realization that the virulence of periodontopathic bacteria relates to their leukaggressive properties, allowing them to evade neutrophil protective mechanisms. Invasion of the periodontal tissues by bacterial products may be inhibited by the complexing of these products with antibody with the formation of antigen-antibody complexes that are phagocytosed and digested, particularly by scavenger phagocytes such as the macrophage. With respect to the destructive phase of periodontal disease, it is clear that the direct effect of lymphocytes mediated either through direct cytotoxic activity, or through biologically-active destructive lymphokines (such as alpha-lymphotoxin and osteoclast activating factor), can lead to tissue destruction. Macrophages, through the production of monokines, collagenase, and reactive oxygen species, can also lead to tissue destruction. The direct effects of bacterial toxins or enzymes which can lead to tissue destruction can be inhibited by complexing with antitoxic or enzyme-neutralizing antibodies. With respect to healing and fibrosis, very little direct information is available; however, it is possible that the lymphocytes and macrophages affect fibrosis by the production of chemotactic factors for fibroblasts which would be expected to bring them to the area of periodontal inflammation and also by production of fibroblast-activating factors, which then cause the fibroblasts to proliferate and produce collagen which replaces lost collagen or results in fibrosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Antimicrobial properties of hydrogen peroxide and sodium bicarbonate individually and in combination against selected oral, gram-negative, facultative bacteria.

The topical application of hydrogen peroxide (H2O2) and sodium bicarbonate (NaHCO3), individually and in combination, has been used empirically in the treatment of periodontal diseases. In this study, we examined both minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of these disinfectants individually and in combination against selected facultative, Gram-negative oral bacteria in a microtiter dilution assay. The bacteria studied included Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Eikenella corrodens, and Capnocytophaga gingivalis. These bacteria exhibited MBC (one hr) values ranging from 75 mumol/L to greater than 10 mmol/L and MIC from less than 5 to 500 mumol/L for H2O2. The tested bacteria exhibited MIC values for NaHCO3 of from 23 to 182 mmol/L, and the MBC (one hr) exceeded 728 mmol/L for most of the strains examined. At sublethal (sub-MIC) concentrations, sodium bicarbonate antagonized the ability of H2O2 to inhibit bacterial growth in MIC assays, but sublethal concentrations of H2O2 had no effect on the MIC values of NaHCO3. Lethal concentrations of H2O2 and NaHCO3 exhibited synergistic antimicrobial activity in combination in one-hour bactericidal assays. Since the bactericidal properties of these antimicrobial agents are synergistic, we conclude that it may be rational to use them in combination to treat certain forms of periodontal disease. Also, lower and perhaps safer concentrations of H2O2 can be used in combination with NaHCO3 when oxidative antimicrobial chemotherapy is indicated.

Genetic heterogeneity of Porphyromonas (Bacteroides) gingivalis by genomic DNA fingerprinting.

This study describes the use of total genomic DNA fingerprinting with the use of restriction endonucleases to characterize clinical isolates of Porphyromonas gingivalis (Bacteroides gingivalis) obtained from patients with periodontitis or with root-canal infections. The majority of independent isolates had a unique DNA fingerprint, indicating extensive genetic heterogeneity within this species. Twenty-nine distinct DNA fingerprints were found among the 33 isolates investigated. This is in contrast to biotyping and serotyping, where only one type and three types, respectively, have been reported. The observed heterogeneity indicates that DNA fingerprinting is a sensitive measure of genetic dissimilarity between P. gingivalis isolates and is able to characterize individual isolates. These results have ecological implications, indicating that there is considerable natural diversity in the global population of P. gingivalis, and that there are likely to be relatively large numbers of genetically distinct clonal lines. Furthermore, DNA fingerprinting is a sensitive and powerful tool for longitudinal and cross-sectional epidemiological studies. This technique provides far greater discrimination between isolates than either biotyping or serotyping, and will be most helpful in, for example, the analysis of distribution of clonal lines within one periodontal patient, or the analysis of the transmission to and turnover of strain populations within a patient population, since the probability of two strains with the same DNA fingerprint being found by chance is small.